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Supplementation of oligosaccharide-based polymer enhanced growth and disease resistance of weaned pigs by modulating intestinal integrity and systemic immunity

Supplementation of oligosaccharide-based polymer enhanced growth and disease resistance of weaned... Background: There is a great demand for antibiotic alternatives to maintain animal health and productivity. The objective of this experiment was to determine the efficacy of dietary supplementation of a blood group A6 type 1 antigen oligosaccharides-based polymer (Coligo) on growth performance, diarrhea severity, intestinal health, and systemic immunity of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli (ETEC), when compared with antibiotics. Results: Pigs in antibiotic carbadox or Coligo treatment groups had greater (P < 0.05) body weight on d 5 or d 11 post-inoculation (PI) than pigs in the control group, respectively. Supplementation of antibiotics or Coligo enhanced (P < 0.05) feed efficiency from d 0 to 5 PI and reduced (P < 0.05) frequency of diarrhea throughout the experiment, compared with pigs in the control group. Supplementation of antibiotics reduced (P < 0.05) fecal β- hemolytic coliforms on d 2, 5, and 8 PI. Pigs in antibiotics or Coligo groups had reduced (P < 0.05) neutrophil counts and serum haptoglobin concentration compared to pigs in the control group on d 2 and 5 PI. Pigs in Coligo had reduced (P < 0.05) total coliforms in mesenteric lymph nodes on d 5 and 11 PI, whereas pigs in antibiotics or Coligo groups had reduced (P < 0.05) total coliforms in spleen on d 11 PI compared with pigs in the control group. On d 5 PI, pigs in the Coligo group had greater (P < 0.05) gene expression of ZO1 in jejunal mucosa, but less (P < 0.05) mRNA expression of IL1B, IL6, and TNF in ileal mucosa, in comparison with pigs in the control group. Supplementation of antibiotics enhanced (P < 0.05) the gene expression of OCLN in jejunal mucosa but decreased (P < 0.05) IL1B and IL6 gene expression in ileal mucosa, compared with the control. On d 11 PI, supplementation of antibiotics or Coligo up-regulated (P < 0.05) gene expression of CLDN1 in jejunal mucosa, but Coligo reduced (P < 0.05) IL6 gene expression in ileal mucosa compared to pigs in the control group. Conclusions: Supplementation of Coligo improved growth performance, alleviated diarrhea severity, and enhanced gut health in weaned pigs infected with ETEC F18 in a manner similar to in-feed antibiotics. Keywords: Enterotoxigenic E. coli, Growth rate, Intestinal barrier function, Oligosaccharide-based polymer, Systemic immunity, Weaned pigs * Correspondence: yahliu@ucdavis.edu Department of Animal Science, University of California, Davis, CA 95616, USA Full list of author information is available at the end of the article © The Author(s). 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 2 of 14 Background combination of blood group oligosaccharides and ε-PL Enterotoxigenic E. coli (ETEC) strains expressing F4 or may enhance the resistance of pigs against F18 ETEC in- F18 fimbriae are major causes of post-weaning diarrhea fection by inhibiting bacterial attachment and/or directly in nursery pigs [1]. Attachment of ETEC to the specific killing bacteria. The overall objective of this study was to receptors on intestinal epithelium leads to colonization investigate the efficacy of blood group A6 type 1-based and secretion of enterotoxins, resulting in secretory diar- polymer on gut integrity and disease resistance of wean- rhea in weanling pigs [2]. To prevent post-weaning diar- ling pigs challenged with F18 ETEC. rhea and improve production of pigs, antibiotics were commonly added to the diet over the past decades. Materials and methods However, frequent use of in-feed antibiotics in livestock Animals, housing, experimental design, and diet production has been shown to contribute to the in- A total of 48 weanling pigs (crossbred; initial body creased prevalence of antibiotic-resistant bacteria and weight (BW): 7.23 ± 1.14 kg; 21 days old) with an equal raised public health concerns [3]. With these issues, the number of gilts and barrows were used in this study. Food and Drug Administration (FDA) banned the use of They were selected from the Swine Teaching and Re- in-feed antibiotics for growth promoting purposes in search Center at the University of California, Davis. The livestock production in the U.S. [4], thus alternative nu- sows and piglets used in this experiment did not receive tritional strategies are highly demanded to enhance dis- E. coli vaccines, antibiotic injections, or antibiotics in ease resistance and production of weanling pigs. creep feed. Before weaning, feces were collected from Many nutritional approaches have been applied to pre- sows and all their piglets destined for this study to verify vent post-weaning diarrhea associated with ETEC and the absence of β-hemolytic E. coli. The F18 ETEC recep- enhance the production of pigs. Among these, the pre- tor status was also tested based on the methods of Kreu- vention of bacterial attachment to the small intestine is zer et al. [18], and all piglets used in this study were one of the most effective defense strategies against ETEC susceptible to F18 ETEC. infection [5]. Oligosaccharides have been reported to After weaning, all pigs were randomly assigned to one possess ETEC receptor activity for bacterial adhesions of the four dietary treatments in a randomized complete [6, 7]. Especially, N-acetylgalactosamine (GalNAc) con- block design with body weight within sex and litter as taining glycans that could enhance the binding affinity the blocks and pig as the experimental unit. There were of various ETEC strains, including K99 [8], F4 [9], and 12 replicate pigs per treatment. Pigs were individually F18 [10–12]. Coddens et al. [11] identified the blood housed (pen size: 0.61m × 1.22 m) in environmental con- group H type 1 determinant (Fuca2Galß3GlcNAc) as the trol rooms at the Cole Facility at the University of Cali- minimal binding epitope of F18 fimbriae. Based on that, fornia, Davis for 18 d, including 7 d before and 11 d after an optimal binding epitope was created by adding the the first F18 ETEC challenge (d 0). The piglets had ad terminal 3-linked galactose or N-acetylgalactosamine of libitum access to feed and water. Environmental enrich- the blood group B type 1 determinant (Galα3(Fucα2)- ment was provided for each pig. The light was on at 07: Galß3GlcNAc) and the blood group A type 1 determin- 00 h and off at 19:00 h daily in the environmental control ant (GalNAcα3(Fucα2)- Galß3GlcNAc). The purified rooms. soluble blood group oligosaccharides were able to greatly The 4 dietary treatments included: 1) Positive control: reduce binding of F18 positive E. coli to intestinal villi of control diet; 2) Low dose oligosaccharide-based polymer F18 receptor-positive pigs in concentrations of 1 to 10 (LOW): control diet supplemented with 10 mg/kg mg/mL [11]. Moreover, multimerizing blood group A on oligosaccharide-based polymer active substance (Coligo); human serum albumin reduced the amount of blood 3) High dose oligosaccharide-based polymer (HIGH): group oligosaccharides needed 1000 times [13]. There- control diet supplemented with 20 mg/kg fore, multimerizing the blood group A oligosaccharides oligosaccharide-based polymer active substance (Coligo); may efficiently prevent enterotoxin-induced secretory and 4) CAR: control diet supplemented with 50 mg/kg diarrhea. Recently, grafted polymers that combine mul- carbadox. Spray-dried plasma and high levels of zinc tiple substances have been proposed for their potential oxide exceeding recommendation and normal practice synergistic effects on preventing human and animal dis- were not included in the diets. The experimental diets eases [14, 15]. Epsilon-poly-lysin (ε-PL) has been re- were fed to pigs throughout the experiment. ported to use as a carrier in the membrane transport of Oligosaccharide-based polymer active substance was a proteins and drugs [16]. Due to its excellent heat stabil- glycoconjugate composed of blood group A6 type 1 anti- ity, biodegradability, and lack of toxicity, ε-PL has gener- gen oligosaccharides grafted on a single peptide of ally been regarded as safe status (GRAS) and been epsilon-poly-lysine. Coligo was designed and synthesized interested in the food and medicine industries as a deliv- by Elicityl (France) in cooperation with Ghent University ery vehicle targeting the desired location [17]. Thus, the (Dr. Eric Cox’s laboratory) and was provided by Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 3 of 14 Pancosma (Geneva, Switzerland). The mean rate of con- response parameters that were reported in previously jugation is 15 mol of oligosaccharide for 1 mol of ε-PL. published research using the same ETEC strain and in- The oligosaccharide part represents 25% of the molecu- oculation dose [21, 22]. lar weight of the oligosaccharide-based polymer. All di- To achieve proper restraint and positioning for ets were formulated to meet pig nutritional blood sample collection, pig was placed on a V- requirements (Table 1)[19] and provided as mash form shaped table restrained in dorsal recumbence. Blood throughout the experiment. samples were collected from the jugular vein of all After 7 days of adaptation, all pigs were orally inoc- pigs with or without ethylenediaminetetraacetic acid ulated with 3 mL of F18 ETEC for 3 consecutive days (EDTA) to yield whole blood and serum, respectively, from d 0 post-inoculation (PI). The F18 ETEC was before ETEC challenge (d 0), and on d 2, 5, and 11 originally isolated from a field disease outbreak by the PI. Serum samples were collected and immediately University of Illinois Veterinary Diagnostic Lab (iso- stored at − 80 °C before further analysis. Before eu- late number: U.IL-VDL # 05–27,242). The F18 ETEC thanasia, pigs were anesthetized with a 1-mL mixture expresses heat-labile toxin (LT), heat-stable toxin b of 100 mg telazol, 50 mg ketamine, and 50 mg xylazine (STb), and Shiga-like toxin (Stx2e). The inoculums (2:1:1) by intramuscular injection. After anesthesia, in- were prepared by the laboratory of the Western Insti- tracardiac injection with 78 mg sodium pentobarbital tute for Food Safety and Security at the University of (Vortech Pharmaceuticals, Ltd., Dearborn, MI, USA) California, Davis, and were provided at 10 colony- per 1 kg of BW was used to euthanize each pig. forming unit (CFU) per 3 mL dose in phosphate- Three 3-cm segments from the duodenum, the middle buffered saline (PBS). This dose caused mild diarrhea of the jejunum, and the ileum (10 cm close to the in the current study, consistent with our previous ileocecal junction) were collected and fixed in Car- published researches [20–22]. noy’s solution (ethanol, chloroform, and glacial acetic acid, 6:3:1 v/v/v) for intestinal morphology analysis. Clinical observations and sample collections Mesenteric lymph nodes were aseptically collected The procedures for this study were adapted from previ- and then pooled within the pig, grounded, diluted, ous research methods of Liu et al. [20] and Kim et al. and plated on brain heart infusion agar for measure- [21, 22]. Clinical observations (diarrhea score and alert- ment of total bacteria, and the results were expressed ness score) were recorded twice daily throughout the as CFU per g of lymph node [23, 24]. Spleen samples study. The diarrhea score of each pig was assessed each were analyzed in the same method as mesenteric day visually by two independent evaluators, with the lymph nodes for bacterial translocation. score ranging from 1 to 5 (1 = normal feces, 2 = moist feces, 3 = mild diarrhea, 4 = severe diarrhea, and 5 = Detection of β-hemolytic coliforms watery diarrhea). The frequency of diarrhea was calcu- Briefly, fecal samples were plated on Columbia Blood lated as the percentage of the counting pig days with a Agar with 5% sheep blood to identify hemolytic coli- diarrhea score 4 or greater. The alertness score of each forms, which can lyse red blood cells surrounding the pig was assessed visually with a score from 1 to 3 (1 = colony. Fecal samples were also plated on MacConkey normal, 2 = slightly depressed or listless, and 3 = severely agar to enumerate total coliforms. Hemolytic colonies depressed or recumbent). All pigs had an alertness score from the blood agar were sub-cultured on MacConkey 1 throughout the study, therefore, data are not reported. agar to confirm that they were lactose-fermenting bac- Pigs were weighed on weaning day (d − 7), d 