López-Colom et al. Journal of Animal Science and Biotechnology (2019) 10:89 https://doi.org/10.1186/s40104-019-0393-y RESEARCH Open Access Efficacy of medium-chain fatty acid salts distilled from coconut oil against two enteric pathogen challenges in weanling piglets Paola López-Colom1, Lorena Castillejos1* , Agustina Rodríguez-Sorrento1, Mónica Puyalto2, Juan José Mallo2 and Susana María Martín-Orúe1 Abstract Background: The search for alternatives to antibiotics in pig production has increased the interest in natural resources with antimicrobial properties, such as medium-chain fatty acids (MCFA) as in-feed additives. This study evaluated the potential of a novel blend of MCFA salts (DIC) from distilled coconut oil with a lauric acid content to reduce enteropathogens and control intestinal diseases around weaning. Two experimental disease models were implemented in early-weaned piglets, consisting of two oral challenges: Salmonella Typhimurium (1.2 × 108 CFU) or enterotoxigenic Escherichia coli (ETEC) F4 (1.5 × 109 CFU). The parameters assessed were: animal performance, clinical signs, pathogen excretion, intestinal fermentation, immune-inflammatory response, and intestinal morphology. Results: The Salmonella challenge promoted an acute course of diarrhea, with most of the parameters responding to the challenge, whereas the ETEC F4 challenge promoted a mild clinical course. A consistent antipathogenic effect of DIC was observed in both trials in the hindgut, with reductions in Salmonella spp. plate counts in the cecum (P = 0.03) on d 8 post-inoculation (PI) (Salmonella trial), and of enterobacteria and total coliform counts in the ileum and colon (P < 0.10) on d 8 PI (ETEC F4 trial). When analyzing the entire colonic microbiota (16S rRNA gene sequencing), this additive tended (P = 0.13) to reduce the Firmicutes/Bacteroidetes ratio and enriched Fibrobacteres after the Salmonella challenge. In the ETEC F4 challenge, DIC prompted structural changes in the ecosystem with increases in Dialister, and a trend (P = 0.14) to increase the Veillonellaceae family. Other parameters such as the intestinal fermentation products or serum pro-inflammatory mediators were not modified by DIC supplementation, nor were the histological parameters. Only the intraepithelial lymphocyte (IEL) counts were lowered by DIC in animals challenged with Salmonella (P = 0.07). With ETEC F4, the IEL counts were higher with DIC on d 8 PI (P = 0.08). Conclusions: This study confirms the potential activity of this MCFA salts mixture to reduce intestinal colonization by opportunistic pathogens such as Salmonella or E. coli and its ability to modulate colonic microbiota. These changes could explain to some extent the local immune cell response at the ileal level. Keywords: Enteric pathogen, Gut microbiota, Intestinal immunity, Medium-chain fatty acids, Weaning pig * Correspondence: lorena.castillejos@uab.cat 1 Animal Nutrition and Welfare Service, Animal and Food Science Department, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Spain Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. López-Colom et al. Journal of Animal Science and Biotechnology (2019) 10:89 Page 2 of 17 Background MCFA in challenged pigs; one tested the control of Sal- In modern pig production, early weaning around 4 wk monella in fatteners under commercial conditions [22] of life is associated with an abrupt dietary change and and the other assessed the efficacy against an LPS chal- immature organ function, with a consequent reduction lenge in an experimental trial with weanlings [23]. in growth. Thus, piglets around the post-weaning period Within this context, the objective of this work was to are highly susceptible to enteric bacterial infections evaluate the potential of a combination of sodium salts caused by opportunistic pathogens. The use of antibi- of medium-chain fatty acids distilled from coconut oil to otics and other medications to circumvent this critical enhance the gut health of weaned piglets in the face of period has been common in intensive systems; however, diarrheic enteric diseases caused by Salmonella or ETEC it has contributed to the development of antibiotic- F4 in controlled clinical assays. resistant strains. Indeed, the last report from the Euro- pean Food Safety Authority (EFSA) for the situation in Methods 2015 documented an increase in multi-drug resistant Two different experiments were performed to evaluate (MDR) strains of Escherichia coli and Salmonella Typhi- the effect of the coconut distillates against an oral chal- murium [1]. There is therefore an urgent need to search lenge with Salmonella Typhimurium (Trial 1) or ETEC for alternatives that will diminish diarrhea outbreaks and F4 (Trial 2). Both trials were performed at the Servei de help to reduce the use of therapeutic and prophylactic Granges i Camps Experimentals of the Universitat antibiotics. Autònoma de Barcelona (UAB). The treatment, manage- Feeding strategies are one of the most commonly-used ment, housing, husbandry and slaughtering conditions management factors for the improvement of gut health conformed to European Union Guidelines (Directive and function in newly weaned pigs. A wide range of bio- 2010/63/EU). active ingredients have been proposed as tools to control enteric pathogens, to help reduce the incidence and se- Animals, housing, and experimental design verity of digestive problems associated with weaning [2]. The two trials were conducted following Biosafety Level Among them, medium-chain fatty acids (MCFA), con- 2 requirements with appropriate training of the involved sidered fatty acids with a chain length of 6- to 12- car- personnel. For the first trial, 56 male piglets from high bon atoms, have been proposed as a potential alternative sanitary status farms were used. The (Landrace × Large to antibiotics based on their long-known antibacterial White) × Piétrain piglets originated from mothers with activity [3]. In contrast to antimicrobial agents, MCFA negative Salmonella serology results; they were weaned have not shown evidence of acquiring resistance [4, 5]. at 28 d of age and had an average body weight (BW) of Besides this, MCFA are an immediate source of energy 8.1 ± 1.16 kg. For the second trial, 72 male piglets due to their rapid passive absorption and digestion, (Landrace × Large White) × Piétrain that originated which is of particular interest for the nutrition of young from mothers that were not vaccinated against E. coli animals [6, 7]. They occur naturally as medium-chain were used; they were weaned at 21 d of age and had an triglycerides (MCT) in milk fat, as well as in other vege- average BW of 5.6 ± 0.95 kg. table sources, such as coconut or palm oils and Cuphea The piglets were transported to the UAB facilities and seed oil [8, 9]. The most abundant MCFA in coconut is for each trial, 48 animals were placed in 16 pens (three lauric acid (C12), representing up to 45% of the coconut animals per pen). In each pen, the animals were distrib- fat content, followed by capric (C10) and caprylic (C8) uted according to weight (a low-, an intermediate-, and acids [10]. C12 acid has been proven to have the greatest a high-weighted animal) to obtain a final homogenous antimicrobial function among all MCFA [4, 11]. weight among pens. The experimental treatments were Several published works can be found substituting fat evenly distributed among the pens. The experimental sources with purified MCFA or alternative natural treatments included a control treatment (CTR) consist- sources rich in MCFA, leading to improvements in the ing of a plain diet without additives and the same diet performance of piglets around weaning [12, 13]. Studies including a commercial blend of salts of MCFA distilled with healthy pigs [14–16] have demonstrated positive ef- from coconut oil (Dicosan) at 3 kg/t (DIC). The rest of fects on growth performance, digestibility, and intestinal the animals were kept in a separate room and used as microbiota. The antibacterial effect has also been widely controls for the challenges (placebo groups, PLB). For demonstrated in vitro [3, 17], particularly against Sal- the first trial, eight piglets were distributed among four monella or enterotoxigenic E. coli (ETEC), among other pens (two animals per pen), and for the second trial 24 pathogens [18, 19]. However, to our knowledge, most of piglets were distributed among eight pens (three animals the in vivo studies testing the effect of MCFA against per pen). Both placebo groups received the same plain pathogens were performed in rodents [5, 20] and chick- diet given to the CTR groups and were euthanized at d ens [21]. Only two studies have tested the potential of 4 post-inoculation (PI). López-Colom et al. Journal of Animal Science and Biotechnology (2019) 10:89 Page 3 of 17 Each pen (3 m2) had a feeder and a water nipple to Table 1 Ingredient composition and chemical analysis of the provide feed and water for ad libitum consumption. The basal diet on an as-fed basis weaning rooms were equipped with automatic heating Ingredients, g/kg and forced ventilation. The experiments were conducted Maize 205 during the autumn-winter seasons (September–October Wheat 180 and February–March for Trial 1 and 2, respectively) with Barley 2 row 170 a mean room temperature of 30.8 ± 5.28 °C. Both trials Extruded soybean 150 were maintained under a 13 h light/11 h dark lighting regimen. Sweet whey-powder (cattle) 112 Fishmeal LT 60.0 Experimental products and diets Soybean meal 47 70.0 The evaluated feed-additive is commercially available Whey powder 50% fat 25.0 (Dicosan) and was supplied by Norel S.A. (Madrid, Mono-calcium phosphate 6.5 Spain). It consisted of a mixture of sodium salts of fatty Calcium carbonate (CaCO3) 3.8 acid obtained from the distillates of coconut oil (67% crude fat and a fatty acid profile with 48.4% lauric acid, L-Lysine HCl 4.5 18.6% myristic acid, 9.9% palmitic acid, 6.8% oleic acid, Vitamin-Mineral premixa 4.0 6.2% caprylic acid, 5.8% capric acid, 3.6% stearic acid DL-Methionine 99 2.6 and 1.3% linoleic acid). Sodium chloride (marine salt) 2.4 The basal diet (Table 1) was