Collinsella urealyticum sp. nov., a urease-positive bacterial strain isolated from swine faeces.

A novel actinobacterial strain, designated AGMB00827T, was isolated from swine faeces. Strain AGMB00827T was obligately anaerobic, Gram-stain-positive, non-motile, non-spore-forming and rod-shaped bacterium. Comparative analyses based on the 16S rRNA gene and whole genome sequence revealed that strain AGMB00827T was affiliated to the genus Collinsella, and was most closely related to Collinsella vaginalis Marseille-P2666T (= KCTC 25056T). Biochemical analysis showed strain AGMB00827T was negative for catalase and oxidase. Interestingly, strain AGMB00827T possessed urease activity, which was determined by traditional methods (API test and Christensen's urea medium), unlike related strains. Furthermore, the major cellular fatty acids (> 10%) of the isolate were C18:1 ω9c, C16:0, C16:0 DMA and C18:2 ω9,12c DMA. Based on the whole genome sequence analysis, the DNA G + C content of strain AGMB00827T was 52.3%, and the genome size and numbers of rRNA and tRNA genes were 1,945,251 bp, 3 and 46, respectively. The average nucleotide identity and digital DNA-DNA hybridization values between strain AGMB00827T and C. vaginalis KCTC 25056 T were 71.0 and 23.2%, respectively. Additionally, the genome analysis revealed that strain AGMB00827T possesses urease gene cluster including ureABC and ureDEFG while the related strains do not have those genes, which is consistent with the urease activity. On the basis of polyphasic taxonomic approach, strain AGMB00827T represents a novel species within the genus Collinsella, for which the name Collinsella urealyticum sp. nov. is proposed. The type strain is AGMB00827T (= KCTC 25287T = GDMCC 1.2724T).

Dietary yeast cell wall enhanced intestinal health of broiler chickens by modulating intestinal integrity, immune responses, and microbiota.

This study was conducted to determine the effects of dietary yeast cell wall (YCW) on growth performance, intestinal health, and immune responses of broiler chickens. In a randomized completely block design (block: initial body weight), a total of 800 broilers (Ross 308; 45.18 ± 3.13 g of initial body weight) were assigned to 2 dietary treatments (40 birds/pen; 10 replicates/treatment) and fed for 5 wk: 1) a basal broiler diet based on corn-soybean meal (CON) and 2) CON + 0.05% dietary YCW. Growth performance was measured at intervals in 3 phase feed program. On the final day of the study, one bird per pen was randomly selected and euthanized for sample collection. Broilers fed YCW had decreased (P < 0.05) feed conversion ratio during the grower phase compared with those fed CON. The YCW increased (P < 0.05) villus height to crypt depth ratio in the duodenum, jejunum, and ileum compared with the CON. In addition, the YCW tended to higher (P < 0.10) number of goblet cells in the duodenum than in the CON. Broilers fed YCW had increased (P < 0.05) serum TGF- ß1, ileal gene expression of the claudin family, and relative abundance of Lactobacillus, Prevotella, and Enterococcus compared with the CON, but decreased serum TNF-α (P < 0.05), IL-1ß (P < 0.05), and IL-6 (P < 0.10), ileal gene expression of IL-6 (P < 0.05), and relative abundance of Clostridium (P < 0.05). The present study demonstrated that the addition of dietary YCW in broiler diets enhanced the intestinal health of broiler chickens and may be associated with modulated intestinal morphology and integrity by upregulating tight junction-related protein gene expression and modifying the ileal microbiota. In addition, dietary YCW modulated immune responses and inflammatory cytokine gene expression in the ileum.

Phytogenic feed additives alleviate pathogenic Escherichia coli-induced intestinal damage through improving barrier integrity and inhibiting inflammation in weaned pigs.

