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Our previous studies demonstrated that trace amounts of antibiotic supplements exacerbated growth performance and systemic inflammation of weaned pigs infected with a pathogenic Escherichia coli(E. coli). The objective of this study was to investigate the influence of in-feed trace amounts of antibiotic on distal colon microbiota of pigs experimentally infected with E. coliF18. Thirty-four weaned pigs (6.88 ± 1.03 kg body weight) were individually and randomly allotted to 1 of 3 dietary treatments (9 to 13 pigs/treatment). The 3 dietary treatments were basal nursery diet (control), and 2 additional diets supplemented with trace amounts (TRA; 0.5 mg/kg) or label-recommended dose (REC; 50 mg/kg) of antibiotic (carbadox), respectively. The experiment lasted 18 d [7 d before and 11 d after the first inoculation (day 0)]. The E. coliF18 inoculum (1010cfu/3 mL) was orally provided to all pigs for 3 consecutive days. Distal colon digesta samples were collected on d 5 and 11 post-inoculation (PI), and microbial profiles were analyzed using 16S rRNA gene sequencing at the V4 hypervariable region. All compositional data were statistically analyzed using R software with QIIME2 plugin. Bray-Curtis PCoA analysis displayed those pigs in both control and TRA groups were separately clustered from REC on d 5 and 11 PI. Pigs in TRA group had a lowest (P< 0.05) relative abundance of Lactobacillaceaewhereas REC group tended to have increased (P< 0.10) relative abundance of Lachnospiraceaethan other treatments on day 5 PI. On day 11 PI, pigs in REC had reduced (P< 0.05) relative abundance of Prevotellaceaethan pigs in CON group. In conclusion, in-feed trace amounts of antibiotic could modify gut microbiota of weaned pigs challenged with E. coliF18.

The objective of this study was to investigate the intestinal microbiota of weaned pigs when supplemented with different botanical blends while being experimentally infected with a pathogenic E. coli. A total of 60 weaned pigs (7.17 ± 0.97 kg) were individually housed and randomly assigned to 1 of the 5 treatments (12 pigs/treatment): sham control (CON-), challenged control (CON+), challenged botanical blend 1 with 100 ppm (BB1_100), challenged BB2 with 50 ppm (BB2_50), and challenged BB2 with 100 ppm (BB2_100). Both botanical blends were composed of capsicum oleoresin but different garlic extract varieties. The experiment lasted for 28 d including a 7-day habituation period followed by an E. colioral inoculation of 1010CFU/dose for 3 consecutive days. Ileal, cecal, and fecal samples were collected on d 5 and 21 post inoculation (PI) to perform 16S rRNA sequencing at the V4 hypervariable region followed by a downstream analysis using QIIME2 (v. 2020.8) and R. No difference was observed in alpha diversity among treatments and sites on d 21 PI; however, CON- had the least Shannon and Chao1 indices in ileal digesta on d 5 PI. Bray-Curtis PCoA displayed distinct clusters by treatments in the ileum and cecum on d 5 and 21 PI. On d 5 PI, Bacteroidotawas more abundant (P< 0.05) in feces of BB1_100 but was the most abundant (P< 0.05) in ileum of CON-. Pigs in BB2 supplementation were more abundant (P< 0.05) in Proteobacteriain feces than in pigs in CON- at d 5 PI. On d 21 PI, Streptococcaceae was more abundant (P< 0.05) in the ileum of CON+ than of BB2_50 and Lachnospiraceae was more (P< 0.05) abundant in feces of pigs in BB2_100 than in BB1_100. In conclusion, supplementing botanical blend can modify the intestinal microbiota in weaned pigs challenged with a pathogenic E. coli.