formulated to satisfy the L-Threonine 2.3 nutrient requirement standards for pigs [24]. For each L-Valine 1.5 trial, the diet was manufactured in the same batch and the additive was mixed with the corresponding amount L-Tryptophan 0.7 of feed to obtain the DIC diet. Dicosan was included ac- Calculated nutrients, g/kg cording to the manufacturer’s recommended dosage (3 Metabolizable energy, kcal/kg 3381 kg/t). The intended dose of the additive was not analyt- Calcium 6.5 ically confirmed in the diet, however, it was expected to Phosphorus 6.6 provide 1.11 kg/t of lauric acid. Sodium 2.3 Bacterial strains Chloride 5.7 For the first trial, the Salmonella strain used for the oral Potassium 9.1 challenge was a monophasic variant of Salmonella Typhi- Magnesium 1.4 murium (4,5,12:i:-) with a ACSSuT-Ge resistance profile Analyzed nutrients, g/kg Salmonella trial ETEC F4 trial and phage type U302; it was isolated from a salmonellosis Dry matter 902 915 outbreak in fattening pigs in Spain (mainly enteric with Ash 54.2 48.2 sporadic septicemia) and provided by the Infectious Diseases Laboratory of UAB (ref. 301/99). The oral inocu- Crude fat 59.4 66.5 lum was prepared by overnight incubation at 37 °C and Crude protein 198 195 250 r/min in buffered peptone water (BPW; Oxoid, Neutral detergent fiber 84.3 81.7 Hampshire, UK) and diluted (1:20) with sterile phosphate Acid detergent fiber 31.1 31.5 buffered saline (PBS; Sigma-Aldrich, Madrid, Spain). The a Provided per kilogram of complete diet: 10,200 IU vitamin A, 2,100 IU vitamin final inoculum concentration was 5.8 × 107 CFU/mL. To D3, 39.9 mg vitamin E, 3 mg vitamin K3, 2 mg vitamin B1, 2.3 mg vitamin B2, 3 confirm the doses, serial dilutions of the inoculum were mg vitamin B6, 0.025 mg vitamin B12, 20 mg calcium pantothenate, 60 mg nicotinic acid, 0.1 mg biotin, 0.5 mg folic acid, 150 mg Fe, 156 mg Cu, 0.5 mg cultured in tryptic soy agar (TSA; BD Difco, Heidelberg, Co, 120 mg Zn, 49.8 mg Mn, 2 mg I, and 0.3 mg Se Germany) by overnight incubation at 37 °C. For the second trial, the enterotoxigenic Escherichia the inoculum were cultured in Luria agar (LA; Labora- coli (ETEC) F4 strain used (positive for virulence factors torios Conda, Torrejón de Ardoz, Spain) by overnight F4ab, F4ac, LT, STb, and EAST1 and negative for F6, incubation at 37 °C. F18, F41, STa, VT1, VT2, and EAE) was isolated from In both trials, the placebo animals received the same 14-wk-old pigs and provided by the Diseases Laboratory amount of confirmed sterile broth inocula. of UAB (ref. 30/14-3). The oral inoculum was prepared by overnight incubation at 37 °C and 250 r/min in brain Experimental procedure heart infusion (BHI; Oxoid). The final inoculum was 2.5 The animals received the experimental diets ad libitum × 108 CFU/mL. To confirm the doses, serial dilutions of over 14 d in the Salmonella trial and 15 d in the ETEC López-Colom et al. Journal of Animal Science and Biotechnology (2019) 10:89 Page 4 of 17 F4 trial. After 6 and 7 d of adaptation in the first and Euthasol, Esteve, Barcelona, Spain). Once dead, the ani- second trials, respectively, the animals were orally chal- mals were bled, the abdomen was immediately opened, lenged with the pathogen or the sterile broth. One pig and the intestinal tract was excised. per pen was euthanized on d 4 and 8 PI. The PLB ani- The digesta content from the ileum (5 cm from the mals were maintained until d 4 PI and thereafter eutha- ileal-cecal junction) and proximal colon (5 cm from the nized following the same procedures performed on the ceco-colic junction) was collected and homogenized, and challenged groups. the pH was immediately determined using a pH-meter Fecal samples for microbiological analysis were asep- calibrated on each day of use (Crison 52-32 electrode, tically collected from the heaviest piglet from each pen Net Interlab, Madrid, Spain). Different aliquots were col- (n = 16) at arrival and after the adaptation period (d 0 lected for different determinations. Samples of approxi- PI), after spontaneous defecation or by rectal stimula- mately 5 g were kept immediately on dry ice until being tion. After the adaptation period, the pathogenic bacteria stored at -20°C for the further analysis of short-chain inocula were administered by oral gavage to all animals fatty acids (SCFA) and lactic acid, and similar aliquots as a single dose of Salmonella Typhimurium (1.2 × 108 were stored at - 80°C for the high-throughput sequen- CFU) or ETEC F4 (1.48 × 109 CFU) on d 7 and 8, re- cing (HTS) of colonic microbiota. Another set of sam- spectively. In order to ensure that the stomach was full ples were preserved in a H2SO4 solution (3 mL of at the time of inoculation and to facilitate bacterial digesta plus 3 mL of 0.2 mol/L H2SO4) and stored at colonization, feed was withdrawn at 21:00 the previous -20°C until ammonia (NH3) determination. day and provided once again 30 min before inoculation In the ETEC F4 trial, additional aliquots (approxi- the following morning. mately 2 g) from the ileum and colon digesta were also Individual BW and pen feed consumption were regis- kept on ice for microbiological analysis and were proc- tered during the adaptation period. Body weight was fur- essed in less than 4 h; other similar aliquots were stored ther recorded on d 0, 4, and 8 PI in both trials, whereas at -80°C for E. coli F4 quantification by real-time quanti- feed consumption was recorded on d 0, 2, 4, and 8 PI tative PCR (qPCR) and for the HTS of colonic micro- for the Salmonella trial and d 0, 1, 2, 3, 4, 5, 7, and 8 PI biota. In parallel, to analyze the bacteria attached to the for the ETEC F4 trial. The average daily gain (ADG) and ileal mucus and epithelium, 15-cm long sections of the average daily feed intake (ADFI) were calculated per ileum were excised, washed thoroughly three times with pen, which was considered the experimental unit. sterile PBS, opened longitudinally, and scraped with a After the oral challenge, animals were checked daily microscope glass slide to obtain mucosa samples. One for clinical signs to evaluate their status (i.e. dehydration, aliquot was kept on ice for microbial analysis. In the case apathy, fecal score), always by the same individual. Mor- of the Salmonella trial, additional cecum digesta samples tality was also registered. No antibiotic treatment was were collected, kept on ice, and processed in less than administered to any of the animals in any of the experi- 4 h for Salmonella counts. ments. The fecal score was assessed using a scale: 1 = For the histological study, 3-cm long sections from the solid and cloddy, 2 = soft with shape, 3 = very soft or distal ileum were removed, opened longitudinally, viscous liquid, and 4 = watery or with blood. A fecal washed thoroughly with sterile PBS, and fixed by score was registered individually on d 0, 1, 2, 3, and 7 PI immersion in a formaldehyde solution 3.7–4% (PanReac, in the Salmonella trial and on d 0, 1, 2, 3, 5, and 7 PI in Castellar del Vallès, Spain). the ETEC F4 trial. Rectal temperature was assessed using Blood samples were centrifuged (1500 × g for 15 min) a digital thermometer (Accu-Vet, Import Vet S.A., Cen- and the obtained serum was divided into different telles, Spain) on d 0 and 2 PI. Within the Salmonella aliquots and stored at - 20°C. trial, additional fecal samples for microbiological analysis were collected from the same animals on d 1, 3, and Analytical procedures 7 PI. Chemical analyses of the diets including the content of On d 4 and 8 PI, one pig per pen was euthanized dry matter (DM), ash, crude protein, and diethyl ether sequentially during the morning (between 08:00 and 14: extract were performed according to the Association of 00) for sampling. On d 4 PI, the animal from each pen Official Agricultural Chemists standard procedures [25]. with the mid-weight at the beginning of the experiment Neutral detergent fiber and acid detergent fiber were was selected, and on d 8 PI, the animal with the highest determined according to the method of Van Soest weight was selected. A 10-mL blood sample was ob- et al. [26]. tained by venipuncture of the cranial vena cava using For microbial counts of Salmonella spp. (Trial 1), the 10-mL tubes without anticoagulant (Aquisel, Madrid, cecum content and feces were suspended in buffered Spain). Immediately, the animals received an intravenous peptone water (BPW; 1:10). The quantitative determin- injection of sodium pentobarbital (200 mg/kg BW; ation consisted of 10-fold serial dilutions in PBS seeded López-Colom et al. Journal of Animal Science and Biotechnology (2019) 10:89 Page 5 of 17 in Xylose-Lactose-Tergitol-4 agar (XLT4; Merck, was performed at a maximum unacceptable Phred qual- Madrid, Spain) and a count of H2S positive colonies and ity score of Q20. The resulting reads were clustered to morphology compatible with Salmonella spp. after 24 h operational taxonomic units (OTUs) using uclust with incubation at 37 °C. With this scheme, animals were 97% sequence similarity and a subsampling pick open given a count level as follows: negative, for animals with reference method [29] at 10%. Representative sequences no Salmonella growth at 102 dilution (< 103 CFU/g were assigned to taxonomy against the bacterial 16S fresh matter [FM]); low, for animals with counts of GreenGenes v.13.8 reference database [30] at a 90% con- 103 to 104 CFU/g FM; medium, for animals with fidence threshold, and sequence alignment and phylo- counts of 105 to 106 CFU/g FM; and high, for animals with genetic tree building were performed using uclust and counts of 107 to 108 CFU/g FM. FastTree, respectively. Thereafter, chimeric sequences For microbial counts of enterobacteria and total coli- were removed with ChimeraSlayer [31] using the default forms (Trial 2), the ileum and colon contents and feces settings and further quality filtering consisted of remov- were suspended in PBS (1:10), serially diluted in Lac- ing singletons and OTUs with a relative abundance tated Ringer’s Solution (Sigma-Aldrich), and seeded in below 0.005% across all samples, as recommended by MacConkey agar and Chromogenic agar (Oxoid), re- Bokulich et