0 before teria and flat pink colonies. All plates were incubated at inoculation, d 5, and 11 PI. Feed intake was recorded 37 °C for 24 h in an air incubator. Populations of both throughout the study. Average daily gain (ADG), average total coliforms and β-hemolytic coliforms on blood agar daily feed intake (ADFI), and feed efficiency (gain:feed) were assessed visually, with a score from 0 to 8 (0 = no was calculated for each interval from d −7to0,d0to5 bacterial growth, 8 = very heavy bacterial growth). The PI, and d 5 to 11 PI. Fecal samples were collected from ratio of scores of β-hemolytic coliforms to total coli- the rectum of all pigs throughout the experiments using forms was calculated. Questionable colonies were sub- a fecal loop or cotton swap on d 2, 5, 8, and 11 PI to test sub-cultured on new MacConkey and blood agar plates for β-hemolytic coliforms and percentage [20–22]. to verify if they were β-hemolytic E. coli by using triple Twenty-four pigs (3 barrows and 3 gilts from each treat- sugar iron agar and lysine iron agar, then verify if they ment) were euthanized on d 5 PI near the peak of infec- were F18 positive E. coli using PCR [25]. tion, and the remaining pigs were euthanized at the end of the experiment (d 11 PI) that was the recovery period Complete blood count of the infection. The selection of necropsy time was Whole blood samples were used for measuring total based on the results of clinical observations and immune and differential blood cell counts by the Comparative Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 4 of 14 Table 1 Ingredient compositions of experimental diets Pathology Laboratory at the University of California, Ingredient, % Control diet Davis. A multiparameter, automated programmed hematology analyzer (Drew/ERBA Scientific 950 FS Corn 44.51 Hematological Analyzer, Drew Scientific Inc., Miami, Dried whey 15.00 FL) was used for the assay to differentiate porcine Soybean meal 14.00 blood optimally. Fish meal 10.00 Soy protein concentrate 7.00 Measurements of serum cytokine and acute-phase Lactose 6.00 proteins Soybean oil 2.00 Serum samples were analyzed for a pro-inflammatory Limestone 0.56 cytokine (Tumor necrosis factor-α; TNF-α; R&D System Inc., Minneapolis, MN, USA) and acute-phase proteins L-Lysine·HCl 0.15 (C-reactive protein and haptoglobin; GenWay Biotech DL-Methionine 0.06 Inc., San Diego, CA, USA) using porcine-specific L-Threonine 0.02 enzyme-linked immunosorbent assay (ELISA) kits. All Salt 0.40 samples were analyzed in duplicate, including standard Vit-mineral, Sow 6 0.30 and control. The intra-assay coefficients of variation for Total 100.00 TNF-α, C-reactive protein, and haptoglobin were 6.2%, Calculated energy and nutrient 4.1%, and 2.7%, respectively. The inter-assay coefficients Metabolizable energy, kcal/kg 3487 of variation for TNF-α, C-reactive protein, and haptoglo- bin were 10.0%, 5.6%, and 6.2%, respectively. The results Net energy, kcal/kg 2615 of TNF-α, C-reactive protein, and haptoglobin were Crude protein, % 22.97 expressed in picograms, micrograms, or milligrams per Ile, % 0.86 milliliter based on the standard curves. Leu, % 1.68 Lys, % 1.35 Intestinal morphology Met, % 0.44 The fixed intestinal tissues were embedded in paraffin, Thr, % 0.79 sectioned at 5 μm, and stained with high iron diamine Trp, % 0.23 and alcian blue. The slides were photographed by an Olympus BX51 microscope at 100 × amplification, and Val, % 0.95 all measurements were conducted in the image process- Met + Cys, % 0.74 ing and analysis software (Image J, NIH). Fifteen straight Ca, % 0.80 and integrated villi and their associated crypts and sur- Total P, % 0.69 rounded area were selected to analyze villi height, crypt Digestible P, % 0.47 depth, the number of goblet cells per villus, and cross- Analyzed nutrients, % sectional area of sulfo- and sialomucin as described by Dry matter 89.6 Deplancke and Gaskins [26], and Kim et al. [22]. Crude protein 22.58 Intestinal barrier and innate immunity ADF 2.87 Jejunal and ileal mucosa samples were analyzed for gene NDF 6.99 expression by quantitative real-time PCR (qRT-PCR). Ca 1.04 Briefly, approximately 100 mg of mucosa sample was ho- P 0.70 mogenized using TRIzol reagent (Invitrogen; Thermo Three additional diets were formulated by adding 10 mg/kg of group A6 Fisher Scientific, Inc., Waltham, MA, USA). Then total type 1-based polymer, 20 mg/kg of group A6 type 1-based polymer (Coligo), or 50 mg/kg of Carbadox to the control diet, respectively RNA was extracted following RNA extraction procedural Provided the following quantities of vitamins and micro minerals per guidelines provided by the reagent manufacturer. The kilogram of complete diet: Vitamin A as retinyl acetate, 11,136 IU; vitamin RNA quality and quantity were assessed by Agilent Bioa- D as cholecalciferol, 2208 IU; vitamin E as DL-alpha tocopheryl acetate, 66 IU; vitamin K as menadione dimethylprimidinol bisulfite, 1.42 mg; thiamin as nalyzer 2100 (Agilent, Santa Clara, CA, USA). The thiamine mononitrate, 0.24 mg; riboflavin, 6.59 mg; pyridoxine as pyridoxine cDNA was produced from 1 μg of total RNA per sample hydrochloride, 0.24 mg; vitamin B , 0.03 mg; D-pantothenic acid as D- calcium pantothenate, 23.5 mg; niacin, 44.1 mg; folic acid, 1.59 mg; biotin, using the High-Capacity cDNA Reverse Transcription 0.44 mg; Cu, 20 mg as copper sulfate and copper chloride; Fe, 126 mg as Kit (Applied Biosystems; Thermo Fisher Scientific, Inc., ferrous sulfate; I, 1.26 mg as ethylenediamine dihydriodide; Mn, 60.2 mg as Waltham, MA, USA) in a total volume of 20 μL. The manganese sulfate; Se, 0.3 mg as sodium selenite and selenium yeast; and Zn, 125.1 mg as zinc sulfate mRNA expression of Claudin 1 (CLDN1), Mucin 2 Amino acids were indicated as standardized ileal digestible AA (MUC2), Occludin (OCLN), and Zonula occludens-1 Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 5 of 14 (ZO-1) in jejunal mucosa and the mRNA expression of among all dietary treatments (Table 3; Fig. 1). Compared Interleukin 1 beta (IL1B), Interleukin 6 (IL6), Cyclooxy- with pigs in control group, pigs supplemented with genase 2 (PTGS2), and Tumor necrosis factor-alpha LOW had lower (P < 0.05) average diarrhea score of (TNF) in ileal mucosa were analyzed by qRT-PCR. Data weaned pigs from d 0 to 5 PI, but this was not the case normalization was accomplished using beta-actin from d 5 to 11 PI. Supplementation of CAR or any dose (ACTB) and ribosomal protein L4 (RPL4) as housekeep- of Coligo had lower (P < 0.05) frequency of diarrhea of ing genes. Primers were designed based on published lit- weaned pigs from d 0 to 11 PI. erature and commercially synthesized by Integrated No β-hemolytic coliform was observed in the feces of DNA Technologies, Coralville, IA. All primers were veri- all pigs before ETEC inoculation. Pigs supplemented fied prior to qRT-PCR (Table S1). The qRT-PCR reac- with CAR had the lowest (P < 0.05) β-hemolytic coliform tion conditions followed the published research [27]. percentage in feces on d 2 and 5 PI among all dietary -ΔΔCT The 2 method was used to analyze the relative ex- treatments (Fig. 2). The percentage of β-hemolytic coli- pression of genes compared with control [28]. form in feces was not different between Coligo groups and CAR on d 8 PI. There were no differences observed Statistical analysis in fecal culture on d 11 PI among the treatments. The normality of data was verified with the Shapiro- Wilk test, and outliers were identified using the UNI- Systemic immunity and red blood cell profile VARIATE procedure (SAS Inst. Inc., Cary, NC, USA). Lymphocyte counts were greater (P < 0.05) in pigs fed All data were analyzed by ANOVA using the PROC CAR on d 0 before ETEC inoculation (Table 4). Pigs in MIXED of SAS (SAS Institute Inc., Cary, NC, USA) in a the LOW group had lower (P < 0.05) neutrophils, lym- randomized complete block design with the pig as the phocytes, and basophils on d 2 PI and lower (P < 0.05) experimental unit. The statistical model included inde- neutrophil counts on d 5 PI, compared with pigs in the pendent variables treatment group, sampling day, and control group. Supplementation of HIGH also had lower interactions as the fixed effect and blocks as random ef- (P < 0.05) white blood cell counts, neutrophils, lympho- fects. Treatment means were separated by using the cytes, and basophils on d 2 PI. Pigs in the CAR group LSMEANS statement and the PDIFF option of PROC had lower (P < 0.05) neutrophils and basophils on d 2 PI MIXED. Contrast statements were used to analyze the and lower (P < 0.05) neutrophils on d 5 PI, but higher dose effects of Coligo. The Chi-square test was used for (P < 0.05) eosinophils on d 5 PI, compared with pigs in analyzing the frequency of diarrhea. Statistical signifi- control group. No difference was observed in white cance and tendency were considered at P < 0.05 and blood cell profiles among treatments on d 11 PI. 0.05 ≤ P < 0.10, respectively. No difference was observed in serum TNF-α concen- tration among dietary treatments throughout the experi- Results ment. Compared with the pigs fed control diet, pigs Growth performance, diarrhea score, β-hemolytic supplemented with LOW had lower (P < 0.05) haptoglo- coliforms bin on d 5 PI, while pigs fed CAR had lower (P < 0.05) No difference was observed in the initial BW and d 0 C-reactive protein on d 2, 5, and 11 PI and had lower BW of pigs among dietary treatments (Table 2). Pigs (P < 0.05) haptoglobin on d 5 PI. No differences in serum supplemented with CAR had greater (P < 0.05) BW on d C-reactive protein and haptoglobin were observed be- 5 PI than pigs in the control and HIGH groups. Pigs tween the control and HIGH groups. supplemented with LOW had the greatest (P < 0.05) Before ETEC inoculation, pigs in the CAR group had BW, but pigs supplemented with HIGH had the lowest the lowest (P < 0.05) mean corpuscular volume and total (P < 0.05) BW on d 11 PI among all dietary treatments. platelets among all dietary treatments on d 0 (Table S2). Supplementation of LOW had greater (P < 0.05) ADFI of Supplementation of LOW had lower (P < 0.05) red blood pigs from d 5 to 11 PI, compared with control and cells and packed cell volume on d 2 PI, while supple- HIGH groups. Supplementation of Coligo had greater mentation of HIGH had lower (P < 0.05) packed cell vol- (P < 0.05) feed efficiency from d 0 to 5 PI compared with ume on d 5 PI, compared with pigs in the control. Pigs pigs in the control group regardless of dose. Supplemen- supplemented with CAR had lower (P < 0.05) red blood tation of HIGH also had greater (P < 0.05) feed efficiency cells and packed cell volume, but higher (P < 0.05) mean of weaned pigs from d 5 to 11 PI, compared with pigs in corpuscular hemoglobin and mean corpuscular the control. Pigs fed with CAR had better (P < 0.05) feed hemoglobin concentration on d 2 and 5 PI, compared efficiency than pigs fed with the control diet from d 0 to with pigs in the control. Supplementation of CAR also 5 PI, but this was not the case from d 5 to 11 PI. had greater (P < 0.05) total protein concentration on d Pigs supplemented with CAR had the lowest (P < 0.05) 11 PI in comparison to pigs in the other treatments. average diarrhea score from d 0 to 5 PI and d 5 to 11 PI Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 6 of 14 Table 2 Growth performance of ETEC-infected pigs fed diets supplemented with oligosaccharide-based polymer (Coligo) or antibiotics Diet P-value 1 2 3 4 5 Item Control LOW HIGH CAR SEM Diet Linear BW, kg d − 7 7.21 7.24 7.15 7.34 0.36 0.99 0.90 d 0 8.93 9.27 8.69 9.03 0.36 0.42 0.47 b ab b a d 5 PI 10.89 11.57 10.97 11.96 0.35 < 0.05 0.85 ab a b ab d 11 PI* 15.14 16.58 14.76 16.02 0.75 < 0.05 0.55 ADG, g d − 7 to 0 235 296 211 260 55.3 0.29 0.57 d 0 to 5 PI 393 453 456 533 50.6 0.29 0.35 d 5 to 11 PI* 622 727 705 690 32.9 0.25 0.13 ADFI, g d − 7 to 0 424 404 377 331 45.9 0.30 0.35 d 0 to 5 PI 635 631 597 677 34.3 0.49 0.44 b a b ab d 5 to 11 PI* 803 930 806 893 57.3 < 0.05 0.94 G:F d − 7 to 0 0.59 0.77 0.59 0.71 0.101 0.43 0.97 b a a a d 0 to 5 PI 0.57 0.76 0.75 0.77 0.054 < 0.05 < 0.05 b ab a ab d 5 to 11 PI* 0.72 0.79 0.85 0.83 0.043 0.07 < 0.05 a,b Within a row, means without a common superscript differ (P < 0.05) BW body weight, ADG average daily gain, ADFI average daily feed intake, G:F gain:feed, and PI post-inoculation. Each least squares mean represents 12 observations, except the *, which has 6 observations LOW Low dose blood group A6 type 1-based polymer (Coligo) HIGH High dose blood group A6 type 1-based polymer (Coligo) CAR carbadox Linear effects of