The objective of this study was to investigate the fecal microbiota of weaned pigs when supplemented with different botanical blends while being experimentally infected with a pathogenic E. coli. A total of 60 weaned pigs (7.17 ± 0.97 kg) were individually housed and randomly assigned to 1 of the 5 treatments (12 pigs/treatment): sham control (CON-), challenged control (CON+), challenged botanical blend 1 with 100 ppm (BB1_100), challenged BB2 with 50 ppm (BB2_50), and challenged BB2 with 100 ppm (BB2_100). Both botanical blends were composed of capsicum oleoresin but different garlic extract varieties. The experiment lasted for 28 d including a 7-d habituation period followed by an E. colioral inoculation of 1010CFU/dose for 3 consecutive days. Fecal samples were collected on d -7, 0, 5 and 21 post inoculation (PI) to perform 16S rRNA sequencing at the V4 hypervariable region. Downstream analysis was performed using QIIME2 (v. 2020.8) and R. No difference was observed among treatments throughout the experiment in alpha diversity. Bray-Curtis PCoA displayed pronounced clusters by days but also displayed overlaps when looking into day and treatment interaction. Firmicuteswas more (P< 0.05) abundant in CON- than the botanical treatments while Bacteroidetesand Proteobacteriawere more (P< 0.05) abundant in BB1_100 and BB2_50 than in CON- on d 5 PI. Firmicutes was less (P< 0.05) abundant in BB2_50 and Proteobacteriawas more (P< 0.05) abundant in BB1_100 than in CON+ on d 21 PI. Lachnospiraceaewas less abundant (P< 0.05) in BB2_50 than in CON- on d 5 PI and less (P< 0.05) abundant in BB1_100 than in CON- on d 21. In conclusion, the botanical treatments have modified the fecal microbiota of weaned pigs challenged with an enteropathogenic E. coli.

The objective of this experiment was to investigate the effects of dietary supplementation of Bacillus amyloquefaciens on total and differential blood cell count in weaned pigs experimentally infected with a pathogenic E. coli. A total of 50 weaned pigs (7.41 ± 1.35 kg) were individually housed in disease containment rooms and randomly assigned to one of the 5 treatments: sham control (CON-), sham B. amyloquefaciens (BAM-), challenged control (CON+), challenged B. amyloquefaciens (BAM+) and challenged carbadox (CAR+). The experiment lasted 28 days with 7 days’ adaptation and 21 days after the first E. coli inoculation. The doses of F18 E. coli inoculum were 1010CFU/3 mL oral dose daily for 3 consecutive days. Whole blood samples were collected from all pigs on d -7, and d 0, 7, 14, and 21 post infection (PI) to measure total and differential blood cell count by complete blood count (CBC) analysis. Supplementation of BAM or CAR increased (P < 0.05) either the percentage or the number of lymphocytes on d 0 before E. coli inoculation. E. coli challenge increased (P < 0.05) white blood cell (WBC) count on d 7 and 21 PI, while supplementation of BAM tended (P < 0.10) to have low WBC on d 7 PI and had lower (P < 0.05) WBC on d 21 PI compared with CON+. Pigs in BAM+ also had lower (P < 0.05) neutrophil count on d 14 PI, pigs fed with CAR had lower (P < 0.05) neutrophil count on d 14 and 21 PI, compared with pigs in CON+. No difference was observed in red blood cell profile among all treatments throughout the experiment. In conclusion, pigs fed with B. amyloquefaciens have similar systemic immune response to pigs in antibiotic group and have relatively lower systemic inflammation caused by E. coli compared with control group.

The objective of this experiment was to investigate dietary supplementation of oligosaccharide-based polymer on blood profiles of weaned pigs experimentally infected with a pathogenic F18 Escherichia coli (E. coli). Forty-eight pigs (7.23 ± 1.11 kg BW) were individually housed in disease containment rooms and randomly allotted to one of four treatments with 12 replicate pigs per treatment. The four dietary treatments were a nursery basal diet (control), and 3 additional diets supplemented with 50 mg/kg Mecadox (AGP), 10 or 20 mg/kg of oligosaccharide-based polymer. The experiment lasted 18 d [7 d before and 11 d after the first inoculation (d 0)]. The doses of F18 E. coli inoculum were 1010cfu/3 mL oral dose daily for 3 days. Blood samples were collected before E. coli inoculation (d 0), and on d 2, 5, 8, and 11 post-inoculation (PI). Total and differential blood cell count were analyzed by CBC test. All data were analyzed by ANOVA using the PROC MIXED of SAS with pig as the experimental unit. Supplementation of oligosaccharide-based polymer linearly (P < 0.05) reduced white blood cell counts, neutrophils, eosinophils, and basophils on d 2 PI, and neutrophils on d 5 PI, compared with the control. No differences were observed in total and differential white blood cell counts among AGP and two oligosaccharide-based polymer treatments except that pigs fed with AGP had greater (P < 0.05) lymphocytes on d 2 PI compared with pigs fed with oligosaccharide-based polymer diets. Supplementation of low dose oligosaccharide-based polymer or AGP reduced (P < 0.05) red blood cell count and packed cell volume on d 2 PI, whereas inclusion of high dose oligosaccharide-based polymer or AGP reduced (P < 0.05) packed cell volume on d 5 PI, compared with the control. In conclusion, supplementation of oligosaccharide-based polymer may alleviate the systemic inflammation caused by F18 E. coli infection.