al. [32]. spectively. The counts were read after 24 h of incubation The ammonia (NH3) concentration was determined at 37 °C. with the aid of a gas-sensitive electrode (Hach Co., Love- In addition, for microbial molecular analysis, DNA land, CO), combined with a digital voltmeter (Crison was extracted from samples (approximately 250 mg) and GLP 22, Crison Instruments S.A., Barcelona, Spain), purified using the commercial QIAamp DNA Stool Mini modified from Barba-Vidal et al. [33]. Three grams of Kit (Qiagen, West Sussex, UK). Subsequently, its con- digesta preserved in H2SO4 (1:2) was centrifuged at 1372 centration and purity were checked using a NanoDrop × g for 10 min. A supernatant was obtained and neutral- 1000 Spectrophotometer (Thermo Fisher, Wilmington, ized with 1 mL of 10 mol/L NaOH to reach a pH of 11, DE). The protocol was followed, applying all recom- while stirring and measuring the ammonia released as mended optimization steps, in order to improve bacterial different voltages in mV. cell rupture and purity. The DNA was finally eluted in The SCFA and lactic acid concentrations were deter- 200 mL of Qiagen buffer AE and stored at - 80 °C until mined based on the method described by Richardson use. et al. [34] and modified by Jensen et al. [35] using gas E. coli F4 was quantified in colonic digesta and ileal chromatography after submitting the samples to an scrapings by qPCR using SYBR green dye. qPCR target- acid-base treatment, followed by a diethyl ether ex- ing the gene coding the F4 fimbria of ETEC F4 was per- traction and derivatization with N-(tertbutyldimethyl- formed by modifying the procedure described by silyl)-N-methyl-trifluoroacetamide (MBTSTFA) plus Gustavo Hermes et al. [27]; the changes consisted of 1% tert-butyldimethylchlorosilane (TBDMCS) agent using SYBR Green PCR Master Mix PCR (Applied Bio- (Sigma-Aldrich). systems, Carlsbad, CA) and reducing the total volume of Tissue samples for morphological measures were the reaction to 20 μL, which included 10 μL of 2× SYBR dehydrated and embedded in paraffin wax, sectioned to Green PCR Master Mix PCR buffer, 0.88 μL of each pri- 4-μm thick slices and stained with hematoxylin and mer (12.5 μmol/L), and 5 μL of the DNA sample. eosin. The morphological measurements (villus height In the case of the HTS of the colonic microbiota in and crypt depth, counts of intra-epithelial lymphocytes both trials, the V3-V4 region of 16S rRNA was targeted and goblet cells in the villus, and counts of mitoses in using MiSeq® Reagent Kit v2 (500 cycles; MiSeq from the crypt) of 10 different villus-crypt pairs were per- Illumina, San Diego, CA). The primers used in the formed using a light microscope (Leica DM5000B, construction of the libraries with amplicons of putative Jenoptik, Barcelona, Spain) fitted to CapturePro software 460 bp were as follows: (ProgRes®, Jenoptik, Barcelona, Spain), using the tech- nique described by Nofrarías et al. [36]. F-5′-TCGTCGGCAGCGTCAGATGTGTATAAGA Serum concentrations of tumor-necrosis factor-α GACAGCCTACGGGNGGCWGCAG (TNF-α) were determined using Quantikine Porcine R-5′-GTCTCGTGGGCTCGGAGATGTGTATAAG TNF-α kits (R&D Systems, Minneapolis, MN); the pig- AGACAGGACTACHVGGGTATCTAATCC major acute-phase protein (Pig-MAP) concentration was determined using a sandwich-type ELISA (Pig MAP Kit The sequence reads of the 16S rRNA gene generated ELISA, Pig CHAMP Pro Europe S.A., Segovia, Spain). In from the MiSeq Illumina system were processed using the Salmonella trial, serological antibodies of Salmonella the QIIME v.1.9.1 pipeline [28] with default settings. were tested using ELISA Herdchek Swine Salmonella The quality filter of the already demultiplexed sequences (IDEXX, Hoofdorp, Netherlands). López-Colom et al. Journal of Animal Science and Biotechnology (2019) 10:89 Page 6 of 17 Statistical analysis OTU and taxa relative abundances under a zero-altered The effect of the experimental treatments on the per- negative binomial or negative binomial model with pscl formance and slaughter measurements was determined [44] and mass [45] packages, respectively, and corrected using the free software R v.3.4.3, using the stats package for the false-discovery rate (FDR). [37] lm function for a one-way ANOVA, with diet as a fixed effect. For the microbial analysis, in Trial 2, entero- Results bacteria and coliform counts were log10-transformed In both studies, the animals showed a good health status and submitted to the same one-way ANOVA analysis. In on arrival, with most of the animals adapting to the facil- the case of Trial 1, Salmonella spp. count levels were an- ities and feed positively. Both challenges promoted a alyzed with the stats package likelihood.test function for clinical course of diarrhea in most of the animals, who frequency analysis; the number of positive animals for were recovering spontaneously at the end of the study. Salmonella spp. followed the stats package glm function Some casualties were registered in both trials. After the under a binomial distribution. The Pig-MAP concentra- Salmonella challenge, one animal from the DIC group tion levels were also analyzed with the same frequency was found dead on d 4 PI and two animals from the analysis, considering the normal range (0.3–1 mg/mL), DIC and CTR groups, respectively, were found dead on borderline (1–2 mg/mL), and high levels (> 2 mg/mL) d 6 PI. Within the ETEC F4 challenge, one animal from [38], and the number of animals with detectable copies the CTR group was found dead on d 1 PI and a second of the pathogen F4 gene or quantifiable levels of fermen- animal from the DIC group had to be euthanized follow- tation products (SCFA and lactic acid) were analyzed ing the clinical flowchart on d 3 PI. There were no stat- under a binomial distribution. istical differences in the number of causalities between The average daily feed intake, rectal temperature, and the treatments (P > 0.05) in any of the trials. daily fecal consistency were assessed using the lme4 package [39] lmer function for an adjusted linear mixed Animal performance model, with a treatment-by-time interaction term. The effects of the experimental treatments on animal For all analyzed data, the pen was the experimental performance are shown in Table 2. There were no sig- unit. The alpha level for the determination of signifi- nificant effects on the final BW, ADFI, or ADG with the cance for all the analyses was 0.05. Statistical trends administration of DIC in any of the trials, except for a were also considered for 0.05 < P < 0.10, unless other- lower ADG (P = 0.032) for the DIC group than the CTR wise indicated. Data are presented as means and residual standard error (RSE). Table 2 Effect of the experimental diets on animal performance Biostatistics of quality-filtered sequences were per- in the Salmonella and ETEC F4 trials formed using the open source software R v.3.4.3. Firstly, Items Salmonella Trial ETEC F4 Trial an OTU table was imported into R with the phyloseq CTR DIC RSE P CTR DIC RSE P package [40]; only OTUs that were shared among ex- BW, kg perimental groups were included and unique OTUs were Initial 8.1 8.1 0.14 0.900 5.6 5.6 0.10 0.239 excluded. Diversity and ordination (non-multidimen- Final 10.8 9.9 0.12 0.164 7.8 7.6 0.69 0.553 sional scaling, NMDS) were analyzed at the OTU level ADFI, g/d using the vegan package [41]. The richness and alpha di- PreIa 233 226 28.1 0.598 178 162 38.4 0.431 versity were calculated with raw counts based on the b Chao1 estimator [42] to estimate the number of taxa in 4 PI 218 193 46.7 0.302 219 211 45.8 0.729 the community, and the Shannon index [43], which con- 8 PIc 428 443 79.5 0.713 282 276 82.0 0.894 siders the richness as well as the evenness. For beta di- Overall d 285 278 41.5 0.766 221 211 46.6 0.664 versity, the Whittaker distance matrix was calculated ADG, g/d based on the relative abundances using the betadisper PreIa 122 109 60.7 0.675 77 78 41.6 0.986 function. To compare any differential effects from treat- 4 PIb -4 -38 108.7 0.541 129 117 90.5 0.796 ments, an ANOVA was performed for richness and di- versity. For ordination analysis, a dissimilarity matrix 8 PIc 326 316 162.6 0.903 260 164 80.6 0.032 based on the Bray-Curtis distances was also calculated Overalld 144 126 70.9 0.616 140 111 43.3 0.207 with relative abundances and two different fitting model ADFI Average daily feed intake, ADG Average daily gain, BW Body weight, PI analyses were applied. The function envfit, which fits Post-inoculation day, RSE Residual standard error a Pre-inoculation period from 0–7 d or 0–6 d before inoculation in the centroids of class variable levels defined as a factor onto Salmonella and ETEC F4 trials, respectively b an ordination, was used, and the anosim function was c Period from 0–4 d post-inoculation Period from 4–8 d post-inoculation used for the analysis of similarities (ANOSIM). Finally, a d Period from experimental day 1–14 or 1–15 in the Salmonella and ETEC F4 differential abundance analysis was performed with the trials, respectively López-Colom et al. Journal of Animal Science and Biotechnology (2019) 10:89 Page 7 of 17 group during the second PI period (4–8 d PI) in the than the PLB groups. After the ETEC challenge, a higher ETEC F4 trial. Regarding the performance response in response of both biomarkers was also observed in CTR the PLB groups, in the Salmonella trial the ADG was 33 groups than the PLB groups (156 ± 52.1 pg/mL in CTR ± 76.0 g/d during the adaptation week, and 306 ± 92.3 vs. 123 ± 17.4 pg/mL in PLB, and 1.56 ± 1.545 mg/mL g/d during the 4-PI period, whereas in the ETEC F4, in CTR vs. 1.22 ± 1.055 mg/mL in PLB, for TNF-α and these values were 75 ± 42.9 g/d and 174 ± 63.8 g/d, re- Pig-MAP, respectively). As expected, from day 4 to 8 PI, spectively. The ADFIs observed in the first trial for the the TNF-α (149 ± 69.2 pg/mL and 113 ± 20.3 pg/mL for PLB group were 184 ± 26.4 g/d and 379 ± 71.2 g/d dur- Trial 1 and 2, respectively) and Pig-MAP concentrations ing the pre- and post-inoculation days, respectively, and (0.96 ± 0.469 mg/mL and 1.46 ± 0.417 mg/mL for Trial those in the second trial for the PLB groups were 187 ± 1 and 2, respectively) were decreased in the CTR groups. 