BACKGROUND: This study was conducted to investigate the effects of each phytogenic feed additive (PFA; PFA1, bitter citrus extract; PFA2, a microencapsulated blend of thymol and carvacrol; PFA3, a mixture of bitter citrus extract, thymol, and carvacrol; PFA4, a premixture of grape seed, grape marc extract, green tea, and hops; PFA5, fenugreek seed powder) on the growth performance, nutrient digestibility, intestinal morphology, and immune response in weaned pigs infected with Escherichia coli (E. coli). RESULTS: A total of 63 4-week-old weaned pigs were placed in individual metabolic cages and assigned to seven treatment groups. The seven treatments were as follows: 1) NC; basal diet without E. coli challenge, 2) PC; basal diet with E. coli challenge, 3) T1; PC + 0.04% PFA1, 4) T2; PC + 0.01% PFA2, 5) T3; PC + 0.10% PFA3, 6) T4; PC + 0.04% PFA4, 7) T5; PC + 0.10% PFA5. The experiments lasted in 21 d, including 7 d before and 14 d after the first E. coli challenge. In the E. coli challenge treatments, all pigs were orally inoculated by dividing a total of 10 mL of E. coli F18 for 3 consecutive days. The PFA-added groups significantly increased (P < 0.05) average daily gain and feed efficiency and decreased (P < 0.05) the fecal score at d 0 to 14 post-inoculation (PI). Tumor necrosis factor α was significantly lower (P < 0.05) in the PFA-added groups except for T1 in d 14 PI compared to the PC treatment. The T3 had a higher (P < 0.05) immunoglobulin G and immunoglobulin A concentration compared to the PC treatment at d 7 PI. Also, T3 showed significantly higher (P < 0.05) villus height:crypt depth and claudin 1 expression in ileal mucosa, and significantly down-regulated (P < 0.05) the expression of calprotectin compared to the PC treatment. CONCLUSIONS: Supplementation of PFA in weaned pigs challenged with E. coli alleviated the negative effects of E. coli and improved growth performance. Among them, the mixed additive of bitter citrus extract, thymol, and carvacrol showed the most effective results, improving immune response, intestinal morphology, and expression of tight junctions.

Olsenella intestinalis sp. nov., isolated from cow feces.

A Gram-stain-negative, anaerobic, non-motile, rod-shaped bacterium, designated as BGYT1T, was isolated from the feces of a cow in Andong, Republic of Korea. It was studied using a polyphasic method to determine its taxonomic position. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain BGYT1T formed a lineage within the genus Olsenella and was most closely related to O. umbonate KCTC 15140T (98.2%). The complete genome sequence of strain BGYT1T was 2,476,083 bp long with a G + C content of 66.9 mol% and contained 1835 genes and 8 contigs. The N50 value was 604,117 bp. There were 50 tRNAs, 6 rRNAs (5S, 16S, 23S), 1778 CDSs and 2 BGCs and 1 tmRNA. The values for ANI (76.8%), AAI (67.3%), and dDDH (22.2%) compared to the closest related species were all below the threshold for bacterial species delineation. In addition, genes encoding the cell wall degrading enzymes such as chitinases, ß-1,3 glucanases, and proteases were also detected. The strain was able to grow at pH 6.0-8.0 (optimum, pH 7.0), in the presence of 0.5-1.5% NaCl (optimum, 0.5%, w/v) and at the temperature range of 35-40 °C (optimum, 35 °C). The predominant fatty acids were C16:0 DMA (20.2%), C16:0 (20.2%), C18:0 (10.5%) and C18:1 cis 9 (17.0%). The polar lipids consisted of an unidentified phospholipid, four unidentified glycolipids and three unidentified lipids. Based on its phenotypic analyses, phylogenetic and physiological characteristics, strain BGYT1T represented a novel species within the genus Olsenella, for which the name Olsenella intestinalis sp. nov. is proposed. The type strain is BGYT1T (= KCTC 25379T = GDMCC 1.3011T).

Effects of different Bacillus licheniformis and Bacillus subtilis ratios on nutrient digestibility, fecal microflora, and gas emissions of growing pigs.

