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PROBIOTICS AND BOTANICAL SUPPLEMENTATIONS ON GUT MICROBIOTA OF WEANED PIGS UNDER DIARRHEAL STRESS

Jinno, Cynthia N.
Advisor(s): Liu, Yanhong

UC Davis Electronic Theses and Dissertations (2022)

Probiotics and botanical extracts are investigated as potential feed additives for weaned pigs to strengthen the intestinal function or overall immunity by improving nutrient digestibility and immunity in weaned pigs. Under proper manipulation, gut microbiota can additionally offer great physiological and immunological benefits to growing pigs, however, gut microbiota yet tends to be overlooked when assessing the viability of potential feed additives to weaned pigs under diarrheal stress. Therefore, three experiments were conducted to determine the impacts of various feed additives (Bacillus (B.) subtilis, B. amyloliquefaciens, and botanical blends) on gut microbiota of newly weaned pigs using a disease challenge model. The first study investigated modifications in fecal and intestinal microbiota of weaned pigs challenged with enterotoxigenic Escherichia coli (ETEC) and compared the gut microbiota when supplemented with B. subtilis DSM 25841 or carbadox, an antibiotic growth promoter. ETEC infection decreased (P < 0.05) microbial richness in feces on d 7 post-inoculation (PI) compared with d 0 before ETEC infection. Relative abundance of Firmicutes was decreased (P < 0.05) while Proteobacteria and Enterobacteriaceae were increased (P < 0.05) in fecal samples of pigs challenged with ETEC on d 7 PI than on d 0. Supplementation of carbadox reduced (P < 0.05) the relative abundance of Lactobacillaceae in feces on d 0 and 21 PI. Lactobacillus was more (P < 0.05) abundant on d 7 PI and Lactobacillaceae was greater (P < 0.05) in relative abundance on d 21 PI in feces from pigs supplemented with B. subtilis than with carbadox. Among intestinal segments, Enterobacteriaceae was more (P < 0.05) abundant in ileum than in jejunum and colon of weaned pigs on d 21 PI. Pigs supplemented with B. subtilis increased (P < 0.05) the relative abundance of gram-positive bacteria than pigs supplemented with carbadox, including Lactobacillaceae and Bifidobacteriaceae in ileal digesta on d 21 PI. Overall, the present study indicated that B. subtilis supplementation modulated the fecal and intestinal microbiota differently from carbadox. The modulation of gut microbiota might contribute to the reduced diarrhea and enhanced intestinal health of ETEC infected pigs. The second study investigated the supplementation of B. amyloliquefaciens on growth performance, diarrhea, systemic immunity, and intestinal microbiota of weaned pigs infected with ETEC and compared the efficacy of B. amyloliquefaciens vs. carbadox on weaned pigs. ETEC infection reduced (P < 0.05) average daily gain (ADG) from d 0 and 21 PI, gain:feed from d 14 and 21 PI, increased (P < 0.05) frequency of diarrhea during the entire experiment and white blood cell and lymphocyte counts on d 7 and 21 PI when positive control was compared with negative control. B. amyloliquefaciens or carbadox supplementation did not affect diarrheal frequency throughout the experiment. However, carbadox supplementation enhanced (P < 0.05) ADG from d 0 to 21 PI than positive control. Pigs fed with carbadox had greater (P < 0.05) monocyte percentage in