adding Coligo to the control diet Bacterial translocation Intestinal morphology Supplementation of HIGH had lower (P < 0.05) bacterial On d 5 PI, supplementation of Coligo dose-dependently translocation in lymph nodes on d 5 and 11 PI compared had greater (linear, P < 0.05) villi height, the ratio of villi with control group (Fig. 3). Pigs supplemented with Coligo height to crypt depth, villi width, and villi area in duode- or CAR had lower (P < 0.05) bacterial translocation in the num, had greater (linear, P < 0.05) the ratio of villi height spleen than pigs in the control on d 11 PI. to crypt depth in jejunum, and had greater (linear, P < Table 3 Diarrhea score and frequency of diarrhea of ETEC-infected weaned pigs fed diets supplemented with oligosaccharide-based polymer (Coligo) or antibiotics Diarrhea score Diet P-value 1 2 3 4 Control LOW HIGH CAR SEM Diet Linear 5 a b ab c d0–5 2.88 2.38 2.62 1.78 0.17 < 0.01 0.15 6 a a a b d5–11 2.60 2.17 2.01 1.28 0.31 < 0.01 0.06 Pig days 120 109 120 105 7 a b b b Frequency of diarrhea 27.50 13.76 14.17 7.62 – < 0.01 – a,b,c Within a row, means without a common superscript differ (P < 0.05) LOW Low dose blood group A6 type 1-based polymer (Coligo) HIGH High dose blood group A6 type 1-based polymer (Coligo) CAR carbadox Linear effects of adding Coligo to the control diet Each least squares mean represents 12 observations Each least squares mean represents 6 observations Frequency = number of pen days with fecal score ≥ 4 Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 7 of 14 crypt depth ratio in jejunum, and bigger (P < 0.05) sialo- mucin area in duodenum than pigs in the control group. In addition, pigs in the CAR group also had greater (P < 0.05) villi height:crypt depth in all intestinal segments on d 5 PI, and greater (P < 0.05) villi height in ileum, in comparison to pigs in the Coligo treatment group. Intestinal barrier and innate immunity No difference was observed in the mRNA expression of MUC2 in jejunal mucosa among pigs in all dietary treat- ment groups (Fig.4). On d 5 PI, supplementation of Fig. 1 Daily diarrhea score of ETEC-infected weaned pigs fed diets supplemented with oligosaccharide-based polymer (Coligo) or HIGH up-regulated (P < 0.05) the mRNA expression of antibiotics. Diarrhea score = 1, normal feces, 2, moist feces, 3, mild ZO1 and addition of CAR had greater (P < 0.05) mRNA diarrhea, 4, severe diarrhea, 5, watery diarrhea. Each least squares expression of OCLN, compared with pigs in control mean from d 0 to d 5 post-inoculation (PI) represents 12 group. On d 11 PI, supplementation of LOW or CAR observations. Each least squares mean from d 6 to d 11 PI had higher (P < 0.05) mRNA expression of CLDN1 in je- represents 6 observations. *Significant differences were observed among dietary treatment: P < 0.05. LOW = Low dose blood group A6 junal mucosa of weaned pigs, compared with the control type 1-based polymer (Coligo); HIGH = High dose blood group A6 and HIGH groups. On d 5 PI, supplementation of LOW type 1-based polymer (Coligo); CAR = Carbadox down-regulated (P < 0.05) the mRNA expression of IL6, supplementation of HIGH had lower (P < 0.05) mRNA 0.05) villi height, the ratio of villi height to crypt depth, expression of IL1B, IL6, and TNF, and supplementation and villi area in ileum, compared with the control group of CAR had lower (P < 0.05) IL1B and IL6 gene expres- (Table S3). Supplementation of Coligo also had greater sion in ileal mucosa of weaned pigs in comparison to (linear, P < 0.05) duodenal and jejunal villi height and je- control pigs (Fig. 5). Supplementation of HIGH also had junal and ileal villi area, and tended to have greater (lin- lower (P < 0.05) IL6 mRNA expression on d 11 PI in ileal ear, P < 0.10) the ratio of villi height to crypt depth in mucosa, compared with the control group. However, no jejunum and ileal villi height on d 11 PI. Pigs fed with differences were observed in the gene expression of in- CAR had greater (P < 0.05) villi height in duodenum and flammatory mediators among LOW, HIGH, and CAR ileum, the ratio of villi height to crypt depth in all three groups. intestinal segments, and villi area in duodenum than pigs in the control group on d 5 PI. On d 11 PI, pigs supple- Discussion mented with CAR had higher (P < 0.05) villi height in all ETEC infection is initiated by bacterial attachment to three intestinal segments, greater (P < 0.05) villi height to specific receptors on the intestinal epithelium by fimbrial adhesins, followed by colonization of ETEC in the small intestine [29]. Once colonization is established, ETEC rapidly proliferate and produce one or more entero- toxins, which can stimulate water and electrolyte secre- tion and reduce fluid absorption in the small intestine and induce diarrhea [30]. Diarrhea caused by ETEC is one of the most prevalent diseases during the weaning stage, which is responsible for anorexia, slower growth, or even the death of pigs. Results of the present study demonstrated that supplementation of Coligo improved growth rate, and reduced frequency of diarrhea and sys- temic inflammation of weaned pigs experimentally chal- lenged with F18 ETEC. The potential mechanisms of Fig. 2 The percentage (%) of β-hemolytic coliform in fecal samples action include inhibition of binding of bacteria and as of ETEC-infected pigs fed diets supplemented with oligosaccharide- such colonization of the gut by the F18 ETEC [11, 13], based polymer (Coligo) or antibiotics. Each least squares mean from enhancing gut barrier function and reducing local and d 0 to d 5 post-inoculation (PI) represents 12 observations. Each systemic inflammation. least squares mean from d 6 to d 11 PI represents 6 observations. a,b Means without a common superscript differ (P < 0.05). LOW = Low In the current study, pigs in the control group grew dose blood group A6 type 1-based polymer (Coligo); HIGH = High slower and had a high frequency of diarrhea compared dose blood group A6 type 1-based polymer to pigs without ETEC challenge in our previous research (Coligo); CAR = Carbadox [20, 22]. These observations, combined with the Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 8 of 14 Table 4 Total and differential white blood cells, and serum cytokine and acute-phase proteins in ETEC-infected weaned pigs fed diets supplemented with oligosaccharide-based polymer (Coligo) or antibiotics Diet P-value 1 2 3 4 Item Control LOW HIGH CAR SEM Diet Linear5 d 0 before infection WBC, 10 /μL 15.28 16.91 15.70 16.85 1.00 0.58 0.77 Neu, 10 /μL 7.93 9.03 8.43 7.56 0.67 0.46 0.60 3 b b b a Lym, 10 /μL 6.39 6.72 6.08 8.26 0.48 < 0.05 0.65 Mono, 10 /μL 0.66 0.83 0.79 0.75 0.11 0.73 0.41 Eos, 10 /μL 0.24 0.27 0.35 0.20 0.10 0.64 0.33 Baso, 10 /μL 0.057 0.072 0.051 0.075 0.018 0.074 0.81 Serum TNF-α, pg/mL 63.56 59.67 61.51 72.80 1.62 0.98 0.99 C-reactive protein, μg/mL 7.37 6.86 8.57 8.08 1.39 0.86 0.46 Haptoglobin, g/mL 0.984 0.730 1.211 1.072 0.135 0.065 0.031 d2 PI 3 a ab b ab WBC, 10 /μL 21.16 18.04 17.78 18.53 1.01 < 0.05 < 0.05 3 a b b b Neu, 10 /μL 12.25 9.83 9.79 8.34 0.96 < 0.05 < 0.05 3 ab b b a Lym, 10 /μL 7.27 6.97 6.79 8.54 0.41 < 0.05 0.41 Mono, 10 /μL 1.16 1.05 0.85 1.33 0.16 0.29 0.21 Eos, 10 /μL 0.40 0.25 0.19 0.23 0.10 0.09 < 0.05 3 a b b b Baso, 10 /μL 0.106 0.036 0.039 0.029 0.017 < 0.05 < 0.05 Serum TNF-α, pg/mL 107.92 67.06 66.28 66.28 32.89 0.63 0.28 a a a b C-reactive protein, μg/mL 20.42 22.74 22.88 12.38 2.693 0.018 0.65 ab b a b Haptoglobin, g/mL 1.306 1.142 1.561 1.066 0.127 < 0.05 0.11 d5 PI WBC, 10 /μL 21.00 18.56 18.67 18.19 1.09 0.14 0.15 3 a b ab b Neu, 10 /μL 10.93 9.17 9.54 7.89 0.54 < 0.05 < 0.05 Lym, 10 /μL 9.03 8.22 8.02 8.35 0.65 0.69 0.25 Mono, 10 /μL 0.86 0.86 0.79 1.21 0.15 0.29 0.74 3 b ab ab a Eos, 10 /μL 0.13 0.26 0.25 0.55 0.14 < 0.05 0.41 Baso, 10 /μL 0.059 0.043 0.078 0.057 0.016 0.49 0.41 Serum TNF-α, pg/mL 90.64 68.36 54.39 32.99 32.88 0.25 0.16 a ab ab b C-reactive protein, μg/mL 21.61 17.46 19.84 15.96 1.72 0.082 0.93 a b ab b Haptoglobin, g/mL 1.655 1.084 1.348 1.154 0.128 0.081 0.93 d11 PI WBC, 10 /μL 16.15 17.40 17.32 14.92 1.58 0.64 0.57 Neu, 10 /μL 8.68 10.71 9.67 8.12 1.22 0.16 0.40 Lym, 10 /μL 6.33 6.69 7.21 6.39 0.63 0.76 0.35 Mono, 10 /μL 0.66 1.12 0.99 1.39 0.19 0.10 0.21 Eos, 10 /μL 0.18 0.31 0.24 0.21 0.13 0.74 0.57 Baso, 10 /μL 0.058 0.083 0.048 0.042 0.025 0.51 0.70 Serum TNF-α, pg/mL 82.58 84.62 72.71 59.39 28.03 0.96 0.79 Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 9 of 14 Table 4 Total and differential white blood cells, and serum cytokine and acute-phase proteins in ETEC-infected weaned pigs fed diets supplemented with oligosaccharide-based polymer (Coligo) or antibiotics (Continued) Diet P-value 1 2 3 4 Item Control LOW HIGH CAR SEM Diet Linear5 a ab ab b C-reactive protein, μg/mL 21.95 17.71 18.29 12.61 2.89 0.091 0.65 Haptoglobin, g/mL 0.819 0.599 0.538 0.412 0.178 0.24 0.31 a,b Within a row, means without a common superscript differ (P < 0.05) WBC white blood cell, Neu neutrophil, Lym lymphocyte, Mono monocyte, Eos eosinophil, Baso basophil, PI post-inoculation. Each least squares mean represents 12 observations, except d 11 PI that has 6 observations LOW Low dose blood group A6 type 1-based polymer (Coligo) HIGH High dose blood group A6 type 1-based polymer (Coligo) CAR carbadox Linear effects of adding Coligo to the control diet presence of β-hemolytic coliforms in feces, confirmed hemolytic coliforms than pigs fed with antibiotics. These that pigs were successfully infected with F18 ETEC. In observations indicated that the beneficial effects of agreement with our previous research, the peak of F18 Coligo and antibiotics on reducing weaned pigs’ diarrhea ETEC infection was present approximately 5 days post- were through different mechanisms. It has been reported inoculation, and most pigs moved into the recovery that heat-labile toxin expressed by ETEC binds blood stage on day 11 to 12 post-inoculation. Results of the group antigens, with a preference for A-epitope [33]. current study have demonstrated that pigs supplemented Moreover, it has been hypothesized that blood group A with Coligo or antibiotics had reduced frequency of diar- antigen might disturb the toxin activity by interfering rhea and enhanced growth performance than pigs in with ETEC binding to the receptors in the small intes- control group, indicating both supplements could pro- tine of pigs [34]. Coddens et al. [10] also observed a high tect pigs against F18 ETEC infection. correlation between blood group A antigen and F18 In agreement with the diarrhea severity results, pigs ETEC adherence in the small intestine of young pigs supplemented with antibiotics had less β-hemolytic coli- in vitro. Moreover, it has been demonstrated that F18 E. forms compared with pigs in the control group, indicat- coli specifically interact with glycosphingolipids possess ing lowered F18 ETEC shedding in pig’s feces during the blood group ABH determinants on a type 1 core, which peak infection period. The exact mechanisms of action were identified as the cell surface receptors for F18 fim- of carbadox are not fully understood, but it has been brial binding to the small intestinal epithelium [11]. suggested that virulence activity was disabled by interfer- With these specific features, the addition of extra blood ing with DNA synthesis in Gram-negative bacteria, in- group A antigen could enhance the binding affinity of cluding E. coli [31, 32]. However, pigs supplemented F18 ETEC to polymers. Thus, fecal culture results sug- with Coligo had a relatively higher percentage of β- gest that the Coligo polymer may reduce the ETEC at- tachment to the small intestine and accelerate the excretion of these pathogenic bacteria from their gastro- intestinal tract. Taken altogether, antibiotics or Coligo may help pigs recover from ETEC infection through dif- ferent mechanisms, which we attempted to explore in the current research. Tight junctions play critical roles in maintaining the integrity of intestinal structure and barrier, and regulat- ing intestinal paracellular permeability [35, 36]. Several multi-protein complexes, including zonulae occludens (ZO), occludins, and claudins, are involved in the tight junction barrier. Previous studies have reported that Fig. 3 Bacterial counts (CFU/g) in lymph node and spleen of ETEC- ETEC infection impairs intestinal barrier function by infected weaned pigs fed diets supplemented with oligosaccharide- down-regulating tight junction protein expression, lead- based polymer (Coligo) or antibiotics. Each least squares mean from ing to intestinal inflammation [22, 37]. Morphological