59.8 g/d and 255 ± 24.6 g/d, respectively. We were not able to detect statistical differences related to the experimental diets for any of the variables on any Clinical signs sampling day. The evolution of the fecal consistency throughout the post-inoculation (PI) period is presented in Fig. 1. After Microbiological analysis the oral challenge, fecal consistencies were impaired in In the Salmonella trial, none of the animals seeded Sal- both trials. The inoculation of Salmonella led to an monella in their feces on arrival nor after the 6 d of evident liquid diarrhea (time P < 0.001), with scores adaptation. The serological analysis from day 4 PI con- reaching level 3 in most of the animals on d 2 PI. After firmed that the animals had not been exposed to Sal- the ETEC inoculation, diarrhea was observed (time monella prior to the inoculation. PLB group remained P = 0.015) in a limited number of animals (barely negative for Salmonella spp. until d 4 PI. Only one piglet reaching scores of 1.8). There were no significant differ- per group (CTR and DIC) seroconverted on d 8 PI by ences related to the experimental diets for any of the trials applying a cut-off of an optical density percentage (P > 0.31). (OD%) ≥ 40. Regarding the pathogen loads, all animals The Salmonella challenge increased the rectal became positive, however, not all animals seeded count- temperature (40.6 ± 0.27 °C on d 2 PI vs. 39.4 ± 0.22 °C able numbers of the pathogen (> 103 CFU/g FM). Sup- on d 0 PI; day P < 0.001). The ETEC F4 challenge did not plementation with DIC reduced the number of animals affect this variable (39.1 ± 0.35 °C on d 2 PI vs. 39.0 with countable numbers in their cecum content, particu- ± 0.24 °C on d 0 PI; day P = 0.21). In relation to the larly on d 8 PI (Table 3). effect of diets, DIC had no significant influence on Figure 2 shows the different levels of Salmonella spp. rectal temperature (P > 0.6). excretion in the feces and cecal content for the experi- mental diets. Although the differences were not statisti- Inflammatory response cally significant, there was a trend for DIC to increase After the Salmonella challenge, the CTR group pre- the Salmonella counts in feces shortly after inoculation sented higher concentrations of TNF-α (312 ± 129.2 pg/ (d 1 PI, P = 0.10; and d 3 PI, P = 0.09), but to clear the mL vs. 106 ± 15.9 pg/mL in PLB) and Pig-MAP (3.08 ± pathogen more efficiently from the cecum on day 4 PI 2.812 mg/mL vs. 0.53 ± 0.140 mg/mL in PLB) on d 4 PI than the CTR group (P = 0.07). At the end of the study Fig. 1 Fecal scores throughout the post-inoculation (PI) period in the Salmonella (a) and ETEC F4 (b) trials. The fecal score was measured using a scale from 0 (hard and cloddy) to 4 (watery or with blood). CTR control group, DIC Dicosan-supplemented group, PI post-inoculation day, PLB placebo group López-Colom et al. Journal of Animal Science and Biotechnology (2019) 10:89 Page 8 of 17 Table 3 Effect of the experimental diets on the numbers of enterobacteria and coliforms, respectively. The attached Salmonella spp.a in the Salmonella trial enterobacteria in the ileal mucosa also tended to be re- Items Salmonella Trial duced with DIC on d 8 PI compared to the CTR diets (P = CTR DIC P 0.08). Feces The pathogen was not always present at quantifiable 1 PI 7/8 8/8 0.228 levels (limit of detection 3.83 log10 F4 gene copies/g FM). A slight trend was found between the diets on d 4 3 PI 8/8 6/8 0.080 PI in terms of the prevalence of the pathogen (P = 0.10), 7 PI 3/8 2/8 0.589 with all the CTR animals (8/8 animals) but not all from Cecum digesta the DIC treatment (6/8 animals) being positive for E. coli 4 PI 8/8 5/7 0.065 F4 in the colon digesta. On day 8 PI, the pathogen pres- 8 PI 7/8 3/8 0.033 ence was reduced in both groups, with no significant dif- PI post-inoculation day ferences between them (P = 0.30; 2/8 vs. 4/8 animals for a Number of animals with countable numbers of Salmonella spp. (< 103 log10 CTR and DIC, respectively), and similar loads (5.11 and CFU/g FM) 5.24 log10 F4 gene copies/g FM for the CTR and DIC groups, respectively). In the PLB group, the number of (d 8 PI), there was also an increase in the number of ani- animals positive for E. coli F4 on day 4 PI was 5/8 with mals with unquantifiable counts (very low) in the cecum mean count of 3.23 log10 F4 gene copies/g FM, which is with the DIC treatment (63% vs. 12% for DIC and CTR, below the limit of detection. respectively; P = 0.26). Regarding the ETEC F4 trial, there were no significant differences related to the experimental trials in the entero- Microbiota 16S rRNA gene analysis bacteria nor coliform plate counts in the feces upon arrival With the objective of assessing changes promoted by the or from after the week of adaptation (9.77 ± 0.282 and 9.70 additives to the intestinal microbiota, we analyzed co- ± 0.360 log10 CFU/g FM, respectively). Table 4 shows the lonic digesta samples taken on d 8 PI by high- microbiological analysis on d 4 and 8 PI of the ileal and co- throughput sequencing (HTS) of the V3-V4 regions of lonic digesta, and also of the ileal mucosa scrapings. On d 4 the 16S rRNA gene. PI, the counts of both bacterial groups were not different The microbiota analysis was performed using a more between the diets. However, on d 8 PI, animals receiving conservative approach, by using the OTU core, including DIC consistently had reduced numbers of enterobacteria only those species shared between experimental treat- and coliforms in their ileum and colon content compared ments, within each experiment (836/843 OTUs in Trial to those who received the CTR treatment. This effect was 1 and 833/848 OTUs in Trial 2). Regarding the evenness particularly apparent in the ileum, with decreases of more of the reads, similar values were obtained when compar- than one log10 unit (5.38 vs. 6.71 log10 CFU/g FM, P = ing treated groups to CTR ones (P > 0.34; 4.94 ± 0.375 0.004; and 5.33 vs. 6.51 log10 CFU/g FM, P = 0.05) for and 4.82 ± 0.243 log10 no. reads/sample in Trial 1 and Fig. 2 Number of animals with different levels of Salmonella spp. counts in feces and cecal contents. Feces sampled on d 1 (a), 3 (b), and 7 (c) post-inoculation (PI), and cecal content on d 4 (d) and 8 (e) PI (Salmonella trial). Range levels: negative (0 CFU/g), very low (< 103 CFU/g), low (103–104 CFU/g), high (105–106 CFU/g), and very high (107–108 CFU/g) López-Colom et al. Journal of Animal Science and Biotechnology (2019) 10:89 Page 9 of 17 Table 4 Effect of the experimental diets on enterobacteria and coliform counts in the ETEC F4 trial Items ETEC F4 trial CTR DIC RSE P Ileum digesta Enterobacteria, log10 CFU/g FM 4 PI 6.81 6.76 0.616 0.872 8 PI 6.71 5.38 0.783 0.004 Coliforms, log10 CFU/g FM 4 PI 6.02 6.08 0.923 0.889 8 PI 6.51 5.33 0.829 0.013 Colon digesta Enterobacteria, log10 CFU/g FM 4 PI 10.7 9.9 1.16 0.213 8 PI 10.9 9.9 1.03 0.083 Coliforms, log10 CFU/g FM 4 PI 8.9 8.9 0.99 0.974 8 PI 10.8 9.8 0.98 0.052 Ileum mucosa Enterobacteria, log10 CFU/g FM 4 PI 5.94 6.32 0.860 0.383 8 PI 6.17 5.16 0.786 0.082 Coliforms, log10 CFU/g FM 4 PI 5.53 5.47 1.117 0.921 8 PI 5.48 5.09 1.093 0.489 CFU Colony-forming units, FM Fresh matter, PI Post-inoculation day, RSE Residual standard error Trial 2, respectively). The Chao1 index did not differ between the groups (P > 0.74). Regarding the microbial diversity or structure, DIC administration did not alter the alpha or beta diversity (Whittaker index) after Salmonella inoculation. After Fig. 3 Non-metric dimensional scaling (NMDS) plot of the ETEC F4 inoculation, however, DIC administration did dissimilarity matrix based on Bray-Curtis distance. Clustering by change the Bray–Curtis distances (microbial structure) experimental diets for the Salmonella (a) and ETEC trial (b) from as shown in Fig. 3 for the NMDS results (envfit P = colon digesta samples collected on day 8 post-inoculation (PI). CTR 0.035 and ANOSIM P = 0.177). control group, DIC Dicosan-supplemented group Focusing on the aggregated taxa counts, Firmicutes and Bacteroidetes were the two major phyla (≥ 40%), 41 and 42 different families were detected per trial, re- with Proteobacteria following thereafter (7.3 ± 4.25% in spectively, representing approximately 5 % of the sum of the Salmonella trial and 9.1 ± 5.30% in the ETEC F4 the least abundant ones (< 1%, 31 families per trial). trial). A few minor phyla (< 1%): Elusimicrobia, Fibro- Among the genera detected, Prevotella was the most bacteres, Lentisphaerae, and Verrumicrobia, were en- prevalent (28.0 ± 10.7% and 29.0 ± 7.52% in Trial 1 and 2, countered in the Salmonella-challenged animals. respectively). A total of 55 genera were found in the Sal- The majority of the families observed in both trials were, monella and 54 in the ETEC F4 trials, and 40 and 42 gen- by order of abundance: Prevotellaceae (27.9 ± 10.68% and era were underrepresented (< 1%) in Trial 1 and 2, 29.0 ± 7.52% in Trial 1 and 2, respectively), Veillonellaceae respectively, accounting for approximately 8 % of the total (22.7 ± 9.55 % and 15.4 ± 5.32 % in Trial 1 and 2, respect- abundance. Unknown taxa represented 7.2 ± 2.87% and ively), Rumminococcaceae (11.4 ± 3.42% and 11.4 ± 2.63% 6.7 ± 2.05 % of the families in the Salmonella and ETEC in Trial 1 and 2, respectively) and Lachnospiraceae (8.29 ± F4 trials, respectively, whereas they represented 24.8 ± 3.555 % and 9.25 ± 3.307 % in Trial 1 and 2, respectively); 6.63 % and 27.2 ± 4.72% of the genera, respectively. López-Colom et al. Journal of Animal Science and Biotechnology (2019) 10:89 Page 10 of 17 Regarding the influence of DIC, the Firmicutes:Bacteroi- When considering the changes produced at the OTU detes (F/B) ratio showed a trend of a numerical decrease level, i.e., species, only those found in at least half of the (P = 0.132) in the Salmonella trial with DIC (1.14 vs. 1.47 samples were considered. Table 6 presents the differ- in the CTR group). In the ETEC F4 trial, F/B ratio was un- ences promoted by DIC (P < 0.05). As observed in the affected (1.23 vs. 1.05 in the CTR group, P = 0.335). aggregated taxa, in the Salmonella trial, several OTUs The effects of DIC on specific populations are also assigned to Lachnospiraceae (new refs. OTU-2808 and presented in Table 5, considering only the taxa found in 2343, and OTU-296082) were proportionally lower in at least half of the samples. At the phylum level, under DIC animals than CTR animals, including the Lachno- the Salmonella challenge, only the minor phylum Fibro- bacterium genus (OTU-584463). In addition, a bacteres (< 1%) was increased in DIC-supplemented ani- Spirochaetes-associated OTU was promoted in DIC ani- mals compared to the CTR (P = 0.02) animals. In mals (OTU-300859) and, in addition, several OTUs as- parallel, the corresponding family and genus, Fibrobac- sociated with Prevotella (within Bacteroidetes) were teraceae and Fibrobacter, respectively, were enriched in increased in DIC animals, possibly explaining the previ- the DIC animals compared to the CTR animals (P < ously mentioned reduction in the F/B ratio. Nonetheless, 0.05). Other changes in the Salmonella trial were also other OTUs also corresponding to Bacteroidetes were in observed in minor families (< 1%), such as lower num- lower numbers, which highlights the high variability bers of the candidate Barnesiellaceae and also in genera within a taxonomic group and the importance of consid- such as Dialister, Lachnobacterium and Butyrivibrio (P < ering the strain. 