The objective of this study was to evaluate the effects of different mixing ratios of Bacillus licheniformis and Bacillus subtilis in diets on nutrient digestibility, fecal microflora, and odor gas emissions of growing pigs. A total of four crossbred ([Landrace × Yorkshire] × Duroc) barrows with average body weight (BW) of 41.2 ± 0.7 kg were randomly allotted four diets over four periods in a 4 × 4 Latin square design. Treatments were as follows: Control (CON, basal diet), CON + 0.2% probiotic complex (L4S6, B. licheniformis and B. subtilis at a 4:6 ratio), CON + 0.2% probiotic complex (L5S5, B. licheniformis and B. subtilis at a 5:5 ratio), CON + 0.2% probiotic complex (L6S4, B. licheniformis and B. subtilis at a 6:4 ratio). Dietary probiotic supplementation showed higher crude protein (CP) digestibility values and lower Escherichia coli counts in fecal samples than the CON group (p < 0.05). There was no significant difference in NH3 or H2S emission until day 3. The positive effect of H2S and NH3 emissions was detected earlier with the L4S6 and L5S5 compared to the L6S4, which had a lower ratio of B. subtilis. Both the L4S6 and L5S5 probiotic complexes significantly decreased the fecal H2S and NH3 emission in days 4 and 6 (p < 0.05). On day 7, all probiotic complexes decreased (p < 0.05) H2S and NH3 emissions than the CON group. Our results agreed that the dietary supplementation of Bacillus licheniformis and Bacillus subtilis complexes in growing pigs can significantly improve CP digestibility and reduce fecal E. coli counts, NH3 and H2S emissions. Notably, the higher mixing ratio of Bacillus subtilis in probiotic supplementation is more effective in reducing the odor of manure.

Faecalicatena faecalis sp. nov., a moderately alkaliphilic bacterial strain isolated from swine faeces.

An obligately anaerobic, Gram-stain-positive, non-motile, non-spore-forming and rod-shaped strain AGMB00832T was isolated from swine faeces. Phylogenetic analysis based on the 16S rRNA gene, together with the housekeeping genes, gyrB and rpoD, revealed that strain AGMB00832T belonged to the genus Faecalicatena and was most closely related to Faecalicatena orotica KCTC 15331T. In biochemical analysis, strain AGMB00832T was shown to be negative for catalase, oxidase and urease. Furthermore, the isolate was positive for ß-glucosidase, ß-glucuronidase, glutamic acid decarboxylase, proline arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase. The major cellular fatty acids (> 10%) of the isolate were C14:0, C16:0 and C18:1ω11t DMA. Based on the whole genome sequence analysis, the DNA G + C content of strain AGMB00832T was 44.2 mol%, and the genome size and numbers of rRNA and tRNA genes were 5,175,159 bp, 11 and 53, respectively. The average nucleotide identity and digital DNA-DNA hybridization values between strain AGMB00832T and related strains were ≤ 77.4 and 22.5%, respectively. Furthermore, the genome analysis revealed the presence of genes for alkaline shock protein 23 and cation/proton antiporters, which may facilitate growth of strain AGMB00832T in alkaline culture condition. On the basis of polyphasic taxonomic approach, strain AGMB00832T represents a novel species within the genus Faecalicatena, for which the name Faecalicatena faecalis sp. nov. is proposed. The type strain is AGMB00832T (= KCTC 15946T = NBRC 114613T).

Potential use of ground brown rice for weanling pigs.