The objective of this study was to compare the dietary effects of Bacillus subtilis and antibiotics in intestinal microbiota of pigs experimentally infected with F18 E. coli. Forty-eight weaned pigs (6.17 ± 0.36 kg BW) were individually housed and randomly allotted in one of four treatments with 12 replicates per treatment: negative control (NC), positive control (PC), antibiotics, and B. subtilis (probiotics). Pigs in NC and PC were fed with basal diet without or with E. coli, respectively. Pigs with antibiotics and probiotics were challenged with E. coli and supplemented with 50 mg/kg of carbadox or 500 mg/kg of B. subtilis, respectively. After 7 days habituation period, pigs were inoculated with F18 E. coli at 1010CFU/3 mL dose for three consecutive days. All pigs were euthanized to collect feces and digesta from jejunum, ileum, and colon on d 21 post-infection to perform 16S rRNA sequencing at the V4 hypervariable region. Downstream analysis was performed using QIIME2 (2019.4) and R. Kruskal-Wallis test followed by Conover test was used to analyze data in R. Colon digesta and feces have greater (P < 0.05) alpha diversity than ileal and jejunal digesta. No difference was observed among treatments at different intestinal sites. Bray-Curtis PCoA plots displayed pronounced clusters of all treatment groups throughout all sites. Bacteroidetes and Proteobacteria were more (P < 0.05) abundant but Firmicutes were less (P < 0.05) abundant in ileal digesta of pigs fed with antibiotics than pigs in probiotics. Firmicutes were more (P < 0.05) abundant in colon and feces of NC than of antibiotics. Bifidobacterium was least (P < 0.05) abundant throughout all sites and Prevotella 1 was most (P < 0.05) abundant in colon of pigs fed with antibiotics compared with other treatments. In conclusion, both B. subtilis and carbadox supplementation modified gut microbiota of weaned pigs challenged with F18 E. coli. However, the impacts are different and need further investigation.

The objective of this experiment was to investigate the effects of dietary supplementation of Bacillus amyloquefaciens on diarrhea and growth performance of weaned pigs experimentally infected with a pathogenic E. coli. A total of 50 weaned pigs (7.41 ± 1.35 kg) were individually housed in disease containment rooms and randomly assigned to one of the 5 treatments: sham control (CON-), sham B. amyloquefaciens (BAM-), challenged control (CON+), challenged B. amyloquefaciens (BAM+) and challenged carbadox (CAR+). The experiment lasted 28 days with 7 days’ adaptation and 21 days after the first E. coli inoculation. The doses of F18 E. coli inoculum were 1010CFU/3 mL oral dose daily for 3 consecutive days. Pigs were weighed on d -7, and d 0, 7, 14, and 21 PI and feed intake was recorded weekly to calculate growth performance. Diarrhea score was recorded twice daily ranging score from 1 (normal feces) to 5 (watery diarrhea). Fecal swabs were performed on each pig on d 2, 7, 14, and 21 PI to test the percentage of β-hemolytic coliforms in total coliforms. All data were analyzed with PROC MIXED of SAS. E. coli challenge reduced (P < 0.05) growth rate through the entire experiment. However, supplementation of CAR enhanced (P < 0.05) growth performance, while inclusion of BAM tended (P < 0.10) to increase growth performance of weaned pigs. No differences were observed in growth performance of pigs between BAM+ and CAR+ throughout the experiment. E. coli challenge did not change the percentage of β-hemolytic coliforms but increased (P < 0.05) frequency of diarrhea. However, no difference was observed in diarrhea among 3 dietary treatments. In conclusion, supplementing B. amyloquefaciens tended to enhance growth performance but had limited effects on diarrhea of weaned pigs challenged with E. coli.