le- d 0 to d 5 post-inoculation (PI) represents 12 observations. Each sions, such as loss of villus absorptive cells, villus least squares mean from d 6 to d 11 PI represents 6 observations. a,b Means without a common superscript differ (P < 0.05). LOW = Low atrophy, and intestinal permeability disturbances are also dose blood group A6 type 1-based polymer (Coligo); HIGH = High observed in the small intestine of pigs with ETEC infec- dose blood group A6 type 1-based polymer tion [1, 38]. In the present study, pigs supplemented (Coligo); CAR = Carbadox with Coligo or antibiotics had greater mRNA expression Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 10 of 14 Fig. 4 Gene expression profiles in jejunal mucosa of ETEC-infected weaned pigs fed diets supplemented with oligosaccharide-based polymer a,b (Coligo) or antibiotics on d 5 or 11 post-inoculation (PI). Means without a common superscript differ (P < 0.05). Each least squares mean represents 6 observations. LOW = Low dose blood group A6 type 1-based polymer (Coligo); HIGH = High dose blood group A6 type 1-based polymer (Coligo); CAR = Carbadox; MUC2 = Mucin-2; CLDN1 = Claudin-1; ZO-1 = Zonula occludens-1; OCDN = Occludin of ZO1 or OCDN at the peak of ETEC infection, respect- normally sterile tissues, such as mesenteric lymph ively, and greater CLDN1 expression on d 11 PI than nodes and other internal organs, including the spleen pigs in control group. ZO-1 protein connects and inter- [40, 41]. The major mechanisms promoting bacterial acts with junctional proteins, such as occludins and clau- translocation are intestinal bacterial overgrowth, defi- dins, to form the physical barrier, which determines the ciencies in host immune defenses by disturbed gut in- permselectivity of the paracellular diffusion pathway tegrity, and increased permeability or mucosal injury [39]. The up-regulation of mRNA expression of tight [42]. It was previously reported that bacterial trans- junction proteins in this study suggests the protective ef- location to mesenteric lymph nodes was increased in fects of Coligo or antibiotics on intestinal barrier func- pigs challenged with ETEC [43, 44]. In the current tion against ETEC infection. Consistently, pigs in Coligo study, pigs supplemented with antibiotics lowered groups or antibiotics group had higher villi height, bacterial populations in the spleen, and pigs fed with greater villi height to crypt depth ratio, and villi area, Coligo had lower bacterial populations in both mes- demonstrating a preventive effect against intestinal enteric lymph nodes and spleen than pigs in the con- structure disruption. Overall, these results demonstrated trol group. These observations clearly supported that that supplementation of Coligo or antibiotics enhanced supplementation of Coligo or antibiotics reduced the gut integrity and morphology of weaned pigs, which was damage of ETEC infection on the gut integrity of one of the major reasons these pigs grew faster than pigs weaned pigs compared with control pigs. Overall, re- in control group. sults of tight junction protein mRNA expression, in- Bacterial translocation is defined as the passage of testinal morphology, and bacterial translocation imply viable bacteria from the gastrointestinal tract to that pigs in Coligo and antibiotics groups may have Fig. 5 Gene expression profiles in ileal mucosa of ETEC-infected weaned pigs fed diets supplemented with oligosaccharide-based polymer a,b (Coligo) or antibiotics on d 5 or 11 post-inoculation (PI). Means without a common superscript differ (P < 0.05). Each least squares mean represents 6 observations. LOW = Low dose blood group A6 type 1-based polymer (Coligo); HIGH = High dose blood group A6 type 1-based polymer (Coligo); CAR = Carbadox; IL1B: Interleukin-1 beta; IL6: Interleukin-6; TNF = Tumor necrosis factor; PTGS2: Cyclooxygenase-2 Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 11 of 14 better intestinal health, which would be responsible potentially modified intestinal microbiota by Coligo. It for better nutrient digestion and absorption, and was reported that porcine blood type AO could be a hence better performance. possible factor influencing microbiota composition [56] The colonized F18 ETEC could produce large quan- and Priori et al. [57] suggest changes in the intestinal tities of toxins, such as heat-labile toxins, heat-stable microbiota are affected by porcine blood group. Thus, toxins, Shiga toxins, and lipopolysaccharides [45]. Those the blood group A antigen in Coligo may affect the com- toxins induce functional changes in the small intestinal position and function of microbial communities when epithelial cells, as well as stimulate the synthesis of cyto- fed to pigs. Moreover, recent studies demonstrated that kines and acute-phase proteins (e. g. C-reactive protein dietary supplementation of ε-PL altered ileal microbiota and haptoglobin), followed by systemic and local inflam- structure and function in pigs [58] and fecal microbial mation [1, 46]. Our previously published research that community in mice [59]. ε-PL supplementation may used the same bacteria strain, F18 ETEC, reported that promote the growth of beneficial microorganisms in the ETEC infection could induce systemic inflammation, intestinal tract, therefore, reducing the proliferation of such as increasing white blood cell counts, neutrophils, pathogens. The exact mechanisms of ε-PL in the current and lymphocytes, as well as enhancing several pro- study remain unclear, so further research is needed to inflammatory cytokines and acute-phase protein concen- confirm the effects of Coligo on the pigs’ gut microbial trations in serum of weaned pigs [20, 47]. In the current community, intestinal inflammation, and immune re- study, pigs supplemented with antibiotics had reductions sponses against F18 ETEC. Pigs supplemented antibi- in neutrophils and serum concentrations of C-reactive otics also had reductions in mRNA expression of protein and haptoglobin during the peak infection proinflammatory markers in the present study. This period. Similarly, pigs supplemented with Coligo had finding demonstrated that pigs supplemented antibiotics lower numbers of white blood cells, neutrophils, and has less severe intestinal inflammation than pigs in con- lymphocytes on d 2 PI, and lower neutrophils on d 5 PI. trol group. In agreement with previous research, anti- In addition, serum haptoglobin concentrations were also biotic supplements might exert anti-inflammatory relatively lower in the pigs supplemented with Coligo. properties in the intestine or accumulate in phagocytic These findings demonstrated the capacity of antibiotics inflammatory cells, therefore, attenuating inflammatory and Coligo polymer in alleviating ETEC-induced sys- responses in animals [60, 61]. Taken altogether, down- temic inflammation, possibly by reducing the bacterial regulation in mRNA expression of proinflammatory cy- population in pigs’ gut. tokines by Coligo or antibiotics supplementation is In response to an infectious challenge, it is well recog- beneficial for pigs in terms of their intestinal health and nized that innate immune responses have essential roles growth performance. in preventing and suppressing inflammation [48]. Patho- In conclusion, results in the current study suggest that gen recognition receptors initiate the innate immune re- in-feed supplementation of Coligo or antibiotic (carba- sponse on intestinal epithelial cells to detect and dox) enhanced growth performance and reduced the se- recognize the pathogen-associated molecular patterns, verity of diarrhea caused by ETEC F18 infection. such as microbial membranes, resulting in a rapid re- Although the percentage of β-hemolytic coliforms in lease of proinflammatory cytokines [49]. It has been re- fecal samples of pigs fed with Coligo was less diminished ported that secreted proinflammatory cytokines, which than pigs supplemented with antibiotics, enhanced dis- are primarily involved in host responses to disease or in- ease resistance was demonstrated by the improved gut fection, could induce intestinal inflammation, tissue de- barrier integrity and attenuated systemic and intestinal struction, and nutrient and ion malabsorption in pigs inflammation. To further explore the mechanisms of ac- [50, 51]. Moreover, numerous studies have confirmed tion of Coligo, integrated metabolomics and metage- the induction of proinflammatory cytokines in porcine nomics approaches may be considered to provide more intestinal epithelial cells caused by ETEC infection [52– insights into the beneficial effects of Coligo or other 54]. The elevation of proinflammatory cytokines by polymers on pigs’ health. Overall, the current study indi- ETEC challenge has nutrient cost, thus also contributing cates that supplementation with Coligo has promising to the reduced growth performance of pigs [55]. In the impacts on promoting growth and disease resistance of current study, mRNA expression of proinflammatory newly weaned pigs infected with ETEC F18. The efficacy markers (i.e., IL1B, IL6, and TNF) in ileal mucosa were of Coligo is comparable to antibiotic (carbadox) demon- down-regulated by Coligo supplementation, which is strating the potential of Coligo as antibiotic alternative consistent with the results of systemic immunity. These for animal growth performance and disease resistance. observations can be accounted for by assuming not only Large-scale animal trials are recommended to further reduced the attachment of ETEC to the intestinal epithe- evaluate the impacts of Coligo on performance of lium by blood group oligosaccharides, but also weaned pigs under commercial practice conditions. Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 12 of 14 Abbreviations Received: 2 July 2021 Accepted: 21 November 2021 ADFI: Average daily feed intake; ADG: Average daily gain; BW: Body weight; CFU: Colony-forming unit; CLDN1: Claudin-1; ETEC: Enterotoxigenic E. coli; IL1B: Interleukin-1 beta; IL6: Interleukin-6; mRNA: Messenger RNA; MUC2: Mucin-2; OCDN: Occludin; PI: Post-inoculation; qRT-PCR: Quantitative References real-time polymerase chain reaction; TNF: Tumor necrosis factor; ZO-1: Zona 1. Nagy B, Fekete PZ. Enterotoxigenic Escherichia coli in veterinary medicine. occludens-1 Int J Med Microbiol. 2005;295(6-7):443–54. https://doi.org/10.1016/j.ijmm.2 005.07.003. 2. Fairbrother JM, Nadeau É, Gyles CL. Escherichia coli in postweaning diarrhea in pigs: an update on bacterial types, pathogenesis, and prevention Supplementary Information strategies. Anim Health Res Rev. 2005;6(1):17–39. https://doi.org/10.1079/A The online version contains supplementary material available at https://doi. HR2005105. org/10.1186/s40104-021-00655-2. 3. Van Boeckel TP, Brower C, Gilbert M, Grenfell BT, Levin SA, Robinson TP, et al. Global trends in antimicrobial use in food animals. Proc Natl Acad Sci. Additional file 1. Table S1 Gene-specific primer sequences and PCR 2015;112(18):5649–54. https://doi.org/10.1073/pnas.1503141112. conditions. 4. FDA (Food and Drug Administration) New animal drugs and new animal Additional file 2. Table S2 Red blood cell profiles of ETEC-infected drug combination products administered in or on medicated feed or weaned pigs fed diets supplemented with oligosaccharide-based poly- drinking water of food-producing animals: recommendations for drug mer (Coligo) or antibiotics. sponsors for voluntarily aligning product use conditions with FDA Guidance for Industry #213. Center for Veterinary Medicine. Washington, DC: US Additional file 3. Table S3 Intestinal morphology of ETEC-infected Department of Health and Human Services; 2016. https://www.fda.gov/ weaned pigs fed diets supplemented with oligosaccharide-based poly- downloads/AnimalVeterinary/GuidanceComplianceEnforcement/Guida mer (Coligo) or antibiotics. nceforIndustry/UCM299624.pdf. 5. Nollet H, Deprez P, Van Driessche E, Muylle E. Protection of just weaned pigs against infection with F18+ Escherichia coli by non-immune plasma Acknowledgments powder. Vet Microbiol. 1999;65(1):37–45. https://doi.org/10.1016/S0378-113 Not applicable. 5(98)00282-X. 6. Newburg DS. Do the binding properties of oligosaccharides in milk protect human infants from gastrointestinal bacteria. J Nutr. 1997;123:980S–4S. Authors’ contributions https://doi.org/10.1093/jn/127.5.980S. The contributions of the authors were as follows: KK conducted the 7. 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Supplementation of oligosaccharide-based polymer enhanced growth and disease resistance of weaned pigs by modulating intestinal integrity and systemic immunity