serum samples on d 7 and 21 PI but lower (P < 0.05) neutrophil percentage on d 21 PI than pigs fed with B. amyloliquefaciens under ETEC challenge. Pigs supplemented with B. amyloliquefaciens had greater (P < 0.05) Shannon index in ileal digesta than pigs fed with carbadox on d 21 PI. Positive control pigs had a greater (P < 0.05) abundance of Bacteroidota and Proteobacteria and lower (P < 0.05) Firmicutes in ileal digesta than negative control on d 21 PI. Carbadox addition increased (P < 0.05) the relative abundances of Firmicutes and Clostridiaceae but reduced (P < 0.05) Bifidobacterium in ileal digesta compared with B. amyloliquefaciens on d 21 PI. The results of the present study indicated that supplementation of B. amyloliquefaciens tended to increase ADG and had limited effects on diarrhea of ETEC infected pigs. However, pigs fed with B. amyloliquefaciens exhibit milder systemic inflammation than control. B. amyloliquefaciens differently modified intestinal microbiota of weaned pigs, compared with carbadox. The third study characterized the impacts of two types of botanical blends on gut microbiota composition and serum and ileal mucosa metabolites in weaned pigs challenged with ETEC. Botanical blends (BB1 and BB2) were comprised of 0.3% capsicum oleoresin and 12% garlic oil extracted from different sources. Throughout the experiment, the relative abundance of Lachnoclostridium was decreased (P < 0.05) in feces as the age of pigs was increased from d -7 to d 21 PI. ETEC infection reduced (P < 0.05) the relative abundance of fecal Faecalibacterium and Prevotella on d 5 and 21 PI when positive control was compared with negative control. On d 21 PI, the relative abundance of Firmicutes and Lactobacillaceae were lower (P < 0.05) in feces of pigs fed with 100 ppm BB1 than pigs in positive control. Pigs supplemented with 100 ppm BB2 had a greater (P < 0.05) relative abundance of Lachnospiraceae in feces than pigs fed with 100 ppm BB1 on d 21 PI. Pigs in positive control had the highest (P < 0.05) Shannon and Chao1 indices in ileal digesta on d 5 PI. Pigs supplemented with 100 ppm BB1 had greater (P < 0.05) abundances of Enterobacteriaceae and Escherichia-Shigella in ileal digesta than pigs in positive control on d 5 PI. The relative abundance of Prevotellaceae was greater (P < 0.05) in cecum of pigs supplemented with 100 ppm BB1 than positive control pigs on d 5 PI. The metabolomics analysis revealed that ETEC infection downregulated pinitol, malic acid, and methionine on d 4 PI and upregulated methionine and guanosine on d 21 PI when positive control was compared with negative control. Supplementation of 100 ppm BB2 upregulated serum pinitol on d 5 PI and serum cholesterol and aminomalonic acids on d 21 PI compared with positive control. In ileal mucosa, asparagine was downregulated by 50 ppm BB2 in comparison to positive control. The present study indicated that supplementation of botanical blends modulated ileal microbiota and serum metabolomics profiles in weaned pigs under ETEC challenge. Overall, feed additives, including probiotics and botanical blends modified gut microbiota of weaned pigs challenged with ETEC to different extent, which may potentially contribute to reduced diarrhea and enhanced intestinal integrity and health of newly weaned pigs when these feed additives were supplemented into the diet.