The purpose of the current study was to assess the effects of substituting corn with ground brown rice on growth performance, immune status, and gut microbiota in weanling pigs. Seventy-two weanling pigs (28 d old with 6.78 ± 0.94 kg body weight [BW]) were randomly allotted to two dietary treatments with six pens and six pigs (three barrows and gilts) per pen within a randomized complete block design. The control pigs were fed a typical diet for weanling pigs based on corn and soybean meal diet (control diet: CON), and the other pigs were fed a formulated diet with 100% replacement of corn with ground brown rice for 35d (treatment diet: GBR). Growth performance, immune status, and gut microbiota of weanling pigs were measured. The substitution of corn with GBR did not affect growth performance or diarrhea frequency. Additionally, there were no differences in white blood cell number, hematocrit, cortisol, C-reactive protein, and serum tumor necrosis factor-alpha levels between pigs fed CON or GBR for the first 2 wk after weaning. However, weanling pigs fed GBR had lower (P < 0.05) serum transforming growth factor-beta 1 level than those fed CON. Furthermore, weanling pigs fed GBR had increased (P < 0.05) relative abundance of phylum Firmicutes and genus Lactobacillus and Streptococcus and decreased (P < 0.05) relative abundance of phylum Bacteroidetes and genus Clostridium and Prevotella in the gut microbiota compared with those fed CON. In conclusion, there was no significant difference in growth performance when corn was replaced with ground brown rice in diets for weanling pigs. Furthermore, the substitution of corn with ground brown rice in weaning diet modulated immune status and gut microbiota of pigs by increasing beneficial microbial communities and reducing harmful microbial communities. Overall, ground brown rice-based diet is a potential alternative to corn-based diet without negative effects on growth performance, immune status, and gut microbiota changes of weanling pigs.

Rice as an alternative feed ingredient in swine diets.

It has become important to explore alternative feed ingredients to reduce feed costs, which are burdensome for livestock production. In addition, it is desirable to find efficient and functional alternative ingredients for traditional feed ingredients in pig diets, considering the stress and sensitivity of disease of pig. Rice is produced around the world like corn that is the typical energy source in pig diets. Although the nutritional quality varies depending on the degree of milling, rice, except whole grains (paddy rice), contains more starch than corn and its structure and granule size are easier to digest than corn. In addition, the fact that rice has fewer non-starch polysaccharides (NSP) and anti-nutritional factors (ANFs) is also effective in improving digestibility and various polyphenols in rice can help modulation of immune responses, which can be beneficial to the gastrointestinal environment and health of pig. Many studies have been conducted on rice focusing on things such as degree of milling, substitution rates of corn, granule size, and processing methods. Most results have shown that rice can be partially or completely used to replace corn in pig diets without negatively affecting pig growth and production. While further research should focus on the precise biological mechanisms at play, it was confirmed that the use of rice could reduce the use of antibiotics and pig removal and protect pigs from gastrointestinal diseases including diarrhea. From this point of view, rice can be evaluated as a valuable feed ingredient for swine diets.

Effects of dietary inactivated probiotics on growth performance and immune responses of weaned pigs.

This experiment was performed to verify whether dietary heat-killed Lactobacillus rhamnosus (LR) improves growth performance and modulates immune responses of weaned pigs. Ninety-six weaned pigs ([Landrace × Yorkshire] × Duroc; 6.95 ± 0.25 kg body weight [BW]; 28 d old) were randomly allocated to four treatments: 1) a basal diet without heat-killed LR (CON), 2) T1 (CON with 0.1% heat-killed LR), 3) T2 (CON with 0.2% heat-killed LR), and 4) T3 (CON with 0.4% heat-killed LR). Each treatment had six pens with four pigs (6 replicates per treatment) in a randomized completely block design. The heat-killed LR used in this study contained 1 × 109 FU/g of LR in a commercial product. Pigs were fed each treatment for four weeks using a two-phase feeding program to measure growth performance and frequency of diarrhea. During the last week of this study, all diets contained 0.2% chromic oxide as an indigestible marker. Fecal sampling was performed through rectal palpation for the consecutive three days after the four adaptation days to measure apparent total tract digestibility (ATTD) of dry matter, crude protein, and gross energy (GE). Blood sampling was also performed on day 1, 3, 7, and 14 after weaning to measure immune responses such as serum tumor necrosis factor-α (TNF-α), transforming growth factor-ß1 (TGF-ß1), C-reactive protein (CRP), and cortisol. The heat-killed LR increased (p < 0.05) growth rate, feed efficiency, and ATTD of GE for overall experimental period compared with CON, but reduced (p < 0.05) post-weaning diarrhea. In addition, pigs fed diets contained heat-killed had lower concentrations of serum TNF-α (d 7; p < 0.05), TGF-ß1 (d 7; p < 0.10), and cortisol (d 3 and 7; p < 0.05) than pigs fed CON. In conclusion, dietary heat-killed LR improved growth rate, modified immune responses of weaned pigs, and alleviated post-weaning diarrhea.