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Abstract

Background: There is a great demand for antibiotic alternatives to maintain animal health and productivity. The objective of this experiment was to determine the efficacy of dietary supplementation of a blood group A6 type 1 antigen oligosaccharides-based polymer (Coligo) on growth performance, diarrhea severity, intestinal health, and systemic immunity of weaned pigs experimentally infected with an enterotoxigenic Escherichia coli (ETEC), when compared with antibiotics. Results: Pigs in antibiotic carbadox or Coligo treatment groups had greater (P < 0.05) body weight on d 5 or d 11 post-inoculation (PI) than pigs in the control group, respectively. Supplementation of antibiotics or Coligo enhanced (P < 0.05) feed efficiency from d 0 to 5 PI and reduced (P < 0.05) frequency of diarrhea throughout the experiment, compared with pigs in the control group. Supplementation of antibiotics reduced (P < 0.05) fecal β- hemolytic coliforms on d 2, 5, and 8 PI. Pigs in antibiotics or Coligo groups had reduced (P < 0.05) neutrophil counts and serum haptoglobin concentration compared to pigs in the control group on d 2 and 5 PI. Pigs in Coligo had reduced (P < 0.05) total coliforms in mesenteric lymph nodes on d 5 and 11 PI, whereas pigs in antibiotics or Coligo groups had reduced (P < 0.05) total coliforms in spleen on d 11 PI compared with pigs in the control group. On d 5 PI, pigs in the Coligo group had greater (P < 0.05) gene expression of ZO1 in jejunal mucosa, but less (P < 0.05) mRNA expression of IL1B, IL6, and TNF in ileal mucosa, in comparison with pigs in the control group. Supplementation of antibiotics enhanced (P < 0.05) the gene expression of OCLN in jejunal mucosa but decreased (P < 0.05) IL1B and IL6 gene expression in ileal mucosa, compared with the control. On d 11 PI, supplementation of antibiotics or Coligo up-regulated (P < 0.05) gene expression of CLDN1 in jejunal mucosa, but Coligo reduced (P < 0.05) IL6 gene expression in ileal mucosa compared to pigs in the control group. Conclusions: Supplementation of Coligo improved growth performance, alleviated diarrhea severity, and enhanced gut health in weaned pigs infected with ETEC F18 in a manner similar to in-feed antibiotics. Keywords: Enterotoxigenic E. coli, Growth rate, Intestinal barrier function, Oligosaccharide-based polymer, Systemic immunity, Weaned pigs * Correspondence: yahliu@ucdavis.edu Department of Animal Science, University of California, Davis, CA 95616, USA Full list of author information is available at the end of the article © The Author(s). 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 2 of 14 Background combination of blood group oligosaccharides and ε-PL Enterotoxigenic E. coli (ETEC) strains expressing F4 or may enhance the resistance of pigs against F18 ETEC in- F18 fimbriae are major causes of post-weaning diarrhea fection by inhibiting bacterial attachment and/or directly in nursery pigs [1]. Attachment of ETEC to the specific killing bacteria. The overall objective of this study was to receptors on intestinal epithelium leads to colonization investigate the efficacy of blood group A6 type 1-based and secretion of enterotoxins, resulting in secretory diar- polymer on gut integrity and disease resistance of wean- rhea in weanling pigs [2]. To prevent post-weaning diar- ling pigs challenged with F18 ETEC. rhea and improve production of pigs, antibiotics were commonly added to the diet over the past decades. Materials and methods However, frequent use of in-feed antibiotics in livestock Animals, housing, experimental design, and diet production has been shown to contribute to the in- A total of 48 weanling pigs (crossbred; initial body creased prevalence of antibiotic-resistant bacteria and weight (BW): 7.23 ± 1.14 kg; 21 days old) with an equal raised public health concerns [3]. With these issues, the number of gilts and barrows were used in this study. Food and Drug Administration (FDA) banned the use of They were selected from the Swine Teaching and Re- in-feed antibiotics for growth promoting purposes in search Center at the University of California, Davis. The livestock production in the U.S. [4], thus alternative nu- sows and piglets used in this experiment did not receive tritional strategies are highly demanded to enhance dis- E. coli vaccines, antibiotic injections, or antibiotics in ease resistance and production of weanling pigs. creep feed. Before weaning, feces were collected from Many nutritional approaches have been applied to pre- sows and all their piglets destined for this study to verify vent post-weaning diarrhea associated with ETEC and the absence of β-hemolytic E. coli. The F18 ETEC recep- enhance the production of pigs. Among these, the pre- tor status was also tested based on the methods of Kreu- vention of bacterial attachment to the small intestine is zer et al. [18], and all piglets used in this study were one of the most effective defense strategies against ETEC susceptible to F18 ETEC. infection [5]. Oligosaccharides have been reported to After weaning, all pigs were randomly assigned to one possess ETEC receptor activity for bacterial adhesions of the four dietary treatments in a randomized complete [6, 7]. Especially, N-acetylgalactosamine (GalNAc) con- block design with body weight within sex and litter as taining glycans that could enhance the binding affinity the blocks and pig as the experimental unit. There were of various ETEC strains, including K99 [8], F4 [9], and 12 replicate pigs per treatment. Pigs were individually F18 [10–12]. Coddens et al. [11] identified the blood housed (pen size: 0.61m × 1.22 m) in environmental con- group H type 1 determinant (Fuca2Galß3GlcNAc) as the trol rooms at the Cole Facility at the University of Cali- minimal binding epitope of F18 fimbriae. Based on that, fornia, Davis for 18 d, including 7 d before and 11 d after an optimal binding epitope was created by adding the the first F18 ETEC challenge (d 0). The piglets had ad terminal 3-linked galactose or N-acetylgalactosamine of libitum access to feed and water. Environmental enrich- the blood group B type 1 determinant (Galα3(Fucα2)- ment was provided for each pig. The light was on at 07: Galß3GlcNAc) and the blood group A type 1 determin- 00 h and off at 19:00 h daily in the environmental control ant (GalNAcα3(Fucα2)- Galß3GlcNAc). The purified rooms. soluble blood group oligosaccharides were able to greatly The 4 dietary treatments included: 1) Positive control: reduce binding of F18 positive E. coli to intestinal villi of control diet; 2) Low dose oligosaccharide-based polymer F18 receptor-positive pigs in concentrations of 1 to 10 (LOW): control diet supplemented with 10 mg/kg mg/mL [11]. Moreover, multimerizing blood group A on oligosaccharide-based polymer active substance (Coligo); human serum albumin reduced the amount of blood 3) High dose oligosaccharide-based polymer (HIGH): group oligosaccharides needed 1000 times [13]. There- control diet supplemented with 20 mg/kg fore, multimerizing the blood group A oligosaccharides oligosaccharide-based polymer active substance (Coligo); may efficiently prevent enterotoxin-induced secretory and 4) CAR: control diet supplemented with 50 mg/kg diarrhea. Recently, grafted polymers that combine mul- carbadox. Spray-dried plasma and high levels of zinc tiple substances have been proposed for their potential oxide exceeding recommendation and normal practice synergistic effects on preventing human and animal dis- were not included in the diets. The experimental diets eases [14, 15]. Epsilon-poly-lysin (ε-PL) has been re- were fed to pigs throughout the experiment. ported to use as a carrier in the membrane transport of Oligosaccharide-based polymer active substance was a proteins and drugs [16]. Due to its excellent heat stabil- glycoconjugate composed of blood group A6 type 1 anti- ity, biodegradability, and lack of toxicity, ε-PL has gener- gen oligosaccharides grafted on a single peptide of ally been regarded as safe status (GRAS) and been epsilon-poly-lysine. Coligo was designed and synthesized interested in the food and medicine industries as a deliv- by Elicityl (France) in cooperation with Ghent University ery vehicle targeting the desired location [17]. Thus, the (Dr. Eric Cox’s laboratory) and was provided by Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 3 of 14 Pancosma (Geneva, Switzerland). The mean rate of con- response parameters that were reported in previously jugation is 15 mol of oligosaccharide for 1 mol of ε-PL. published research using the same ETEC strain and in- The oligosaccharide part represents 25% of the molecu- oculation dose [21, 22]. lar weight of the oligosaccharide-based polymer. All di- To achieve proper restraint and positioning for ets were formulated to meet pig nutritional blood sample collection, pig was placed on a V- requirements (Table 1)[19] and provided as mash form shaped table restrained in dorsal recumbence. Blood throughout the experiment. samples were collected from the jugular vein of all After 7 days of adaptation, all pigs were orally inoc- pigs with or without ethylenediaminetetraacetic acid ulated with 3 mL of F18 ETEC for 3 consecutive days (EDTA) to yield whole blood and serum, respectively, from d 0 post-inoculation (PI). The F18 ETEC was before ETEC challenge (d 0), and on d 2, 5, and 11 originally isolated from a field disease outbreak by the PI. Serum samples were collected and immediately University of Illinois Veterinary Diagnostic Lab (iso- stored at − 80 °C before further analysis. Before eu- late number: U.IL-VDL # 05–27,242). The F18 ETEC thanasia, pigs were anesthetized with a 1-mL mixture expresses heat-labile toxin (LT), heat-stable toxin b of 100 mg telazol, 50 mg ketamine, and 50 mg xylazine (STb), and Shiga-like toxin (Stx2e). The inoculums (2:1:1) by intramuscular injection. After anesthesia, in- were prepared by the laboratory of the Western Insti- tracardiac injection with 78 mg sodium pentobarbital tute for Food Safety and Security at the University of (Vortech Pharmaceuticals, Ltd., Dearborn, MI, USA) California, Davis, and were provided at 10 colony- per 1 kg of BW was used to euthanize each pig. forming unit (CFU) per 3 mL dose in phosphate- Three 3-cm segments from the duodenum, the middle buffered saline (PBS). This dose caused mild diarrhea of the jejunum, and the ileum (10 cm close to the in the current study, consistent with our previous ileocecal junction) were collected and fixed in Car- published researches [20–22]. noy’s solution (ethanol, chloroform, and glacial acetic acid, 6:3:1 v/v/v) for intestinal morphology analysis. Clinical observations and sample collections Mesenteric lymph nodes were aseptically collected The procedures for this study were adapted from previ- and then pooled within the pig, grounded, diluted, ous research methods of Liu et al. [20] and Kim et al. and plated on brain heart infusion agar for measure- [21, 22]. Clinical observations (diarrhea score and alert- ment of total bacteria, and the results were expressed ness score) were recorded twice daily throughout the as CFU per g of lymph node [23, 24]. Spleen samples study. The diarrhea score of each pig was assessed each were analyzed in the same method as mesenteric day visually by two independent evaluators, with the lymph nodes for bacterial translocation. score ranging from 1 to 5 (1 = normal feces, 2 = moist feces, 3 = mild diarrhea, 4 = severe diarrhea, and 5 = Detection of β-hemolytic coliforms watery diarrhea). The frequency of diarrhea was calcu- Briefly, fecal samples were plated on Columbia Blood lated as the percentage of the counting pig days with a Agar with 5% sheep blood to identify hemolytic coli- diarrhea score 4 or greater. The alertness score of each forms, which can lyse red blood cells surrounding the pig was assessed visually with a score from 1 to 3 (1 = colony. Fecal samples were also plated on MacConkey normal, 2 = slightly depressed or listless, and 3 = severely agar to enumerate total coliforms. Hemolytic colonies depressed or recumbent). All pigs had an alertness score from the blood agar were sub-cultured on MacConkey 1 throughout the study, therefore, data are not reported. agar to confirm that they were lactose-fermenting bac- Pigs were weighed on weaning day (d − 7), d 0 before teria and flat pink colonies. All plates were incubated at inoculation, d 5, and 11 PI. Feed intake was recorded 37 °C for 24 h in an air incubator. Populations of both throughout the