Supplementation of various Bacillus spp.has shown to improve growth performance, immune responses, and gut health of newly weaned pigs. The present study investigated the effects of dietary supplementation of 109 CFU/kg of Bacillus (B.) amyloliquefacienson growth performance, diarrhea, systemic immunity, and intestinal microbiome of weaned pigs with or without enterotoxigenic Escherichia coli(ETEC) challenge. An antibiotic treatment was also included in the experiment to compare the efficacy of B. amyloliquefaciensvs. carbadox (50 mg/kg). ETEC challenge reduced (P < 0.05) growth rate but increased (P < 0.05) the frequency of diarrhea in pigs throughout the experiment. Supplementation of B. amyloliquefacienstended (P < 0.10) to increase growth performance but had limited impacts on diarrhea of weaned pigs. ETEC challenge increased (P < 0.05) white blood cell (WBC) count on d 7 and 21 post-inoculation (PI), while supplementation of B. amyloliquefacienstended (P < 0.10) to reduce WBC on d 7 PI and reduced (P < 0.05) WBC on d 21 PI, compared with control. No differences were observed in performance, diarrhea, and systemic immunity of pigs when comparing B. amyloliquefacienswith carbadox. Supplementation of B. amyloliquefaciensand antibiotics had different impacts on intestinal microbiome. ETEC challenge increased (P < 0.05) relative abundance of Lactobacillusin feces on d 7 PI but reduced (P < 0.05) relative abundance of Prevotellain feces on d 21 PI. Prior ETEC challenge, Lachnospiraceaewas less (P < 0.05) abundant in feces of pigs supplemented with B. amyloliquefaciensthan pigs in carbadox group on d 0 before ETEC infection. The relative abundance of Enterobacteriaceaein fecal samples was the lowest (P < 0.05) in pigs supplemented with carbadox among all treatment on d 0. On d 14 and 21 PI, the relative abundance of Actinobacteria in feces was the lowest (P < 0.05) in carbadox treatment among all treatment of the experiment. Ileal digesta were also collected on d 21 PI to analyze intestinal microbiota diversity changes among treatments. The Shannon diversity index was greater (P < 0.05) in ileal digesta of pigs supplemented with B. amyloliquefaciensthan pigs supplemented with carbadox. ETEC reduced (P < 0.05) the relative abundance of Firmicutes and Lactobacillusand increased (P < 0.05) the relative abundance of Proteobacteria and Lachnospiraceaein ileal digesta. Under ETEC challenge, pigs supplemented with B. amyloliquefacienshad lower (P < 0.05) abundance of Firmicutes and Clostridiaceaeand greater (P < 0.05) abundance of Lachnospiraceaein the ileum than pigs supplemented with carbadox. In conclusion, supplementation of B. amyloliquefacienshad similar impacts on growth performance and systemic immunity but had different impacts on intestinal microbiota of weaned pigs in comparison to carbadox.

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 1010CFU 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 Enterobacteriaceaein 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.This experiment aims to investigate an antimicrobial blend consisting of essential oils, medium-chain fatty acids, and a toxin-adsorbing mineral on diarrhea, growth performance, and gut microbiome of newly weaned pigs experimentally infected with a pathogenic Escherichia coli(F18 E. coli). A total of 36 weaned pigs were randomly allotted to one of three dietary treatments: (1) a complex control diet that met the nutrient requirement of weaned pigs; (2) supplementing 0.25% of the antimicrobial blend; and (3) 0.50% of the antimicrobial blend. The experiment lasted 28 d with 7 d adaptation and 21 d after the first F18 E. coliinoculation. Results of this experiment demonstrate that supplementation of this antimicrobial blend enhanced disease resistance of weaned pigs, as indicated by reduced frequency of diarrhea during the entire experimental period. An improved feed efficiency was also observed in pigs supplemented with antimicrobial blend at the last week of the experiment. In addition, feces collected on day 7 post-E. coliinoculation contained relatively more Lactobacillaceaebut less Enterobacteriaceaewhen pigs were supplemented with this antimicrobial blend. In conclusion, supplementation of antimicrobial blend could reduce diarrhea of E. coli-infected pigs and modify fecal microbiome of weaned pigs during the peak of E. coliinfection.Supplementation of 0.25% or 0.50% of an antimicrobial blend consisting of essential oils, medium-chain fatty acids, and a toxin-adsorbing mineral reduced the incidence and severity of diarrhea in weaned pigs infected with a pathogenic Escherichia coli(E. coli). Supplementation of 0.50% of this antimicrobial blend shaped fecal microbiome of weaned pigs during the peak of E. coliinfection.