Effects of dietary protease on immune responses of weaned pigs.

This experiment was conducted to investigate effects of dietary protease on immune responses of weaned pigs. Weaned pigs (n = 75; 7.06 ± 0.18 kg BW; 28 d old) were randomly assigned to 3 treatments (5 pigs/pen; 5 pens/treatment). Dietary treatments were positive control, a diet with required protein level (PC), negative control, a diet with lower protein level than PC (NC), and NC + 0.02% dietary protease (PRO). The dietary protease used in this experiment was a commercial product containing 75,000 protease units/g derived from Nocardiopsis prasina produced in Bacillus licheniformis. The dietary treatments did not contain any ingredients or additives that may provide antibacterial or physiological effects. Pigs were fed respective dietary treatments for 6 weeks. Blood was collected from randomly selected 2 pigs in each pen on d 1, 3, 7, and 14 after weaning. Measurements were number of white blood cells (WBC), tumor necrosis factor-α (TNF-α), transforming growth factor-ß1 (TGF-ß1), and C-reactive protein (CRP). Pigs fed PRO had lower WBC on d 7 (14.84 vs 20.42 × 103/µL; p < 0.05) and TNF-α on d 7 (618 vs 889 pg/mL; p = 0.085) and 14 (437 vs 576 pg/mL; p = 0.069) than those fed NC, but there were no differences on WBC and TNF-α between PC and PRO. Pigs fed PRO had lower TGF-ß1 on d 3 (630 vs. 1,588 and 1,396 pg/mL; p < 0.05) than those fed PC and NC. However, no differences were found on CRP among dietary treatments. In conclusion, addition of dietary protease reduced inflammatory immune responses of weaned pigs.

Peribacillus faecalis sp. nov., a moderately halophilic bacterium isolated from the faeces of a cow.

A Gram-stain-positive, facultatively anaerobic, endospore-forming, rod-shaped strain, AGMB 02131T, which grew at 20-40 °C (optimum 30 °C), pH 3.0-11.0 (optimum pH 4.0) and in the presence of 0-18 % (w/v) NaCl (optimum 10 %), was isolated from a cow faecal sample and identified as a novel strain using a polyphasic taxonomic approach. The phylogenetic analysis based on 16S rRNA gene sequences along with the whole genome (92 core gene sets) revealed that AGMB 02131T formed a group within the genus Peribacillus, and showed the highest sequence similarity with Peribacillus endoradicis DSM 28131T (96.9 %), following by Peribacillus butanolivorans DSM 18926T (96.6 %). The genome of AGMB 02131T comprised 70 contigs, the chromosome length was 4 038 965 bp and it had a 38.5 % DNA G+C content. Digital DNA-DNA hybridization revealed that AGMB 02131T displayed 21.4 % genomic DNA relatedness with the most closely related strain, P. butanolivorans DSM 18926T. AGMB 02131T contains all of the conserved signature indels that are specific for members of the genus Peribacillus. The major cellular fatty acids (>10 %) of AGMB 02131T were C18 : 1ω9c, C18:0 and C16 : 0. The major polar lipids present were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. On the basis of the phenotypic, phylogenetic, genomic and chemotaxonomic features, AGMB 02131T represents a novel species of the genus Peribacillus, for which the name Peribacillus faecalis sp. nov. is proposed. The type strain is AGMB 02131T (=KCTC 43221T=CCTCC AB 2020077T).

The immunomodulatory effect of antimicrobial peptide HPA3P restricts Brucella abortus 544 infection in BALB/c mice.