study. Average daily gain (ADG), average total coliforms and β-hemolytic coliforms on blood agar daily feed intake (ADFI), and feed efficiency (gain:feed) were assessed visually, with a score from 0 to 8 (0 = no was calculated for each interval from d −7to0,d0to5 bacterial growth, 8 = very heavy bacterial growth). The PI, and d 5 to 11 PI. Fecal samples were collected from ratio of scores of β-hemolytic coliforms to total coli- the rectum of all pigs throughout the experiments using forms was calculated. Questionable colonies were sub- a fecal loop or cotton swap on d 2, 5, 8, and 11 PI to test sub-cultured on new MacConkey and blood agar plates for β-hemolytic coliforms and percentage [20–22]. to verify if they were β-hemolytic E. coli by using triple Twenty-four pigs (3 barrows and 3 gilts from each treat- sugar iron agar and lysine iron agar, then verify if they ment) were euthanized on d 5 PI near the peak of infec- were F18 positive E. coli using PCR [25]. tion, and the remaining pigs were euthanized at the end of the experiment (d 11 PI) that was the recovery period Complete blood count of the infection. The selection of necropsy time was Whole blood samples were used for measuring total based on the results of clinical observations and immune and differential blood cell counts by the Comparative Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 4 of 14 Table 1 Ingredient compositions of experimental diets Pathology Laboratory at the University of California, Ingredient, % Control diet Davis. A multiparameter, automated programmed hematology analyzer (Drew/ERBA Scientific 950 FS Corn 44.51 Hematological Analyzer, Drew Scientific Inc., Miami, Dried whey 15.00 FL) was used for the assay to differentiate porcine Soybean meal 14.00 blood optimally. Fish meal 10.00 Soy protein concentrate 7.00 Measurements of serum cytokine and acute-phase Lactose 6.00 proteins Soybean oil 2.00 Serum samples were analyzed for a pro-inflammatory Limestone 0.56 cytokine (Tumor necrosis factor-α; TNF-α; R&D System Inc., Minneapolis, MN, USA) and acute-phase proteins L-Lysine·HCl 0.15 (C-reactive protein and haptoglobin; GenWay Biotech DL-Methionine 0.06 Inc., San Diego, CA, USA) using porcine-specific L-Threonine 0.02 enzyme-linked immunosorbent assay (ELISA) kits. All Salt 0.40 samples were analyzed in duplicate, including standard Vit-mineral, Sow 6 0.30 and control. The intra-assay coefficients of variation for Total 100.00 TNF-α, C-reactive protein, and haptoglobin were 6.2%, Calculated energy and nutrient 4.1%, and 2.7%, respectively. The inter-assay coefficients Metabolizable energy, kcal/kg 3487 of variation for TNF-α, C-reactive protein, and haptoglo- bin were 10.0%, 5.6%, and 6.2%, respectively. The results Net energy, kcal/kg 2615 of TNF-α, C-reactive protein, and haptoglobin were Crude protein, % 22.97 expressed in picograms, micrograms, or milligrams per Ile, % 0.86 milliliter based on the standard curves. Leu, % 1.68 Lys, % 1.35 Intestinal morphology Met, % 0.44 The fixed intestinal tissues were embedded in paraffin, Thr, % 0.79 sectioned at 5 μm, and stained with high iron diamine Trp, % 0.23 and alcian blue. The slides were photographed by an Olympus BX51 microscope at 100 × amplification, and Val, % 0.95 all measurements were conducted in the image process- Met + Cys, % 0.74 ing and analysis software (Image J, NIH). Fifteen straight Ca, % 0.80 and integrated villi and their associated crypts and sur- Total P, % 0.69 rounded area were selected to analyze villi height, crypt Digestible P, % 0.47 depth, the number of goblet cells per villus, and cross- Analyzed nutrients, % sectional area of sulfo- and sialomucin as described by Dry matter 89.6 Deplancke and Gaskins [26], and Kim et al. [22]. Crude protein 22.58 Intestinal barrier and innate immunity ADF 2.87 Jejunal and ileal mucosa samples were analyzed for gene NDF 6.99 expression by quantitative real-time PCR (qRT-PCR). Ca 1.04 Briefly, approximately 100 mg of mucosa sample was ho- P 0.70 mogenized using TRIzol reagent (Invitrogen; Thermo Three additional diets were formulated by adding 10 mg/kg of group A6 Fisher Scientific, Inc., Waltham, MA, USA). Then total type 1-based polymer, 20 mg/kg of group A6 type 1-based polymer (Coligo), or 50 mg/kg of Carbadox to the control diet, respectively RNA was extracted following RNA extraction procedural Provided the following quantities of vitamins and micro minerals per guidelines provided by the reagent manufacturer. The kilogram of complete diet: Vitamin A as retinyl acetate, 11,136 IU; vitamin RNA quality and quantity were assessed by Agilent Bioa- D as cholecalciferol, 2208 IU; vitamin E as DL-alpha tocopheryl acetate, 66 IU; vitamin K as menadione dimethylprimidinol bisulfite, 1.42 mg; thiamin as nalyzer 2100 (Agilent, Santa Clara, CA, USA). The thiamine mononitrate, 0.24 mg; riboflavin, 6.59 mg; pyridoxine as pyridoxine cDNA was produced from 1 μg of total RNA per sample hydrochloride, 0.24 mg; vitamin B , 0.03 mg; D-pantothenic acid as D- calcium pantothenate, 23.5 mg; niacin, 44.1 mg; folic acid, 1.59 mg; biotin, using the High-Capacity cDNA Reverse Transcription 0.44 mg; Cu, 20 mg as copper sulfate and copper chloride; Fe, 126 mg as Kit (Applied Biosystems; Thermo Fisher Scientific, Inc., ferrous sulfate; I, 1.26 mg as ethylenediamine dihydriodide; Mn, 60.2 mg as Waltham, MA, USA) in a total volume of 20 μL. The manganese sulfate; Se, 0.3 mg as sodium selenite and selenium yeast; and Zn, 125.1 mg as zinc sulfate mRNA expression of Claudin 1 (CLDN1), Mucin 2 Amino acids were indicated as standardized ileal digestible AA (MUC2), Occludin (OCLN), and Zonula occludens-1 Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 5 of 14 (ZO-1) in jejunal mucosa and the mRNA expression of among all dietary treatments (Table 3; Fig. 1). Compared Interleukin 1 beta (IL1B), Interleukin 6 (IL6), Cyclooxy- with pigs in control group, pigs supplemented with genase 2 (PTGS2), and Tumor necrosis factor-alpha LOW had lower (P < 0.05) average diarrhea score of (TNF) in ileal mucosa were analyzed by qRT-PCR. Data weaned pigs from d 0 to 5 PI, but this was not the case normalization was accomplished using beta-actin from d 5 to 11 PI. Supplementation of CAR or any dose (ACTB) and ribosomal protein L4 (RPL4) as housekeep- of Coligo had lower (P < 0.05) frequency of diarrhea of ing genes. Primers were designed based on published lit- weaned pigs from d 0 to 11 PI. erature and commercially synthesized by Integrated No β-hemolytic coliform was observed in the feces of DNA Technologies, Coralville, IA. All primers were veri- all pigs before ETEC inoculation. Pigs supplemented fied prior to qRT-PCR (Table S1). The qRT-PCR reac- with CAR had the lowest (P < 0.05) β-hemolytic coliform tion conditions followed the published research [27]. percentage in feces on d 2 and 5 PI among all dietary -ΔΔCT The 2 method was used to analyze the relative ex- treatments (Fig. 2). The percentage of β-hemolytic coli- pression of genes compared with control [28]. form in feces was not different between Coligo groups and CAR on d 8 PI. There were no differences observed Statistical analysis in fecal culture on d 11 PI among the treatments. The normality of data was verified with the Shapiro- Wilk test, and outliers were identified using the UNI- Systemic immunity and red blood cell profile VARIATE procedure (SAS Inst. Inc., Cary, NC, USA). Lymphocyte counts were greater (P < 0.05) in pigs fed All data were analyzed by ANOVA using the PROC CAR on d 0 before ETEC inoculation (Table 4). Pigs in MIXED of SAS (SAS Institute Inc., Cary, NC, USA) in a the LOW group had lower (P < 0.05) neutrophils, lym- randomized complete block design with the pig as the phocytes, and basophils on d 2 PI and lower (P < 0.05) experimental unit. The statistical model included inde- neutrophil counts on d 5 PI, compared with pigs in the pendent variables treatment group, sampling day, and control group. Supplementation of HIGH also had lower interactions as the fixed effect and blocks as random ef- (P < 0.05) white blood cell counts, neutrophils, lympho- fects. Treatment means were separated by using the cytes, and basophils on d 2 PI. Pigs in the CAR group LSMEANS statement and the PDIFF option of PROC had lower (P < 0.05) neutrophils and basophils on d 2 PI MIXED. Contrast statements were used to analyze the and lower (P < 0.05) neutrophils on d 5 PI, but higher dose effects of Coligo. The Chi-square test was used for (P < 0.05) eosinophils on d 5 PI, compared with pigs in analyzing the frequency of diarrhea. Statistical signifi- control group. No difference was observed in white cance and tendency were considered at P < 0.05 and blood cell profiles among treatments on d 11 PI. 0.05 ≤ P < 0.10, respectively. No difference was observed in serum TNF-α concen- tration among dietary treatments throughout the experi- Results ment. Compared with the pigs fed control diet, pigs Growth performance, diarrhea score, β-hemolytic supplemented with LOW had lower (P < 0.05) haptoglo- coliforms bin on d 5 PI, while pigs fed CAR had lower (P < 0.05) No difference was observed in the initial BW and d 0 C-reactive protein on d 2, 5, and 11 PI and had lower BW of pigs among dietary treatments (Table 2). Pigs (P < 0.05) haptoglobin on d 5 PI. No differences in serum supplemented with CAR had greater (P < 0.05) BW on d C-reactive protein and haptoglobin were observed be- 5 PI than pigs in the control and HIGH groups. Pigs tween the control and HIGH groups. supplemented with LOW had the greatest (P < 0.05) Before ETEC inoculation, pigs in the CAR group had BW, but pigs supplemented with HIGH had the lowest the lowest (P < 0.05) mean corpuscular volume and total (P < 0.05) BW on d 11 PI among all dietary treatments. platelets among all dietary treatments on d 0 (Table S2). Supplementation of LOW had greater (P < 0.05) ADFI of Supplementation of LOW had lower (P < 0.05) red blood pigs from d 5 to 11 PI, compared with control and cells and packed cell volume on d 2 PI, while supple- HIGH groups. Supplementation of Coligo had greater mentation of HIGH had lower (P < 0.05) packed cell vol- (P < 0.05) feed efficiency from d 0 to 5 PI compared with ume on d 5 PI, compared with pigs in the control. Pigs pigs in the control group regardless of dose. Supplemen- supplemented with CAR had lower (P < 0.05) red blood tation of HIGH also had greater (P < 0.05) feed efficiency cells and packed cell volume, but higher (P < 0.05) mean of weaned pigs from d 5 to 11 PI, compared with pigs in corpuscular hemoglobin and mean corpuscular the control. Pigs fed with CAR had better (P < 0.05) feed hemoglobin concentration on d 2 and 5 PI, compared efficiency than pigs fed with the control diet from d 0 to with pigs in the control. Supplementation of CAR also 5 PI, but this was not the case from d 5 to 11 PI. had greater (P < 0.05) total protein concentration on d Pigs supplemented with CAR had the lowest (P < 0.05) 11 PI in comparison to pigs in the other treatments. average diarrhea score from d 0 to 5 PI and d 5 to 11 PI Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 6 of 14 Table 2 Growth performance of ETEC-infected pigs fed diets supplemented with oligosaccharide-based polymer (Coligo) or antibiotics Diet P-value 1 2 3 4 5 Item Control LOW HIGH CAR SEM Diet Linear BW, kg d − 7 7.21 7.24 7.15 7.34 0.36 0.99 0.90 d 0 8.93 9.27 8.69 9.03 0.36 0.42 0.47 b ab b a d 5 PI 10.89 11.57 10.97 11.96 0.35 < 0.05 0.85 ab a b ab d 11 PI* 15.14 16.58 14.76 16.02 0.75 < 0.05 0.55 ADG, g d − 7 to 0 235 296 211 260 55.3 0.29 0.57 d 0 to 5 PI 393 453 456 533 50.6 0.29 0.35 d 5 to 11 PI* 622 727 705 690 32.9 0.25 0.13 ADFI, g d − 7 to 0 424 404 377 331 45.9 0.30 0.35 d 0 to 5 PI 635 631 597 677 34.3 0.49 0.44 b a b ab d 5 to 11 PI* 803 930 806 893 57.3 < 0.05 0.94 G:F d − 7 to 0 0.59 0.77 0.59 0.71 0.101 0.43 0.97 b a a a d 0 to 5 PI 0.57 0.76 0.75 0.77 0.054 < 0.05 < 0.05 b ab a ab d 5 to 11 PI* 0.72 0.79 0.85 0.83 0.043 0.07 < 0.05 a,b Within a row, means without a common superscript differ (P < 0.05) BW body weight, ADG average daily gain, ADFI average daily feed intake, G:F gain:feed, and PI post-inoculation. Each least squares mean represents 12 observations, except the *, which has 6 observations LOW Low dose blood group A6 type 1-based polymer (Coligo) HIGH High dose blood group A6 type 1-based polymer (Coligo) CAR carbadox Linear effects of adding Coligo to the control diet Bacterial translocation Intestinal morphology Supplementation of HIGH had lower (P < 0.05) bacterial On d 5 PI, supplementation of Coligo dose-dependently