0.10). Although not significant, there was a relevant nu- When focusing on populations under the ETEC F4 merical decrease in the abundance of the Enterobacteria- challenge (Table 5), few changes were observed in DIC- ceae family in DIC animals compared to in the CTR supplemented animals; Dialister (P = 0.008) was signifi- animals (P = 0.16). cantly increased and Lachnospira tended to be reduced Table 5 Ln changes in taxa promoted by DIC supplementation in the Salmonella and ETEC F4 trials Taxonomic rank Taxon Ln changea (DIC vs. CTR) Adjusted Pb Salmonella trial Phylum Fibrobacteres 2.229 0.019 Family cand. Barnesiellaceae -4.499 0.003 Fibrobacteraceae 2.229 0.024 Enterobacteriaceae - 1.746 0.157 Spirochaetaceae 1.326 0.192 Alcaligenaceae 0.876 0.192 Genus Dialister - 0.690 0.000 Fibrobacter 2.225 0.033 Lachnobacterium - 3.219 0.035 Butyrivibrio - 1.251 0.095 ETEC F4 trial Family Veillonellaceae 0.363 0.136 Oxalobacteraceae - 0.804 0.151 Deferribacteraceae 1.915 0.151 Turicibacteraceae - 1.384 0.176 Genus Dialister 5.981 0.008 Lachnospira - 0.841 0.069 Megasphaera 1.137 0.149 Coprococcus - 1.051 0.149 Oxalobacter - 0.804 0.173 Mucispirillum 1.915 0.173 Turicibacter - 1.384 0.183 a Positive values and negative values indicate higher and lower abundance, respectively, in treated animals. Taxa are sorted by level of significance (from higher to lower). The presented differences are based only on taxa detected in at least half of the samples per diet b Adjusted P value < 0.20 López-Colom et al. Journal of Animal Science and Biotechnology (2019) 10:89 Page 11 of 17 Table 6 Differentially abundant OTUsa between the DIC and CTR groups in the Salmonella and ETEC F4 trials Taxonomic classificationb OTU code CTR DIC Salmonella trial Bacteroidetes; Prevotellaceae; Prevotella 300859 0.480 0.701 Firmicutes; Lachnospiraceae; Lachnospira 349257 0.273 0.175 Firmicutes; Clostridiales New ref. 2497 0.005 0.089 Unassigned New ref. 6586 0.005 0.062 Firmicutes; Erysipelotrichaceae 225636 0.055 0.002 Bacteroidetes; cand. Barnesiellaceae 315846 0.048 0.003 Bacteroidetes; Bacteroidaceae; Bacteroides ovatus 535375 0.039 0.015 Bacteroidetes; cand. Paraprevotellaceae; cand. Prevotella New ref. 6366 0.065 0.052 Firmicutes; Christensenellaceae 410242 0.001 0.024 Spirochaetes; Sphaerochaetaceae; Sphaerochaeta 1832447 0.003 0.017 Bacteroidetes; Bacteroidales New ref. 2311 0.003 0.025 Firmicutes; Clostridiales 357471 0.012 0.033 Bacteroidetes; Prevotellaceae; Prevotella stercorea New ref. 1872 0.008 0.033 Bacteroidetes; Prevotellaceae; Prevotella New ref. 1748 0.009 0.022 Bacteroidetes; Prevotellaceae; Prevotella 296082 0.008 0.023 Firmicutes; Lachnospiraceae; Lachnobacterium 584463 0.017 0.001 Bacteroidetes; Prevotellaceae; Prevotella stercorea New ref. 2808 0.001 0.008 Bacteroidetes; cand. Paraprevotellaceae; cand. Prevotella New ref. 648 0.002 0.009 Bacteroidetes; Prevotellaceae; Prevotella stercorea 302538 0.025 0.021 Firmicutes; Lachnospiraceae; Blautia 526773 0.014 0.013 ETEC trial Bacteroidetes; Prevotellaceae; Prevotella stercorea 524371 2.430 1.654 Firmicutes; Clostridiales 584083 0.210 0.261 Firmicutes; Veillonellaceae; Megasphaera 298050 0.038 0.220 Firmicutes; Lachnospiraceae; Coprococcus 344804 0.032 0.318 Firmicutes; Veillonellaceae; Dialister 403701 0.001 0.056 Bacteroidetes; Bacteroidaceae; Bacteroides 513445 0.006 0.084 Bacteroidetes; Prevotellaceae; Prevotella 248447 0.049 0.046 Bacteroidetes; S24-7 844589 0.004 0.040 Bacteroidetes; Bacteroidales; cand. Paraprevotellaceae; YRC22 289468 0.034 0.030 Bacteroidetes; Prevotellaceae; Prevotella New ref. 2220 0.021 0.014 Firmicutes; Erysipelotrichaceae 287798 0.026 0.026 Firmicutes; Veillonellaceae; Megasphaera 264967 0.017 0.011 Bacteroidetes; cand. Paraprevotellaceae; cand. Prevotella New ref. 5084 0.008 0.004 Firmicutes; Clostridiales 798164 0.010 0.011 Firmicutes; Ruminococcaceae 583134 0.008 0.013 Firmicutes; Lachnospiraceae; Lachnospira 843553 0.399 0.140 Firmicutes; Lachnospiraceae; Coprococcus 366623 0.282 0.000 Firmicutes; Clostridiales 826624 0.143 0.081 a Mean relative abundances (%) are presented for OTUs with an adjusted P value < 0.05 and detected in at least half of the animals per diet b Order is specified only when it represents the lowest rank classification López-Colom et al. Journal of Animal Science and Biotechnology (2019) 10:89 Page 12 of 17 (P = 0.069) compared to CTR animals. Other bacteria Table 7 Effects of the experimental diets on intestinal were numerically altered (P < 0.20) by DIC, with most of fermentation parameters in the Salmonella and ETEC F4 trials them representing < 1%, with the exception of Veillonel- Items Salmonella Trial ETEC F4 Trial laceae (P = 0.136; approximately 15%) and the genus CTR DIC RSE P CTR DIC RSE P within Megasphaera (P = 0.149; approximately 5%). Ileum digesta pH Intestinal fermentation products 4 PI 7.20 7.00 0.228 0.111 6.81 6.80 0.208 0.891 Table 7 shows the modifications of the pH and the main 8 PI 7.37 7.34 0.190 0.786 6.86 6.77 0.195 0.346 fermentation products in the ileum and colon digesta for NH3, μmol/g both trials. The ileal pH was not significantly modified 4 PI 4.4 2.0 2.77 0.113 0.6 1.4 1.91 0.431 by the experimental treatments, except for a numerical 8 PI 14.6 19.1 8.89 0.326 1.0 1.1 0.39 0.752 reduction (7.00 vs. 7.20; P = 0.11) observed for DIC Lactic acid, μmol/g compared to CTR on d 4 PI in the Salmonella trial. In 4 PI 10.5 29.8 29.17 0.207 23.2 27.7 18.91 0.652 the ileum, the major product of fermentation was lactic 8 PI 41.8 23.4 27.09 0.196 19.1 36.0 25.73 0.209 acid, with ranges of 1.80–117 and 1.98–105 μmol/g FM Colon digesta in Trial 1 and 2, respectively. No differences were de- pH tected between the diets. Acetic acid was found at very 4 PI 6.93 6.70 0.274 0.137 6.26 6.37 0.238 0.360 low levels in the ileum in both trials (average values of 1.61 and 4.44 μmol/g FM for Trial 1 and 2, respectively), 8 PI 7.07 7.04 0.156 0.671 5.97 6.07 0.266 0.464 with several animals below the minimum detection level NH3, μmol/g on d 8 PI (12 for Trial 1 and 3 for Trial 2), and no differ- 4 PI 32.5 24.4 19.94 0.431 10.7 10.8 5.821 0.969 ences between the experimental diets. 8 PI 57.3 39.1 25.86 0.18 18.5 17.6 6.763 0.801 Regarding the colon digesta, there were no obvious Total SCFA, μmol/g changes in pH, despite a slight reduction with DIC on d 4 4 PI 98 88 37.1 0.613 107 102 21.5 0.606 PI in the Salmonella trial (6.70 vs. 6.92; P = 0.14). The am- 8 PI 147 136 17.6 0.244 155 163 21.5 0.461 monia content was also not modified by the experimental Acetic acid, % diets. The total SCFA content was not modified by the ex- 4 PI 55.8 55.2 8.22 0.892 60.5 60.1 6.01 0.907 perimental diets, but showed an increase from d 4 to 8 PI 8 PI 56.0 54.2 7.37 0.629 61.3 60.2 4.06 0.573 in both trials (from 92.7 to 141 and 104 to 159 μmol/g Propionic acid, % FM, respectively). The fermentation profile showed few 4 PI 26.6 24.6 3.86 0.317 22.5 24.7 5.91 0.479 changes with the diets; a numerical trend was observed in the Salmonella trial (P = 0.12) for an increase in the valeric 8 PI 21.7 25.9 6.52 0.218 22.6 23.1 3.32 0.778 acid proportion in the colon with DIC detected on d 4 PI. Butyric acid, % In the ETEC trial, higher valeric concentrations were also 4 PI 12.9 14.3 6.01 0.649 12.6 11.2 3.78 0.492 observed with DIC supplementation than with CTR diets 8 PI 17.0 15.3 3.61 0.373 13.5 12.7 2.09 0.452 on d 8 PI (P = 0.03). Valeric acid, % 4 PI 2.65 4.10 1.804 0.118 2.73 2.55 1.327 0.802 Ileal histomorphometry 8 PI 4.10 3.53 1.467 0.456 1.59 2.84 1.056 0.033 Table 8 shows the changes promoted by the experimen- BCFA, % tal diet in terms of ileal histomorphometry. The villus 4 PI 2.16 1.83 1.186 0.586 1.68 1.37 0.774 0.465 height showed a marked increase from d 4 to 8 PI in 8 PI 1.27 1.11 0.531 0.566 0.95 1.21 0.365 0.176 both trials, but was not modified by the experimental di- BCFA Branched-chain fatty acids, PI Post-inoculation day, RSE Residual standard ets, neither were the crypt depth or the villus:crypt ratio. error, SCFA Short-chain fatty acids There were no differences related to the inclusion of DIC either for the number of mitoses or the number of (MCFA) salts from coconut distillates (DIC) against en- goblet cells. However, intriguingly, the IEL counts dis- teric pathogens, such as Salmonella Typhimurium or played a trend to decrease with DIC in the Salmonella ETEC F4, and the consequent reinforcement of the gut trial on d 8 PI (P = 0.07), whereas these were increased health and general health status of early-life weaned pig- (P = 0.08) with the additive in the ETEC F4 trial. lets. In order to fulfill these objectives, DIC was adminis- tered in feed in two different trials, each with one of the Discussion two pathogens. In both trials, animals exhibited signs of The present study aimed to evaluate the possible anti- diarrhea after the oral challenge and an immediate de- microbial effect of a mixture of medium-chain fatty acids crease in feed intake. López-Colom et al. Journal of Animal Science and Biotechnology (2019) 10:89 Page 13 of 17 Table 8 Effects of the experimental diets on histomorphometry One of the most remarkable effects of this study was in the ileum in the Salmonella and ETEC F4 trials the specific action of supplemented MCFA on popula- Items Salmonella trial ETEC F4 trial tions of Salmonella, coliforms, and enterobacteria. CTR DIC RSE P CTR DIC RSE P Salmonella spp. counts in the cecum tended to be Villus height, μm reduced by DIC, despite a higher initial colonization 4 PI 185 183 65.5 0.962 278 229 89.4 0.289 after the challenge. After the ETEC F4 inoculation, DIC 8 PI 282 276 58.1 0.852 323 321 63.7 0.955 consistently diminished the enterobacteria and total coli- Crypt depth, μm form populations in the luminal content of the ileum 4 PI 315 323 69.6 0.815 196 182 30.9 0.38 and colon (particularly on d 8 PI), and the same trend 8 PI 300 290 37.1 0.596 189 184 19.7 0.600 was observed in the ileal mucosa, despite the effects on the luminal E. coli F4 pathogen being small. As stated in Villus:Crypt the introduction, a higher antibacterial activity has been 4 PI 0.60 0.63 0.257 0.841 1.47 1.29 0.469 0.454 described for MCFA than other free fatty acids [53]. 