The discovery of antimicrobial peptides (AMPs) in recent years has been promising for the treatment of multidrug resistant pathogenic microbes. Brucellosis is still considered one of the most common zoonoses in the world. In this study, we evaluated the effect HPA3P peptide in the bacterial uptake and intracellular growth of Brucella abortus (B. abortus) 544 in murine macrophages RAW 264.7. HPA3P was further utilized in a mouse model for infection and treatment. This peptide did not show cytotoxicity or bactericidal effect to B. abortus. However, it inhibited bacterial internalization at 0, 15 and 30 min incubation at two different doses at 12 and 24 µM as well as reduced intracellular growth after 2, 24 and 48 h incubation. Mice treated with HPA3P demonstrated a significant 1.01-log reduction (P < 0.0001) and spleen weight reduction compared to the nanocarrier control (P < 0.01). Significant increases in key cytokines Interferon-γ (IFN-γ) and Tumor necrosis factor (TNF) at 3, 7 and 14 days post-infection were observed in HPA3P treated mice similar to the antibiotic control group with both compared to the nanocarrier control. Monocyte chemoattractant protein-1 (MCP-1) was also heightened at 14 days post-infection. Histopathological analysis also suggests reduced bacterial granuloma in the liver and spleens of HPA3P treated group compared with the nanocarrier control group. In this study, the modulation of crucial cytokines IFN-γ and TNF might have led to a considerable reduction in the proliferation of B. abortus in a mouse model of brucellosis. Further investigation might be required to maximize the efficacy of HPA3P treatment in murine brucellosis.

A Tetraspecific VHH-Based Neutralizing Antibody Modifies Disease Outcome in Three Animal Models of Clostridium difficile Infection.

Clostridium difficile infection (CDI), a leading cause of nosocomial infection, is a serious disease in North America, Europe, and Asia. CDI varies greatly from asymptomatic carriage to life-threatening diarrhea, toxic megacolon, and toxemia. The incidence of community-acquired infection has increased due to the emergence of hypervirulent antibiotic-resistant strains. These new strains contribute to the frequent occurrence of disease relapse, complicating treatment, increasing hospital stays, and increasing morbidity and mortality among patients. Therefore, it is critical to develop new therapeutic approaches that bypass the development of antimicrobial resistance and avoid disruption of gut microflora. Here, we describe the construction of a single heteromultimeric VHH-based neutralizing agent (VNA) that targets the two primary virulence factors of Clostridium difficile, toxins A (TcdA) and B (TcdB). Designated VNA2-Tcd, this agent has subnanomolar toxin neutralization potencies for both C. difficile toxins in cell assays. When given systemically by parenteral administration, VNA2-Tcd protected against CDI in gnotobiotic piglets and mice and to a lesser extent in hamsters. Protection from CDI was also observed in gnotobiotic piglets treated by gene therapy with an adenovirus that promoted the expression of VNA2-Tcd.

A chimeric protein comprising the glucosyltransferase and cysteine proteinase domains of toxin B and the receptor binding domain of toxin A induces protective immunity against Clostridium difficile infection in mice and hamsters.

Clostridium difficile is the major cause of hospital-acquired infectious diarrhea and colitis in developed countries. The pathogenicity of C. difficile is mainly mediated by the release of 2 large potent exotoxins, toxin A (TcdA) and toxin B (TcdB), both of which require neutralization to prevent disease occurrence. We have generated a novel chimeric protein, designated mTcd138, comprised of the glucosyltransferase and cysteine proteinase domains of TcdB and the receptor binding domain of TcdA and expressed it in Bacillus megaterium. To ensure that mTcd138 is atoxic, 2 point mutations were introduced to the glucosyltransferase domain of TcdB, which essentially eliminates toxicity of mTcd138. Parenteral immunizations of mice and hamsters with mTcd138 induced protective antibodies to both toxins and provided protection against infection with the hyper-virulent C. difficile strain UK6.