translocation in lymph nodes on d 5 and 11 PI compared had greater (linear, P < 0.05) villi height, the ratio of villi with control group (Fig. 3). Pigs supplemented with Coligo height to crypt depth, villi width, and villi area in duode- or CAR had lower (P < 0.05) bacterial translocation in the num, had greater (linear, P < 0.05) the ratio of villi height spleen than pigs in the control on d 11 PI. to crypt depth in jejunum, and had greater (linear, P < Table 3 Diarrhea score and frequency of diarrhea of ETEC-infected weaned pigs fed diets supplemented with oligosaccharide-based polymer (Coligo) or antibiotics Diarrhea score Diet P-value 1 2 3 4 Control LOW HIGH CAR SEM Diet Linear 5 a b ab c d0–5 2.88 2.38 2.62 1.78 0.17 < 0.01 0.15 6 a a a b d5–11 2.60 2.17 2.01 1.28 0.31 < 0.01 0.06 Pig days 120 109 120 105 7 a b b b Frequency of diarrhea 27.50 13.76 14.17 7.62 – < 0.01 – a,b,c Within a row, means without a common superscript differ (P < 0.05) LOW Low dose blood group A6 type 1-based polymer (Coligo) HIGH High dose blood group A6 type 1-based polymer (Coligo) CAR carbadox Linear effects of adding Coligo to the control diet Each least squares mean represents 12 observations Each least squares mean represents 6 observations Frequency = number of pen days with fecal score ≥ 4 Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 7 of 14 crypt depth ratio in jejunum, and bigger (P < 0.05) sialo- mucin area in duodenum than pigs in the control group. In addition, pigs in the CAR group also had greater (P < 0.05) villi height:crypt depth in all intestinal segments on d 5 PI, and greater (P < 0.05) villi height in ileum, in comparison to pigs in the Coligo treatment group. Intestinal barrier and innate immunity No difference was observed in the mRNA expression of MUC2 in jejunal mucosa among pigs in all dietary treat- ment groups (Fig.4). On d 5 PI, supplementation of Fig. 1 Daily diarrhea score of ETEC-infected weaned pigs fed diets supplemented with oligosaccharide-based polymer (Coligo) or HIGH up-regulated (P < 0.05) the mRNA expression of antibiotics. Diarrhea score = 1, normal feces, 2, moist feces, 3, mild ZO1 and addition of CAR had greater (P < 0.05) mRNA diarrhea, 4, severe diarrhea, 5, watery diarrhea. Each least squares expression of OCLN, compared with pigs in control mean from d 0 to d 5 post-inoculation (PI) represents 12 group. On d 11 PI, supplementation of LOW or CAR observations. Each least squares mean from d 6 to d 11 PI had higher (P < 0.05) mRNA expression of CLDN1 in je- represents 6 observations. *Significant differences were observed among dietary treatment: P < 0.05. LOW = Low dose blood group A6 junal mucosa of weaned pigs, compared with the control type 1-based polymer (Coligo); HIGH = High dose blood group A6 and HIGH groups. On d 5 PI, supplementation of LOW type 1-based polymer (Coligo); CAR = Carbadox down-regulated (P < 0.05) the mRNA expression of IL6, supplementation of HIGH had lower (P < 0.05) mRNA 0.05) villi height, the ratio of villi height to crypt depth, expression of IL1B, IL6, and TNF, and supplementation and villi area in ileum, compared with the control group of CAR had lower (P < 0.05) IL1B and IL6 gene expres- (Table S3). Supplementation of Coligo also had greater sion in ileal mucosa of weaned pigs in comparison to (linear, P < 0.05) duodenal and jejunal villi height and je- control pigs (Fig. 5). Supplementation of HIGH also had junal and ileal villi area, and tended to have greater (lin- lower (P < 0.05) IL6 mRNA expression on d 11 PI in ileal ear, P < 0.10) the ratio of villi height to crypt depth in mucosa, compared with the control group. However, no jejunum and ileal villi height on d 11 PI. Pigs fed with differences were observed in the gene expression of in- CAR had greater (P < 0.05) villi height in duodenum and flammatory mediators among LOW, HIGH, and CAR ileum, the ratio of villi height to crypt depth in all three groups. intestinal segments, and villi area in duodenum than pigs in the control group on d 5 PI. On d 11 PI, pigs supple- Discussion mented with CAR had higher (P < 0.05) villi height in all ETEC infection is initiated by bacterial attachment to three intestinal segments, greater (P < 0.05) villi height to specific receptors on the intestinal epithelium by fimbrial adhesins, followed by colonization of ETEC in the small intestine [29]. Once colonization is established, ETEC rapidly proliferate and produce one or more entero- toxins, which can stimulate water and electrolyte secre- tion and reduce fluid absorption in the small intestine and induce diarrhea [30]. Diarrhea caused by ETEC is one of the most prevalent diseases during the weaning stage, which is responsible for anorexia, slower growth, or even the death of pigs. Results of the present study demonstrated that supplementation of Coligo improved growth rate, and reduced frequency of diarrhea and sys- temic inflammation of weaned pigs experimentally chal- lenged with F18 ETEC. The potential mechanisms of Fig. 2 The percentage (%) of β-hemolytic coliform in fecal samples action include inhibition of binding of bacteria and as of ETEC-infected pigs fed diets supplemented with oligosaccharide- such colonization of the gut by the F18 ETEC [11, 13], based polymer (Coligo) or antibiotics. Each least squares mean from enhancing gut barrier function and reducing local and d 0 to d 5 post-inoculation (PI) represents 12 observations. Each systemic inflammation. least squares mean from d 6 to d 11 PI represents 6 observations. a,b Means without a common superscript differ (P < 0.05). LOW = Low In the current study, pigs in the control group grew dose blood group A6 type 1-based polymer (Coligo); HIGH = High slower and had a high frequency of diarrhea compared dose blood group A6 type 1-based polymer to pigs without ETEC challenge in our previous research (Coligo); CAR = Carbadox [20, 22]. These observations, combined with the Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 8 of 14 Table 4 Total and differential white blood cells, and serum cytokine and acute-phase proteins in ETEC-infected weaned pigs fed diets supplemented with oligosaccharide-based polymer (Coligo) or antibiotics Diet P-value 1 2 3 4 Item Control LOW HIGH CAR SEM Diet Linear5 d 0 before infection WBC, 10 /μL 15.28 16.91 15.70 16.85 1.00 0.58 0.77 Neu, 10 /μL 7.93 9.03 8.43 7.56 0.67 0.46 0.60 3 b b b a Lym, 10 /μL 6.39 6.72 6.08 8.26 0.48 < 0.05 0.65 Mono, 10 /μL 0.66 0.83 0.79 0.75 0.11 0.73 0.41 Eos, 10 /μL 0.24 0.27 0.35 0.20 0.10 0.64 0.33 Baso, 10 /μL 0.057 0.072 0.051 0.075 0.018 0.074 0.81 Serum TNF-α, pg/mL 63.56 59.67 61.51 72.80 1.62 0.98 0.99 C-reactive protein, μg/mL 7.37 6.86 8.57 8.08 1.39 0.86 0.46 Haptoglobin, g/mL 0.984 0.730 1.211 1.072 0.135 0.065 0.031 d2 PI 3 a ab b ab WBC, 10 /μL 21.16 18.04 17.78 18.53 1.01 < 0.05 < 0.05 3 a b b b Neu, 10 /μL 12.25 9.83 9.79 8.34 0.96 < 0.05 < 0.05 3 ab b b a Lym, 10 /μL 7.27 6.97 6.79 8.54 0.41 < 0.05 0.41 Mono, 10 /μL 1.16 1.05 0.85 1.33 0.16 0.29 0.21 Eos, 10 /μL 0.40 0.25 0.19 0.23 0.10 0.09 < 0.05 3 a b b b Baso, 10 /μL 0.106 0.036 0.039 0.029 0.017 < 0.05 < 0.05 Serum TNF-α, pg/mL 107.92 67.06 66.28 66.28 32.89 0.63 0.28 a a a b C-reactive protein, μg/mL 20.42 22.74 22.88 12.38 2.693 0.018 0.65 ab b a b Haptoglobin, g/mL 1.306 1.142 1.561 1.066 0.127 < 0.05 0.11 d5 PI WBC, 10 /μL 21.00 18.56 18.67 18.19 1.09 0.14 0.15 3 a b ab b Neu, 10 /μL 10.93 9.17 9.54 7.89 0.54 < 0.05 < 0.05 Lym, 10 /μL 9.03 8.22 8.02 8.35 0.65 0.69 0.25 Mono, 10 /μL 0.86 0.86 0.79 1.21 0.15 0.29 0.74 3 b ab ab a Eos, 10 /μL 0.13 0.26 0.25 0.55 0.14 < 0.05 0.41 Baso, 10 /μL 0.059 0.043 0.078 0.057 0.016 0.49 0.41 Serum TNF-α, pg/mL 90.64 68.36 54.39 32.99 32.88 0.25 0.16 a ab ab b C-reactive protein, μg/mL 21.61 17.46 19.84 15.96 1.72 0.082 0.93 a b ab b Haptoglobin, g/mL 1.655 1.084 1.348 1.154 0.128 0.081 0.93 d11 PI WBC, 10 /μL 16.15 17.40 17.32 14.92 1.58 0.64 0.57 Neu, 10 /μL 8.68 10.71 9.67 8.12 1.22 0.16 0.40 Lym, 10 /μL 6.33 6.69 7.21 6.39 0.63 0.76 0.35 Mono, 10 /μL 0.66 1.12 0.99 1.39 0.19 0.10 0.21 Eos, 10 /μL 0.18 0.31 0.24 0.21 0.13 0.74 0.57 Baso, 10 /μL 0.058 0.083 0.048 0.042 0.025 0.51 0.70 Serum TNF-α, pg/mL 82.58 84.62 72.71 59.39 28.03 0.96 0.79 Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 9 of 14 Table 4 Total and differential white blood cells, and serum cytokine and acute-phase proteins in ETEC-infected weaned pigs fed diets supplemented with oligosaccharide-based polymer (Coligo) or antibiotics (Continued) Diet P-value 1 2 3 4 Item Control LOW HIGH CAR SEM Diet Linear5 a ab ab b C-reactive protein, μg/mL 21.95 17.71 18.29 12.61 2.89 0.091 0.65 Haptoglobin, g/mL 0.819 0.599 0.538 0.412 0.178 0.24 0.31 a,b Within a row, means without a common superscript differ (P < 0.05) WBC white blood cell, Neu neutrophil, Lym lymphocyte, Mono monocyte, Eos eosinophil, Baso basophil, PI post-inoculation. Each least squares mean represents 12 observations, except d 11 PI that has 6 observations LOW Low dose blood group A6 type 1-based polymer (Coligo) HIGH High dose blood group A6 type 1-based polymer (Coligo) CAR carbadox Linear effects of adding Coligo to the control diet presence of β-hemolytic coliforms in feces, confirmed hemolytic coliforms than pigs fed with antibiotics. These that pigs were successfully infected with F18 ETEC. In observations indicated that the beneficial effects of agreement with our previous research, the peak of F18 Coligo and antibiotics on reducing weaned pigs’ diarrhea ETEC infection was present approximately 5 days post- were through different mechanisms. It has been reported inoculation, and most pigs moved into the recovery that heat-labile toxin expressed by ETEC binds blood stage on day 11 to 12 post-inoculation. Results of the group antigens, with a preference for A-epitope [33]. current study have demonstrated that pigs supplemented Moreover, it has been hypothesized that blood group A with Coligo or antibiotics had reduced frequency of diar- antigen might disturb the toxin activity by interfering rhea and enhanced growth performance than pigs in with ETEC binding to the receptors in the small intes- control group, indicating both supplements could pro- tine of pigs [34]. Coddens et al. [10] also observed a high tect pigs against F18 ETEC infection. correlation between blood group A antigen and F18 In agreement with the diarrhea severity results, pigs ETEC adherence in the small intestine of young pigs supplemented with antibiotics had less β-hemolytic coli- in vitro. Moreover, it has been demonstrated that F18 E. forms compared with pigs in the control group, indicat- coli specifically interact with glycosphingolipids possess ing lowered F18 ETEC shedding in pig’s feces during the blood group ABH determinants on a type 1 core, which peak infection period. The exact mechanisms of action were identified as the cell surface receptors for F18 fim- of carbadox are not fully understood, but it has been brial binding to the small intestinal epithelium [11]. suggested that virulence activity was disabled by interfer- With these specific features, the addition of extra blood ing with DNA synthesis in Gram-negative bacteria, in- group A antigen could enhance the binding affinity of cluding E. coli [31, 32]. However, pigs supplemented F18 ETEC to polymers. Thus, fecal culture results sug- with Coligo had a relatively higher percentage of β- gest that the Coligo polymer may reduce the ETEC at- tachment to the small intestine and accelerate the excretion of these pathogenic bacteria from their gastro- intestinal tract. Taken altogether, antibiotics or Coligo may help pigs recover from ETEC infection through dif- ferent mechanisms, which we attempted to explore in the current research. Tight junctions play critical roles in maintaining the integrity of intestinal structure and barrier, and regulat- ing intestinal paracellular permeability [35, 36]. Several multi-protein complexes, including zonulae occludens (ZO), occludins, and claudins, are involved in the tight junction barrier. Previous studies have reported that Fig. 3 Bacterial counts (CFU/g) in lymph node and spleen of ETEC- ETEC infection impairs intestinal barrier function by infected weaned pigs fed diets supplemented with oligosaccharide- down-regulating tight junction protein expression, lead- based polymer (Coligo) or antibiotics. Each least squares mean from ing to intestinal inflammation [22, 37]. Morphological le- d 0 to d 5 post-inoculation (PI) represents 12 observations. Each sions, such as loss of villus absorptive cells, villus least squares mean from d 6 to d 11 PI represents 6 