8 PI 0.99 0.99 0.209 0.944 1.78 1.79 0.364 0.935 Most recent studies have demonstrated their in vitro ac- MIT, cells/100 μm tivity against a wide range of pathogenic bacteria [5, 11, 4 PI 0.251 0.325 0.160 0.39 0.078 0.068 0.103 0.845 53, 54]. Lauric acid (C12) has been shown to have the 8 PI 0.075 0.099 0.082 0.569 0.104 0.091 0.141 0.862 highest antimicrobial activity among the fatty acids GC, cells/100 μm present in coconut oil [54, 55]. However, other medium- 4 PI 2.20 1.93 0.851 0.556 1.75 1.43 0.542 0.262 chain fatty acids, caprylic acid (C8) and capric acid 8 PI 1.49 1.28 0.548 0.457 1.31 1.33 0.519 0.954 (C10), showed the highest antimicrobial activity against IEL, cells/100 μm different E. coli and Salmonella strains tested [3]. Freese 4 PI 2.92 2.76 1.509 0.841 2.07 2.04 0.482 0.917 et al. [56] and Sheu et al. [57] also reported lower mini- 8 PI 3.92 3.06 0.882 0.072 2.39 2.88 0.523 0.080 mum inhibitory concentrations (MIC) of caproic (C6) GC Goblet cells in the villus, IEL Intraepithelial lymphocytes in the villus, MIT and caprylic (C8) acids for 50% growth inhibition of E. Mitoses in the crypt, PI Post-inoculation day, RSE Residual standard error coli than capric acid (C10) and C12. Furthermore, C6 and C8 have been selected for their antimicrobial activ- During the week of adaptation after weaning, ani- ity, rather than longer-chain fatty acids, to be tested in mals from the different diets displayed similar perfor- in vivo trials [23]. Nevertheless, although C12 is the mances, with no influence of DIC supplementation. major compound in the distillates of coconut (48.4% Other authors, however, have described improvements crude fat, CF), these also include important proportions in performance related to the supplementation of of other MCFA (6.2% CF for C8 and 5.8% CF for C10) MCFA. Some works evaluating MCFA as an energy that might support the aforementioned effects. source, at higher doses (2.1–7.75%), registered growth Among the in vivo studies that have evaluated the ef- improvements [6, 13, 46, 47] that could be justified fect of MCFA administration on pathogens, some au- by the more rapid digestion and absorption of these thors have described lower numbers of clostridia or E. fatty acids. Improvements have also been seen in ani- coli [15, 16, 58–60], and also Salmonella [22]. These re- mals challenged with ETEC F4 or LPS [23, 48], how- ported effects on the colonization of the gut by different ever, not necessarily by using high doses. Regarding pathogens could have been mediated by the direct activ- the possible effects of MCFA supplementation on feed ity of MCFA, but also indirectly, by changes promoted intake, we did not detect any impact of the experi- by these additives in the intestinal microbiota. In this re- mental diets. The potential impact of MCFA as gard, different in vivo trials in pigs have described sig- additives on feed intake is controversial. When sup- nificant changes in bacterial populations promoted by plemented at high levels, as a rapid source of energy, the direct supplementation of MCFA supplementation MCFA could prompt satiety due to their rapid oxida- [61, 62], including a transgenerational influence ob- tion in the liver [49, 50]. Moreover, MCFA have also served by the supplementation of sows having an effect been described as rancid and presenting low palatabil- on their offspring [63]. The variability in these effects in ity [51, 52], which could therefore lead to a decline the literature could be the result of differences between in feed intake and impair the adaptation to dry feed MCFA sources, blends, dosages, and methods of admin- after weaning. Nonetheless, in our study, there was istration, making the direct comparison between studies no negative impact of DIC on the feed intake during difficult. In particular, most of the experimental blends the first week post-weaning. The lower dosage (at tested were composed of a combination of C8 and C10 0.3%) than that used in other studies, and the differ- and negligible quantities of C12, or their conjugated ent nature and composition of MCFA blends, could forms, into triacylglycerol (MCT), with lipases needed to explain the discrepancies with other studies. release, not always successfully, the free fatty acids. López-Colom et al. Journal of Animal Science and Biotechnology (2019) 10:89 Page 14 of 17 To our knowledge, very few works have evaluated the the opportunity to show its effects on the current effect of MCFA on intestinal microbiota by performing IEL response. an entire profiling of their populations, particularly when However, within the ETEC F4 trial, the reduction in faced with a pathogen challenge. The results of our work possible specific commensal populations such as entero- show that, in global terms, the microbiota profiles ob- bacteria and coliforms as observed in the ETEC F4- served after the two challenges were not substantially challenged animals, might suggest a more hostile milieu, different from those previously described for piglets of which consequently mobilized more IELs than the CTR this age [64, 65], suggesting that these animals did not group. In turn, the IEL response could also be related to suffer any drastic dysbiosis. In fact, other challenges in a possible readaptation of the community structure, as piglets with Salmonella could not differentiate clusters seen in the NMDS results (envfit P = 0.035). In this re- from unchallenged and Salmonella-challenged animals gard, the impact of the MCFA supplementation was evi- either [66], and were not even able to detect the Salmon- dent at the structural level, with the most relevant ella genus by HTS, as occurred in our animals. Never- change in the bacterial groups observed for the increase theless, it must be taken into account that the activity of in Dialister (P = 0.008) within the Veillonellaceae family. the supplemented MCFA against potential pathogens However, other mechanisms related to a direct modula- was observed mainly in the ileum and cecum, and the tor effect of MCFA on the immune response of the ani- assortment of microbial populations was also affected. mal should not be disregarded. MCFA supplemented to This leads us to hypothesize that the direct impact on pre-weaning piglets as triglycerides (MCT) have been upper parts of the gut is reflected, for instance, on the shown to stimulate the immune response in the stom- gastric barrier or the small intestine in the foregut. ach, regardless of the complexity of the microbiota [71]. Nevertheless, there were still observed microbiota In addition, similar stimulatory effects on intestinal villi changes promoted by DIC diets. In both trials, the and the local immunity have been demonstrated in LPS- DIC diets were able to significantly modify particular challenged weanlings based on C6- and C8-MCT [23]. microbial taxa, although the affected bacterial groups To try and elucidate possible associations between the were different. With the Salmonella challenge, the microbial populations and gut variables assessed in this most relevant change was a significant increase study, particularly those modified by the experimental registered in the Fibrobacteres phylum (P = 0.019). treatments, we performed a correlation analysis; the Accompanied by a (numerical) reduction in Entero- most relevant correlations (r > 0.5 and adjusted P < bacteriaceae, it could be hypothesized that the sup- 0.10) are presented in Table 9. Interestingly, candidate plemented MCFA prompts a more fibrolytic-like Barnesiellaceae and Enterobacteriaceae families were adult microbial profile. In addition, it is interesting positively correlated with the IEL counts in the Salmon- to remark that several OTUs from the Lachnospira- ella trial, whereas in the ETEC F4 trial, no significant ceae family were decreased with the DIC diet com- correlation was found. This could be somehow related pared to the CTR diet and that the Lachnospira to the differential response registered in IEL with DIC genus has been reported to increase with the pres- between both trials. Valeric acid showed only a signifi- ence of Salmonella [67]. This response of the micro- cant high inverse correlation with Clostridiaceae (P = biota to DIC supplementation could also be 0.011) in the Salmonella trial. Under the ETEC F4 associated with the response registered in IEL at the ileal level. On d 8 PI, the animals treated with DIC Table 9 Pearson’s correlationsa between gut variables and and challenged with Salmonella maintained lower bacterial populations for Salmonella and ETEC F4 trials numbers of IEL than the CTR group. A possible hy- Gut variable Microbiotab Pearson (r) Adjusted P pothesis is that the pigs in the DIC group