Cover page: PROBIOTICS AND BOTANICAL SUPPLEMENTATIONS ON GUT MICROBIOTA OF WEANED PIGS UNDER DIARRHEAL STRESS

Article
Peer Reviewed

Dietary supplementation of Bacillus subtilis or antibiotics modified intestinal microbiome of weaned pigs under enterotoxigenic Escherichia coli infection.

UC Davis Previously Published Works (2022)

Our previous research reported that supplementation of Bacillus subtilis DSM 25841 promoted growth and disease resistance of weaned pigs under enterotoxigenic Escherichia coli (ETEC) challenge and its efficacy is comparable to carbadox. This follow-up study aimed to characterize the effects of ETEC infection, supplementing B. subtilis DSM 25841 or carbadox on intestinal microbiota of pigs. Forty-eight weaned pigs (6.17 ± 0.36 kg BW) were randomly allotted to one of four treatments: negative control (NC), positive control (PC), antibiotics (AGP, 50 mg/kg of carbadox), and direct fed microbials (DFM, 2.56 × 10⁹ CFU/kg of B. subtilis). The experiment lasted 28 days with 7 days before and 21 days after first E. coli inoculation (day 0). Pigs in the PC, AGP, and DFM groups were orally inoculated with F18 ETEC for 3 consecutive days with 10¹⁰ CFU per dose per day. Fecal samples were collected on day -7, and day 7 and day 21 post inoculation, digesta samples were collected from jejunum, ileum, and distal colon on day 21 post inoculation to perform 16S rRNA sequencing. Sampling days and locations influenced (p < 0.05) Chao1 index and beta-diversity. Age increased (p < 0.05) the relative abundance of Firmicutes but decreased (p < 0.05) the relative abundance of Bacteroidetes in feces. ETEC infection increased (p < 0.05) the relative abundance of Proteobacteria in feces on day 7 post inoculation. AGP reduced (p < 0.05) relative abundance of Firmicutes and Lactobacillaceae in feces compared with PC and DFM. AGP reduced (p < 0.05) relative abundance of Bifidobacteriaceae in jejunum and ileum, while DFM reduced (p < 0.05) relative abundance of Actinomycetaceae in jejunum and Lachnospiraceae in ileum, compared with PC. Pigs fed with DFM had greater (p < 0.05) relative abundance of Ruminococcaceae, Veillonellaceae, Bifidobacteriaceae in jejunum, Lactobacillaceae in ileum and colon, and Bifidobacteriaceae in colon than pigs in AGP. Current results indicate that carbadox or B. subtilis had stronger influences on microbial diversity and composition in ileum than other intestinal segments and feces. Supplementation of B. subtilis could increase or maintain the relative abundance of beneficial bacteria in ileum compared with carbadox.

Cover page: Dietary supplementation of <i>Bacillus subtilis</i> or antibiotics modified intestinal microbiome of weaned pigs under enterotoxigenic <i>Escherichia coli</i> infection.

Article
Peer Reviewed

Trace amounts of antibiotic altered metabolomic and microbial profiles of weaned pigs infected with a pathogenic E. coli

UC Davis Previously Published Works (2022)

Background

Our previous study has shown that supplementation of trace amounts of antibiotic exacerbated the detrimental effects of enterotoxigenic E. coli (ETEC) infection and delayed the recovery of pigs that may be associated with modified metabolites and metabolic pathways. Therefore, the objective of this study was to explore the impacts of trace levels of antibiotic (carbadox) on host metabolic profiles and colon microbiota of weaned pigs experimentally infected with ETEC F18.

Results

The multivariate analysis highlighted a distinct metabolomic profile of serum and colon digesta between trace amounts of antibiotic (TRA; 0.5 mg/kg carbadox) and label-recommended dose antibiotic (REC; 50 mg/kg carbadox) on d 5 post-inoculation (PI). The relative abundance of metabolomic markers of amino acids, carbohydrates, and purine metabolism were significantly differentiated between the TRA and REC groups (q < 0.2). In addition, pigs in REC group had the highest (P < 0.05) relative abundance of Lactobacillaceae and tended to have increased (P < 0.10) relative abundance of Lachnospiraceae in the colon digesta on d 5 PI. On d 11 PI, pigs in REC had greater (P < 0.05) relative abundance of Clostridiaceae compared with other groups, whereas had reduced (P < 0.05) relative abundance of Prevotellaceae than pigs in control group.

Conclusions

Trace amounts of antibiotic resulted in differential metabolites and metabolic pathways that may be associated with its slow responses against ETEC F18 infection. The altered gut microbiota profiles by label-recommended dose antibiotic may contribute to the promotion of disease resistance in weaned pigs.

Cover page: Trace amounts of antibiotic altered metabolomic and microbial profiles of weaned pigs infected with a pathogenic E. coli

Article
Peer Reviewed

Enzymatically Digested Food Waste Altered Fecal Microbiota But Not Meat Quality and Carcass Characteristics of Growing-Finishing Pigs.