observations. a,b Means without a common superscript differ (P < 0.05). LOW = Low atrophy, and intestinal permeability disturbances are also dose blood group A6 type 1-based polymer (Coligo); HIGH = High observed in the small intestine of pigs with ETEC infec- dose blood group A6 type 1-based polymer tion [1, 38]. In the present study, pigs supplemented (Coligo); CAR = Carbadox with Coligo or antibiotics had greater mRNA expression Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 10 of 14 Fig. 4 Gene expression profiles in jejunal mucosa of ETEC-infected weaned pigs fed diets supplemented with oligosaccharide-based polymer a,b (Coligo) or antibiotics on d 5 or 11 post-inoculation (PI). Means without a common superscript differ (P < 0.05). Each least squares mean represents 6 observations. LOW = Low dose blood group A6 type 1-based polymer (Coligo); HIGH = High dose blood group A6 type 1-based polymer (Coligo); CAR = Carbadox; MUC2 = Mucin-2; CLDN1 = Claudin-1; ZO-1 = Zonula occludens-1; OCDN = Occludin of ZO1 or OCDN at the peak of ETEC infection, respect- normally sterile tissues, such as mesenteric lymph ively, and greater CLDN1 expression on d 11 PI than nodes and other internal organs, including the spleen pigs in control group. ZO-1 protein connects and inter- [40, 41]. The major mechanisms promoting bacterial acts with junctional proteins, such as occludins and clau- translocation are intestinal bacterial overgrowth, defi- dins, to form the physical barrier, which determines the ciencies in host immune defenses by disturbed gut in- permselectivity of the paracellular diffusion pathway tegrity, and increased permeability or mucosal injury [39]. The up-regulation of mRNA expression of tight [42]. It was previously reported that bacterial trans- junction proteins in this study suggests the protective ef- location to mesenteric lymph nodes was increased in fects of Coligo or antibiotics on intestinal barrier func- pigs challenged with ETEC [43, 44]. In the current tion against ETEC infection. Consistently, pigs in Coligo study, pigs supplemented with antibiotics lowered groups or antibiotics group had higher villi height, bacterial populations in the spleen, and pigs fed with greater villi height to crypt depth ratio, and villi area, Coligo had lower bacterial populations in both mes- demonstrating a preventive effect against intestinal enteric lymph nodes and spleen than pigs in the con- structure disruption. Overall, these results demonstrated trol group. These observations clearly supported that that supplementation of Coligo or antibiotics enhanced supplementation of Coligo or antibiotics reduced the gut integrity and morphology of weaned pigs, which was damage of ETEC infection on the gut integrity of one of the major reasons these pigs grew faster than pigs weaned pigs compared with control pigs. Overall, re- in control group. sults of tight junction protein mRNA expression, in- Bacterial translocation is defined as the passage of testinal morphology, and bacterial translocation imply viable bacteria from the gastrointestinal tract to that pigs in Coligo and antibiotics groups may have Fig. 5 Gene expression profiles in ileal mucosa of ETEC-infected weaned pigs fed diets supplemented with oligosaccharide-based polymer a,b (Coligo) or antibiotics on d 5 or 11 post-inoculation (PI). Means without a common superscript differ (P < 0.05). Each least squares mean represents 6 observations. LOW = Low dose blood group A6 type 1-based polymer (Coligo); HIGH = High dose blood group A6 type 1-based polymer (Coligo); CAR = Carbadox; IL1B: Interleukin-1 beta; IL6: Interleukin-6; TNF = Tumor necrosis factor; PTGS2: Cyclooxygenase-2 Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 11 of 14 better intestinal health, which would be responsible potentially modified intestinal microbiota by Coligo. It for better nutrient digestion and absorption, and was reported that porcine blood type AO could be a hence better performance. possible factor influencing microbiota composition [56] The colonized F18 ETEC could produce large quan- and Priori et al. [57] suggest changes in the intestinal tities of toxins, such as heat-labile toxins, heat-stable microbiota are affected by porcine blood group. Thus, toxins, Shiga toxins, and lipopolysaccharides [45]. Those the blood group A antigen in Coligo may affect the com- toxins induce functional changes in the small intestinal position and function of microbial communities when epithelial cells, as well as stimulate the synthesis of cyto- fed to pigs. Moreover, recent studies demonstrated that kines and acute-phase proteins (e. g. C-reactive protein dietary supplementation of ε-PL altered ileal microbiota and haptoglobin), followed by systemic and local inflam- structure and function in pigs [58] and fecal microbial mation [1, 46]. Our previously published research that community in mice [59]. ε-PL supplementation may used the same bacteria strain, F18 ETEC, reported that promote the growth of beneficial microorganisms in the ETEC infection could induce systemic inflammation, intestinal tract, therefore, reducing the proliferation of such as increasing white blood cell counts, neutrophils, pathogens. The exact mechanisms of ε-PL in the current and lymphocytes, as well as enhancing several pro- study remain unclear, so further research is needed to inflammatory cytokines and acute-phase protein concen- confirm the effects of Coligo on the pigs’ gut microbial trations in serum of weaned pigs [20, 47]. In the current community, intestinal inflammation, and immune re- study, pigs supplemented with antibiotics had reductions sponses against F18 ETEC. Pigs supplemented antibi- in neutrophils and serum concentrations of C-reactive otics also had reductions in mRNA expression of protein and haptoglobin during the peak infection proinflammatory markers in the present study. This period. Similarly, pigs supplemented with Coligo had finding demonstrated that pigs supplemented antibiotics lower numbers of white blood cells, neutrophils, and has less severe intestinal inflammation than pigs in con- lymphocytes on d 2 PI, and lower neutrophils on d 5 PI. trol group. In agreement with previous research, anti- In addition, serum haptoglobin concentrations were also biotic supplements might exert anti-inflammatory relatively lower in the pigs supplemented with Coligo. properties in the intestine or accumulate in phagocytic These findings demonstrated the capacity of antibiotics inflammatory cells, therefore, attenuating inflammatory and Coligo polymer in alleviating ETEC-induced sys- responses in animals [60, 61]. Taken altogether, down- temic inflammation, possibly by reducing the bacterial regulation in mRNA expression of proinflammatory cy- population in pigs’ gut. tokines by Coligo or antibiotics supplementation is In response to an infectious challenge, it is well recog- beneficial for pigs in terms of their intestinal health and nized that innate immune responses have essential roles growth performance. in preventing and suppressing inflammation [48]. Patho- In conclusion, results in the current study suggest that gen recognition receptors initiate the innate immune re- in-feed supplementation of Coligo or antibiotic (carba- sponse on intestinal epithelial cells to detect and dox) enhanced growth performance and reduced the se- recognize the pathogen-associated molecular patterns, verity of diarrhea caused by ETEC F18 infection. such as microbial membranes, resulting in a rapid re- Although the percentage of β-hemolytic coliforms in lease of proinflammatory cytokines [49]. It has been re- fecal samples of pigs fed with Coligo was less diminished ported that secreted proinflammatory cytokines, which than pigs supplemented with antibiotics, enhanced dis- are primarily involved in host responses to disease or in- ease resistance was demonstrated by the improved gut fection, could induce intestinal inflammation, tissue de- barrier integrity and attenuated systemic and intestinal struction, and nutrient and ion malabsorption in pigs inflammation. To further explore the mechanisms of ac- [50, 51]. Moreover, numerous studies have confirmed tion of Coligo, integrated metabolomics and metage- the induction of proinflammatory cytokines in porcine nomics approaches may be considered to provide more intestinal epithelial cells caused by ETEC infection [52– insights into the beneficial effects of Coligo or other 54]. The elevation of proinflammatory cytokines by polymers on pigs’ health. Overall, the current study indi- ETEC challenge has nutrient cost, thus also contributing cates that supplementation with Coligo has promising to the reduced growth performance of pigs [55]. In the impacts on promoting growth and disease resistance of current study, mRNA expression of proinflammatory newly weaned pigs infected with ETEC F18. The efficacy markers (i.e., IL1B, IL6, and TNF) in ileal mucosa were of Coligo is comparable to antibiotic (carbadox) demon- down-regulated by Coligo supplementation, which is strating the potential of Coligo as antibiotic alternative consistent with the results of systemic immunity. These for animal growth performance and disease resistance. observations can be accounted for by assuming not only Large-scale animal trials are recommended to further reduced the attachment of ETEC to the intestinal epithe- evaluate the impacts of Coligo on performance of lium by blood group oligosaccharides, but also weaned pigs under commercial practice conditions. Kim et al. Journal of Animal Science and Biotechnology (2022) 13:10 Page 12 of 14 Abbreviations Received: 2 July 2021 Accepted: 21 November 2021 ADFI: Average daily feed intake; ADG: Average daily gain; BW: Body weight; CFU: Colony-forming unit; CLDN1: Claudin-1; ETEC: Enterotoxigenic E. coli; IL1B: Interleukin-1 beta; IL6: Interleukin-6; mRNA: Messenger RNA; MUC2: Mucin-2; OCDN: Occludin; PI: Post-inoculation; qRT-PCR: Quantitative References real-time polymerase chain reaction; TNF: Tumor necrosis factor; ZO-1: Zona 1. Nagy B, Fekete PZ. Enterotoxigenic Escherichia coli in veterinary medicine. occludens-1 Int J Med Microbiol. 2005;295(6-7):443–54. https://doi.org/10.1016/j.ijmm.2 005.07.003. 2. Fairbrother JM, Nadeau É, Gyles CL. Escherichia coli in postweaning diarrhea in pigs: an update on bacterial types, pathogenesis, and prevention Supplementary Information strategies. Anim Health Res Rev. 2005;6(1):17–39. https://doi.org/10.1079/A The online version contains supplementary material available at https://doi. HR2005105. org/10.1186/s40104-021-00655-2. 3. Van Boeckel TP, Brower C, Gilbert M, Grenfell BT, Levin SA, Robinson TP, et al. Global trends in antimicrobial use in food animals. Proc Natl Acad Sci. Additional file 1. Table S1 Gene-specific primer sequences and PCR 2015;112(18):5649–54. https://doi.org/10.1073/pnas.1503141112. conditions. 4. FDA (Food and Drug Administration) New animal drugs and new animal Additional file 2. Table S2 Red blood cell profiles of ETEC-infected drug combination products administered in or on medicated feed or weaned pigs fed diets supplemented with oligosaccharide-based poly- drinking water of food-producing animals: recommendations for drug mer (Coligo) or antibiotics. sponsors for voluntarily aligning product use conditions with FDA Guidance for Industry #213. Center for Veterinary Medicine. Washington, DC: US Additional file 3. Table S3 Intestinal morphology of ETEC-infected Department of Health and Human Services; 2016. https://www.fda.gov/ weaned pigs fed diets supplemented with oligosaccharide-based poly- downloads/AnimalVeterinary/GuidanceComplianceEnforcement/Guida mer (Coligo) or antibiotics. nceforIndustry/UCM299624.pdf. 5. Nollet H, Deprez P, Van Driessche E, Muylle E. Protection of just weaned pigs against infection with F18+ Escherichia coli by non-immune plasma Acknowledgments powder. Vet Microbiol. 1999;65(1):37–45. https://doi.org/10.1016/S0378-113 Not applicable. 5(98)00282-X. 6. Newburg DS. Do the binding properties of oligosaccharides in milk protect human infants from gastrointestinal bacteria. J Nutr. 1997;123:980S–4S. Authors’ contributions https://doi.org/10.1093/jn/127.5.980S. The contributions of the authors were as follows: KK conducted the 7. 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Journal

Journal of Animal Science and Biotechnology – Springer Journals

Published: Jan 12, 2022

Keywords: Enterotoxigenic E. coli; Growth rate; Intestinal barrier function; Oligosaccharide-based polymer; Systemic immunity; Weaned pigs

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