might Salmonella trial have retained a more benign environment within the IEL candidate Barnesiellaceae 0.698 0.055 gastrointestinal tract. Another plausible explanation IEL candidate Odoribacteraceae 0.694 0.057 could be that the clear reduction in the pathogen IEL Odoribacter 0.694 0.057 counts in the cecum by the addition of DIC stopped the recruitment of IELs, since these defense cells IEL Enterobacteriaceae 0.686 0.065 have been found near Salmonella pathogens [68]. In Valeric acid Clostridiaceae -0.782 0.011 fact, the IEL counts were higher within the Salmon- ETEC F4 trial ella challenge than in previous trials in our group Valeric acid Megasphaera 0.837 0.005 [33, 69], reflecting a more acute course of diarrhea. Valeric acid Veillonellaceae 0.784 0.023 If we consider that the IELs remain quiescent until IEL Intraepithelial lymphocytes in the ileal villus facing a real threat [70], it seems reasonable that a Correlations considered: r > 0.5 and adjusted P < 0.10 after the Salmonella challenge, DIC treatment had b Microbial populations identified by high-throughput sequencing (HTS) López-Colom et al. Journal of Animal Science and Biotechnology (2019) 10:89 Page 15 of 17 challenge, valeric acid was positively correlated with Availability of data and materials Megasphaera (r = 0.837) and the corresponding family The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. The dataset generated Veillonellaceae (r = 0.784). This correlation is coincident corresponding to the final 16S rRNA gene sequences is available in the with the higher presence of Dialister on d 8 PI in DIC European Nucleotide Archive (ENA), accession code PRJEB30494. animals and the higher percentage of valeric acid than in CTR animals. Dialister and Megasphaera, among other Ethics approval and consent to participate All procedures with animals received prior approval from the Animal and members within the Veillonellaceae family, have been Human Experimental Ethical Committee of the UAB (permit no. CEAAH reported to produce valeric acid as an end metabolite of 2933). the fermentation of carbohydrates and lactate [72]. Consent for publication Not applicable. Conclusions The results obtained from this study support the activity Competing interests of a blend of sodium salts of coconut oil distillates in re- The authors declare that they have no competing interests. ducing the hindgut colonization by pathogenic popula- Author details tions such as enterobacteria, E. coli, and Salmonella in 1 Animal Nutrition and Welfare Service, Animal and Food Science orally-challenged weaned piglets. The effects could be Department, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Spain. 2Norel S.A, Madrid, Spain. mediated by the changes promoted in the microbiota ecosystem since significant effects were registered in dif- Received: 20 April 2019 Accepted: 6 September 2019 ferent microbial groups. Under a Salmonella challenge, significant increases were registered in the Fibrobacteres References phylum and after an ETEC F4 challenge, the most rele- 1. EFSA, ECDC. The European Union summary report on antimicrobial vant changes were registered in the Dialister genus of resistance in zoonotic and indicator bacteria from humans, animals and the Veillonellaceae family within a diverging microbial food in 2015. EFSA J. 2017;15:4694. 2. Heo JM, Opapeju FO, Pluske JR, Kim JC, Hampson DJ, Nyachoti CM. structure. There was also a differential impact of this Gastrointestinal health and function in weaned pigs: a review of feeding blend on the intestinal local immune response, leading strategies to control post-weaning diarrhoea without using in-feed to higher numbers of ileal intraepithelial lymphocytes antimicrobial compounds. J Anim Physiol Anim Nutr. 2012;97(2):207–37. 3. Skrivanová E, Marounek M, Benda V, Brezina P. Susceptibility of Escherichia after the Salmonella challenge, but lower numbers after coli, Salmonella sp. and Clostridium perfringens to organic acids and the ETEC F4 challenge. The effects of the supplemented monolaurin. Vete Med. 2006;51(3):81–8. MCFA might respond to different complex interactions 4. Petschow BW, Batema RP, Ford LL. Susceptibility of Helicobacter pylori to bactericidal properties of medium-chain monoglycerides and free fatty between the opportunistic pathogens, the commensal acids. Antimicrob Agents Chemother. 1996;40(2):302–6. microbiota and the host response. 5. Manohar V, Echard B, Perricone N, Ingram C, Enig M, Bagchi D, et al. In vitro and in vivo effects of two coconut oils in comparison to monolaurin on Abbreviations Staphylococcus aureus: rodent studies. J Med Food. 2013;16(6):499–503. ADFI: Average daily feed intake; ADG: Average daily gain; ANOSIM: Analysis 6. Li Y, Zhang H, Yang L, Zhang L, Wang T. Effect of medium-chain of similarities; BW: Body weight; CFU: Colony-forming unit; DM: Dry matter; triglycerides on growth performance, nutrient digestibility, plasma ETEC: Enterotoxigenic Escherichia coli; FM: Fresh matter; GC: Goblet cells; metabolites and antioxidant capacity in weanling pigs. Anim Nutr. 2015; HTS: High-throughput sequencing; IEL: Intraepithelial lymphocytes; 1(1):12–8. MCFA: Medium-chain fatty acids; MCT: Medium-chain triglycerides; 7. Schönfeld P, Wojtczak L. Short- and medium-chain fatty acids in energy MIT: Mitoses; OTU: Operational taxonomic unit; PBS: Phosphate buffered metabolism: the cellular perspective. J Lipid Res. 2016;57(6):943–54. solution; PI: Post-inoculation; PreI: Pre-inoculation; qPCR: Quantitative 8. DebMandal M, Mandal S. Coconut (Cocos nucifera L.: Arecaceae): in health polymerase-chain reaction; SCFA: Short-chain fatty acids promotion and disease prevention. Asian Pac J Trop Med. 2011;4(3):241–7. 9. Baltić B, Starčević M, Đorđević J, Mrdović B, Marković R. Importance of medium chain fatty acids in animal nutrition. In: IOP Conf Ser Earth Environ Acknowledgments Sci, vol. 85; 2017. The authors would like to thank Laura Noguera Solans for her support with 10. Zentek J, Buchheit-Renko S, Ferrara F, Vahjen W, Van Kessel AG, Pieper R. the experimental trial. Nutritional and physiological role of medium-chain triglycerides and medium-chain fatty acids in piglets. Anim Health Res Rev. 2011;12(1):83–93. 11. Vande Maele L, Heyndrickx M, Maes D, De Pauw N, Mahu M, Verlinden M, Authors’ contributions et al. In vitro susceptibility of Brachyspira hyodysenteriae to organic acids and LC, MP, JJM, and SMMO conceived and designed the experiments; MP and essential oil components. J Vet Med Sci. 2016;78(2):325–8. JJM contributed reagents and materials; PLC and ARS performed the 12. Hernández A, Pluske J. Using dietary medium-chain triglycerides to improve experiment and the laboratory analyses; PLC received, curated, and post-weaning performance of pigs. Pork CRC 2B-102/103-0506. 2008. url: maintained the animal records; LC and SMMO supervised the experiment; http://researchrepository.murdoch.edu.au/id/eprint/30626. Accessed Nov PLC, LC, and SMMO analyzed and interpreted the data and wrote the paper. 2017. All authors read and approved the final manuscript. 13. Weng RC. Dietary fat preference and effects on performance of piglets at weaning. Asian-Australas J Anim Sci. 2017;30(6):834–42. Funding 14. Dierick NA, Decuypere JA, Molly K, Van Beek E, Vanderbeke E. The This work was funded by the “PORCDIGEST” project (IDI-20140262), funded combined use of triacylglycerols (TAGs) containing medium chain fatty by the CDTI and the Ministerio de Economía y Competitividad (Spain) and acids (MCFAs) and exogenous lipolytic enzymes as an alternative for had support from the pre-doctoral FI grant of the Generalitat de Catalunya nutritional antibiotics in piglet nutrition. II. In vivo release of MCFAs in (Spain) awarded to Paola López-Colom. gastric cannulated and slaughtered piglets by endogenous and exogenous López-Colom et al. Journal of Animal Science and Biotechnology (2019) 10:89 Page 16 of 17 lipases; effects on the luminal gut flora and growth performance. Livest 35. Jensen MT, Cox RP, Jensen BB. Microbial production of skatole in the hind Prod Sci. 2002;76(1-2):1–16. gut of pigs given different diets and its relation to skatole deposition in 15. Hanczakowska E, Szewczyk A, Okoń K. Effects of dietary caprylic and capric backfat. Anim Sci. 1995;61(2):293–304. acid on piglet performance and mucosal epithelium structure of ileum. J 36. Nofrarías M, Manzanilla EG, Pujols J, Gibert X, Majó N, Segalés J, et al. Effects of Anim Feed Sci. 2011;20:556–65. spray-dried porcine plasma and plant extracts on intestinal morphology and 16. Kuang Y, Wang Y, Zhang Y, Song Y, Zhang X, Lin Y, et al. Effects of dietary on leukocyte cell subsets of weaned pigs. J Anim Sci. 2006;84(10):2735–42. combinations of organic acids and medium chain fatty acids as a 37. R Core Team. R: a language and environment for statistical computing. replacement of zinc oxide on growth, digestibility and immunity of weaned Vienna: R Foundation for Statistical Computing; 2013. pigs. Anim Feed Sci Technol. 2015;208:145–57. 38. Piñeiro C, Piñeiro M, Morales J, Andrés M, Lorenzo E, Pozo MD, et al. Pig- 17. Dierick NA, Decuypere JA, Molly K, Van Beek E, Vanderbeke E. The MAP and haptoglobin concentration reference values in swine from combined use of triacylglycerols containing medium-chain fatty acids commercial farms. Vet J. 2009;179(1):78–84. (MCFAs) and exogenous lipolytic enzymes as an alternative for nutritional 39. Douglas B, Mächler M, Bolker B, Walker S. Fitting linear mixed-effects models antibiotics in piglet nutrition. I. In vitro screening of the release of MCFAs using lme4. J Stat Softw. 2015;67(1):1–48. from selected fat sources by selected exogenous lipolytic enzymes under 40. McMurdie PJ, Holmes S. Phyloseq: an R package for reproducible interactive simulated pig gastric conditions and their effects on the gut flora of piglets. analysis and graphics of microbiome census data. PLoS One. 2013;8(4): Livest Prod Sci. 2002;75(2):129–42. e61217. 18. Messens W, Goris J, Dierick N, Herman L, Heyndrickx M. Inhibition of 41. Oksanen J. vegan: community ecology package. 