UC Davis Previously Published Works (2019)

This experiment aimed to evaluate meat quality, fatty acid profile in back-fat, and fecal microbiota of growing-finishing pigs fed with liquid enzymatically digested food waste. Fifty-six crossbred pigs (approximately 32.99 kg body weight) were assigned to one of two treatments with seven replicate pens and four pigs per pen. Pigs were fed with control (corn-soybean meal diets) or food waste from d 0 to 53, while all pigs were fed with the control diet from d 53 to 79. The 16S rRNA sequencing was used to analyze microbiota of feces collected on d 0, 28, 53, and 79. Meat quality and carcass characteristics were measured in one pig per pen at the end of the experiment. Pigs fed with food waste contained more (p < 0.05) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in back-fat. Feeding food waste increased (p < 0.05) the relative abundances of Lachnospiraceae and Ruminococcaceae, but decreased (p < 0.05) the relative abundances of Streptococcaceae and Clostridiaceae in feces on d 29 or d 53. In conclusion, feeding enzymatically digested food waste did not affect pork quality, but provided more beneficial fatty acids to pork consumers and altered the fecal microbiota in growing-finishing pigs.

Cover page: Enzymatically Digested Food Waste Altered Fecal Microbiota But Not Meat Quality and Carcass Characteristics of Growing-Finishing Pigs.

Article
Peer Reviewed

Effects of a blend of essential oils, medium-chain fatty acids, and a toxin-adsorbing mineral on diarrhea and gut microbiome of weanling pigs experimentally infected with a pathogenic Escherichia coli.

UC Davis Previously Published Works (2022)

A proprietary antimicrobial feed additive comprised of essential oils, medium-chain fatty acids, and a toxin-adsorbing mineral showed promising bacteriostatic and bactericidal effects in vitro. This study investigated the impacts of supplementing this blend on growth, gut microbiome, and enteric disease resilience in weaned pigs experimentally challenged with an enterotoxigenic Escherichia coli (ETEC). Thirty-six weanling pigs (6.88 ± 0.30 kg body weight) blocked by weight and gender were assigned to one of three dietary treatments: control or dietary supplementation with 0.25% or 0.50% of the antimicrobial blend. This study lasted 28 d with 7 d before and 21 d after the first ETEC inoculation (day 0). All pigs were orally inoculated with 1010 CFU F18 + ETEC/3-mL dose for 3 consecutive days. Growth performance data and diarrhea scores were recorded throughout the experiment. Fecal samples collected on days -7, 0, 7, and 21 post first inoculation (PI), and ileal digesta and mucosal tissue collected on day 21 PI were further analyzed for gut microbiome using 16S rRNA sequencing. All data, except for frequency of diarrhea and gut microbiome, were analyzed by ANOVA using the PROC MIXED of SAS. The chi-square test was used for analyzing frequency of diarrhea. Gut microbiome data were analyzed using QIIME2 and visualized using the R program. Dietary supplementation of 0.25% or 0.5% of the antimicrobial blend increased (P < 0.05) feed efficiency on days 14 to 21 PI of ETEC and reduced (P < 0.05) frequency of diarrhea during the study. Compared with the control group, adding 0.5% dietary antimicrobial blend increased (P < 0.05) relative abundance of Firmicutes but reduced (P < 0.05) Bacteroidetes and Proteobacteria in feces on day 7 PI. Pigs that received the antimicrobial blend also had higher (P < 0.05) relative abundance of Lactobacillaceae, but lower (P < 0.05) relative abundance of Enterobacteriaceae in feces on day 7 PI than pigs in control. In conclusion, supplementation of this antimicrobial blend at 0.5% reduced incidence of severe diarrhea in weaned pigs challenged with F18 ETEC and enhanced feed efficiency of weaned pigs at the last week of the experiment. Supplementation of this antimicrobial blend also modified the microbiota diversity in feces and ileal mucosa of weaned pigs.

Cover page: Effects of a blend of essential oils, medium-chain fatty acids, and a toxin-adsorbing mineral on diarrhea and gut microbiome of weanling pigs experimentally infected with a pathogenic Escherichia coli.

Article
Peer Reviewed