The objective of this experiment was to investigate dietary supplementation of oligosaccharide-based polymer on growth performance, diarrhea, and fecal β-hemolytic coliforms of weaned pigs experimentally infected with a pathogenic F18 Escherichia coli (E. coli). Forty-eight pigs (7.23 ± 1.11 kg BW) were individually housed in disease containment rooms and randomly allotted to one of four treatments with 12 replicate pigs per treatment. The four dietary treatments were a nursery basal diet (control), and 3 additional diets supplemented with 50 mg/kg Mecadox (AGP), 10 or 20 mg/kg of oligosaccharide-based polymer. The experiment lasted 18 d [7 d before and 11 d after the first inoculation (d 0)]. The doses of F18 E. coli inoculum were 1010cfu/3 mL oral dose daily for 3 days. Growth performance was measured on d -7 to 0 before inoculation, and d 0 to 5 and 5 to 11 post-inoculation (PI). Diarrhea score (DS; 1, normal, to 5, watery diarrhea) was daily recorded for each pig. Fecal samples were collected on d 2, 5, 8, and 11 PI to test the percentage of β-hemolytic coliforms in total coliforms. All data were analyzed by ANOVA using the PROC MIXED of SAS with pig as the experimental unit. Inclusion of oligosaccharide-based polymer linearly increased (P < 0.05) ADFI on d 0 to 5 PI, and feed efficiency on d 0 to 5 PI and d 5 to 11 PI (P = 0.07), compared with the control. Supplementation of AGP or oligosaccharide-based polymer reduced (P < 0.01) frequency of diarrhea of pigs from d 0 to 11 PI. No differences were observed in overall growth performance and percentage of fecal β-hemolytic coliforms on d 8 PI among pigs in AGP and oligosaccharide-based polymer treatments. In conclusion, supplementation of oligosaccharide-based polymer enhanced feed efficiency and reduced diarrhea of weaned pigs infected with a pathogenic E. coli.

The experiment was conducted to investigate the effects of trace amounts of antibiotic on growth performance, diarrhea, systemic immunity, and intestinal health of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli. Weaned pigs (n= 34, 6.88 ± 1.03 kg body weight [BW]) were individually housed in disease containment rooms and randomly allotted to one of the three dietary treatments: nursery basal diet (CON) and two additional diets supplemented with 0.5 or 50 mg/kg carbadox to the nursery basal diet (TRAor REC), respectively. The experiment lasted 18 d with 7 d before and 11 d after the first E. coliinoculation. The E. coliF18 inoculum was orally provided to all pigs with a dose of 1010colony-forming unit (CFU)/3 mL for three consecutive days. Fecal and blood samples were collected on day 0 before inoculation and days 2, 5, 8, and 11 postinoculation (PI) to test the percentage of β-hemolytic coliforms in total coliforms and complete blood cell count, respectively. Sixteen pigs were euthanized on day 5 PI, whereas the remaining pigs were euthanized at the end of the experiment to collect the jejunal and ileal mucosa and mesenteric lymph node for gene expression and bacterial translocation, respectively. Pigs in REC had greater (P< 0.05) final BW and lower (P< 0.05) overall frequency of diarrhea compared with pigs in the CON and TRA groups. Pigs in TRA had the lowest (P< 0.05) average daily gain and feed efficiency from day 0 to 5 PI, highest (P< 0.05) percentage of β-hemolytic coliforms in fecal samples on days 2 and 5 PI, and greatest (P< 0.05) bacterial colonies in mesenteric lymph nodes on day 11 PI compared with pigs in the CON and REC groups. Pigs in TRA had the greatest (P< 0.05) neutrophils on day 5 PI and higher (P< 0.05) white blood cell counts and lymphocytes than other groups on day 11 PI. Pigs in TRA had the greatest (P< 0.05) serum C-reactive protein on days 2 and 5 PI and serum tumor necrosis factor-α on day 5 PI, compared with pigs in the CON and REC groups. Pigs fed REC had increased (P< 0.05) mRNA expression of zona occludens-1(ZO-1) and occludin (OCDN) and reduced (P< 0.05) interleukin-1 beta (IL1B), interleukin-6 (IL6), and tumor necrosis factor-alpha (TNFA) in ileal mucosa on day 5 PI, compared with the CON, whereas TRA upregulated (P< 0.05) mRNA expression of IL1B, IL6, and cyclooxygenase-2 (COX2) in the ileal mucosa on day 11 PI, compared with the REC. In conclusion, trace amounts of antibiotic may exacerbate the detrimental effects of E. coliinfection on pig performance by increasing diarrhea and systemic inflammation of weanling pigs.