2009. https://cran.r-project. Salmonella typhimurium by medium-chain fatty acids in an in vitro org/web/packages/vegan/vegan.pdf. Accessed May 2018. simulation of the porcine cecum. Vet Microbiol. 2010;141(1-2):73–80. 42. Chao A. Nonparametric estimation of the number of classes in a population. 19. Martínez-Vallespín B, Vahjen W, Zentek J. Effects of medium-chain fatty Scand J Stat. 1984;11(4):265–70. acids on the structure and immune response of IPEC-J2 cells. 43. Shannon CE, Weaver W. The mathematical theory of communication. Cytotechnology. 2016;68(5):1925–36. Urbana: University Illinois Press; 1963. p. 117. 20. Kono H, Fujii H, Asakawa M, Maki A, Amemiya H, Hirai Y, et al. Medium-chain 44. Zeileis A, Kleiber C, Jackman S. Regression models for count data in R. J Stat triglycerides enhance secretory IgA expression in rat intestine after administration Softw. 2008. https://doi.org/10.18637/jss.v027.i08. of endotoxin. Am J Physiol Gastrointest Liver Physiol. 2004;286(6):G1081–9. 45. Venables WN, Ripley BD. Modern applied statistics with S. 4th ed. New York: 21. Van Immerseel F, De Buck J, Boyen F, Bohez L, Pasmans F, Volf J, et al. Springer; 2002. Medium-chain fatty acids decrease colonization and invasion through hilA 46. Cera KR, Mahan DC, Reinhart GA. Evaluation of various extracted vegetable suppression shortly after infection of chickens with Salmonella enterica oils, roasted soybeans, medium-chain triglyceride and an animal-vegetable serovar Enteritidis. Appl Environ Microbiol. 2004;70:3582–7. fat blend for postweaning swine. J Anim Sci. 1990;68:2756–65. 22. Rasschaert G, Michiels J, Tagliabue M, Missotten J, De Smet S, Heyndrickx M. 47. Rodas BD, Maxwell CV. The effect of fat source and medium-chain Effect of organic acids on Salmonella shedding and colonization in pigs on triglyceride level on performance of the early-weaned pig. Anim Sci Res a farm with high Salmonella prevalence. J Food Prot. 2016;79(1):51–8. Rep. 1990;129(9):278–87. 23. De Keyser K, Dierick N, Kanto U, Hongsapak T, Buyens G, Kuterna L, et al. 48. Lei XJ, Park JW, Baek DH, Kim JK, Kim IH. Feeding the blend of organic acids Medium-chain glycerides affect gut morphology, immune- and goblet cells in and medium chain fatty acids reduces the diarrhea in piglets orally post-weaning piglets: in vitro fatty acid screening with Escherichia coli and in vivo challenged with enterotoxigenic Escherichia coli K88. Anim Feed Sci consolidation with LPS challenge. J Anim Physiol Anim Nutr. 2019;103:221–30. Technol. 2017;224:46–51. 24. NRC. Nutrient requirements of swine: eleventh revised edition. 11th ed. 49. Guillot E, Vaugelade P, Lemarchal P, Rérat A. Intestinal absorption and liver Washington: The National Academies Press; 2012. uptake of medium-chain fatty acids in non-anaesthetized pigs. Br J Nutr. 25. AOAC. Official methods of analysis. 18th ed. Gaithersburg: Association of 1993;69:431–42. Official Analytical Chemists; 2007. 50. Geng S, Zhu W, Xie X, Li X, Wu J, Liang Z, et al. Medium-chain triglyceride 26. Van Soest PJ, Robertson JB, Lewis BA. Methods for dietary fiber, neutral ameliorates insulin resistance and inflammation in high fat diet-induced detergent fiber, and nonstarch polysaccharides in relation to animal obese mice. EurJ Nutr. 2016;55(3):931–40. nutrition. J Dairy Sci. 1991;74(10):3583–97. 51. Decuypere JA, Dierick NA. The combined use of triacylglycerols containing 27. Gustavo Hermes R, Molist F, Pérez JF, Gómez de Segura A, Ywazaki M, Davin medium-chain fatty acids and exogenous lipolytic enzymes as an alternative R, et al. Casein glycomacropeptide in the diet may reduce Escherichia coli to in-feed antibiotics in piglets: concept, possibilities and limitations. An attachment to the intestinal mucosa and increase the intestinal lactobacilli overview. Nutr Res Rev. 2003;16(2):193–210. of early weaned piglets after an enterotoxigenic E. coli K88 challenge. Br J 52. Desbois AP, Smith VJ. Antibacterial free fatty acids: activities, mechanisms of Nutr. 2013;109(6):1001–12. action and biotechnological potential. Appl Microbiol Biotechnol. 2009;85: 28. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello 1629–42. EK, et al. QIIME allows analysis of high-throughput community sequencing 53. Batovska DI, Todorova IT, Tsvetkova IV, Najdenski HM. Antibacterial study of data. Nat Methods. 2010;7(5):335–6. the medium chain fatty acids and their 1-monoglycerides: Individual effects 29. Rideout JR, He Y, Navas-Molinas JA, Walters WA, Ursell LK, Gibbons SM, et al. and synergistic relationships. Polish J Microbiol. 2009;58(1):43–7. Subsampled open-reference clustering creates consistent, comprehensive 54. Shilling M, Matt L, Rubin E, Visitacion MP, Haller NA, Grey SF, et al. OTU definitions and scales to billions of sequences. PeerJ. 2014;2:e545. Antimicrobial effects of virgin coconut oil and its medium-chain fatty acids 30. DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, et al. on Clostridium difficile. J Med Food. 2013;16(12):1079–85. Greengenes, a chimera-checked 16s rRNA gene database and workbench 55. Tangwatcharin P, Khopaibool P. Activity of virgin coconut oil, lauric acid or compatible with ARB. Appl Environ Microbiol. 2006;72:5069–72. monolaurin in combination with lactic acid against Staphylococcus aureus. 31. Haas BJ, Gevers D, Earl AM, Feldgarden M, Ward DV, Giannoukos G, et al. Southeast Asian J Trop Med Public Health. 2012;43(4):969–85. Chimeric 16S rRNA sequence formation and detection in Sanger and 454- 56. Freese E, Sheu C, Galliers E. Function of lipophilic acids as antimicrobial pyrosequenced PCR amplicons. Genome Res. 2011;21(3):494–504. food additives. Nature. 1973;241:321–5. 32. Bokulich NA, Subramanian S, Faith JJ, Gevers D, Gordon JI, Knight R, et al. 57. Sheu C, Salomon D, Simmons J, Sreevalsan T, Freese E. Inhibitory effects of Quality-filtering vastly improves diversity estimates from Illumina amplicon lipophilic acids and related compounds on bacteria and mammalian cells. sequencing. Nat Methods. 2013;10(1):57–9. Antimicrob Agents Chemother. 1975;7:349–63. 33. Barba-Vidal E, Buttow Roll VF, Castillejos L, Guerra-Ordaz AA, Manteca X, 58. Dierick NA, Michiels J, Van Nevel C. Effect of medium chain fatty acids and Mallo JJ, Martín-Orúe SM. Response to a Salmonella Typhimurium challenge benzoic acid, as alternatives for antibiotics, on growth and some gut in piglets supplemented with protected sodium butyrate or Bacillus parameters in piglets. Commun Agric Appl Biol Sci. 2004;69(2):187–90. licheniformis: effects on performance, intestinal health and behavior. Transl 59. Upadhaya SD, Lee KY, Kim IH. Protected organic acid blends as an Anim Sci. 2017;1(2):186–200. alternative to antibiotics in finishing pigs. Asian-Australasian J Anim Sci. 34. Richardson AJ, Calder AG, Stewart CS, Smith A. Simultaneous determination 2014;27(11):1600–7. of volatile and non-volatile acidic fermentation products of anaerobes by 60. Upadhaya SD, Lee KY, Serpunja S, Song TH, Kim IH. Growth performance, capillary gas chromatography. Lett Appl Microbiol. 1989;9(1):5–8. nutrient digestibility, fecal microbiota and fecal noxious gas emission in López-Colom et al. Journal of Animal Science and Biotechnology (2019) 10:89 Page 17 of 17 weaning pigs fed high and low density diet with and without protected organic acid blends. Anim Feed Sci Technol. 2018;239:1–8. 61. Zentek J, Buchheit-Renko S, Männer K, Vahjen W. Intestinal concentrations of free and encapsulated dietary medium-chain fatty acids and effects on gastric microbial ecology and bacterial metabolic products in the digestive tract of piglets. Arch Anim Nutr. 2012;66(1):14–26. 62. Zentek J, Ferrara F, Pieper R, Tedin L, Meyer W, Vahjen W. Effects of dietary combinations of organic acids and medium chain fatty acids on the gastrointestinal microbial ecology and bacterial metabolites in the digestive tract of weaning piglets. J Anim Sci. 2013;91(7):3200–10. 63. Lan R, Kim I. Effects of organic acid and medium chain fatty acid blends on the performance of sows and their piglets. Anim Sci J. 2018;89(12):1673–9. 64. Xiao L, Estellé J, Kiilerich P, Ramayo-Caldas Y, Xia Z, Feng Q, et al. A reference gene catalogue of the pig gut microbiome. Nat Microbiol. 2016; 19:16161. 65. Holman DB, Brunelle BW, Trachsel J, Allen HK. Meta-analysis to define a core microbiota in the swine gut. mSystems. 2017;2(3):e00004–17. 66. Borewicz KA, Kim HB, Singer RS, Gebhart CJ, Sreevatsan S, Johnson T, et al. Changes in the porcine intestinal microbiome in response to infection with Salmonella enterica and Lawsonia intracellularis. PLoS One. 2015;1010: e0139106. 67. Leite FLL, Singer RS, Ward T, Gebhart CJ, Isaacson RE. Vaccination against Lawsonia intracellularis decreases shedding of Salmonella enterica serovar Typhimurium in co-infected pigs and alters the gut microbiome. Sci Rep. 2018;8:2857. 68. Hoytema van Konijnenburg DP, Reis BS, Pedicord VA, Farache J, Victora GD, Mucida D. Intestinal epithelial and intraepithelial T cell crosstalk mediates a dynamic response to infection. Cell. 2017;171(4):783–94. 69. Barba-Vidal E, Castillejos L, Roll VFB, Cifuentes-Orjuela G, Moreno Muñoz JA, Martín-Orúe SM. The probiotic combination of Bifidobacterium longum subsp. infantis CECT 7210 and Bifidobacterium animalis subsp. lactis BPL6 reduces pathogen loads and improves gut health of weaned piglets orally challenged with Salmonella Typhimurium. Front Microbiol. 2017;8:1570. 70. Cheroutre H. IELs: enforcing law and order in the court of the intestinal epithelium. Immunol Rev. 2005;206:114–31. 71. Trevisi P, Priori D, Motta V, Luise D, Jansman AJM, Koopmans SJ, et al. The effects of starter microbiota and the early life feeding of medium chain triglycerides on the gastric transcriptome profile of 2- or 3-week-old cesarean delivered piglets. J Anim Sci Biotechnol. 2017;8:82. 72. Marchandin H, Teyssier C, Campos J, Jean-Pierre H, Roger F, Gay B, et al. Negativicoccus succinicivorans gen. nov., sp. nov., isolated from human clinical samples, emended description of the family Veillonellaceae and description of Negativicutes classis nov., Selenomonadales ord. nov. and Acidaminococcaceae fam. nov. In the bacterial phylum Firmicutes. Int J SystEvol Microbiol. 2010;60(6):1271–9.