Assessment of the Effects of the Synbiotic Combination of Bifidobacterium longum subsp. infantis CECT 7210 and Oligofructose-Enriched Inulin Against Digestive Bacterial Infections in a Piglet Model.

The use of bifidobacteria as probiotics has proven to be beneficial in gastroenteric infections. Furthermore, prebiotics such as inulin can enhance the survival and growth of these bacteria. Two trials were performed to evaluate the effects of the administration of Bifidobacterium longum subsp. infantis CECT 7210 and oligofructose-enriched inulin against Salmonella enterica serovar Typhimurium or enterotoxigenic Escherichia coli (ETEC) F4. A total of 72 (Salmonella trial) and 96 (ETEC F4 trial) weaned piglets were used in a 2 × 2 design (with or without synbiotic, inoculated or not with the pathogen). After adaptation, animals were orally inoculated. Performance and clinical signs were evaluated. On days 4 and 8 (Salmonella trial) and 3 and 7 (ETEC F4 trial) post-inoculation (PI), one animal per pen was euthanized. Blood, digestive content and tissue samples were collected and microbiological counts, fermentation products, serum inflammatory markers and ileum histomorphometry analysis were performed. Both challenges had an impact on faecal consistency (p < 0.001), including the faecal shedding of Salmonella and increased numbers of enterobacteria and coliforms. The synbiotic administration did not have any effect on pathogen loads but induced changes in the fermentation profile, such as increased valeric acid in both trials as well as decreased acetic acid, except for Salmonella-challenged animals. The effect on propionate varied among trials, increasing in challenged synbiotic-treated pigs and decreasing in non-challenged ones in the Salmonella trial (P interaction = 0.013), while the opposed occurred in the ETEC F4 trial (P interaction = 0.013). The administration of the synbiotic increased intraepithelial lymphocytes (IEL; p = 0.039) on day 8 PI in the Salmonella trial and a similar trend occurred in non-challenged pigs in the ETEC F4 trial (P interaction = 0.086). The results did not provide evidence of reduced pathogen load with the synbiotic, although a modulation in fermentative activity could be identified depending on the challenge. Consistent increases were found in IEL, suggesting that this synbiotic combination has some immunomodulatory properties.

Effectiveness of Two Plant-Based In-Feed Additives against an Escherichia coli F4 Oral Challenge in Weaned Piglets.

This study evaluates the efficacy of two plant-based feed supplementations to fight colibacillosis in weanlings. A total of 96 piglets (32 pens) were assigned to four diets: a control diet (T1) or supplemented with ZnO (2500 ppm Zn) (T2) or two different plant supplements, T3 (1 kg/t; based on essential oils) and T4 (T3 + 1.5 kg/t based on non-volatile compounds). After one week, animals were challenged with ETEC F4, and 8 days after, one animal per pen was euthanized. Performance, clinical signs, microbial analysis, inflammatory response, intestinal morphology, and ileal gene expression were assessed. ZnO improved daily gains 4 days after challenge, T3 and T4 showing intermediate values (96, 249, 170, and 157 g/d for T1, T2, T3, and T4, p = 0.035). Fecal lactobacilli were higher with T3 and T4 compared to ZnO (7.55, 6.26, 8.71, and 8.27 cfu/gFM; p = 0.0007) and T3 increased the lactobacilli/coliforms ratio (p = 0.002). T4 was associated with lower levels of Pig-MAP (p = 0.07) and increases in villus/crypt ratio (1.49, 1.90, 1.73, and 1.84; p = 0.009). Moreover, T4 was associated with an upregulation of the REG3G gene (p = 0.013; pFDR = 0.228) involved in the immune response induced by enteric pathogens. In conclusion, both plant supplements enhanced animal response in front of an ETEC F4 challenge probably based on different modes of action.

Effects of the Administration of Bifidobacterium longum subsp. infantis CECT 7210 and Lactobacillus rhamnosus HN001 and Their Synbiotic Combination With Galacto-Oligosaccharides Against Enterotoxigenic Escherichia coli F4 in an Early Weaned Piglet Model.

We evaluated the potential of multi-strain probiotic (Bifidobacterium longum subsp. infantis CECT 7210 and Lactobacillus rhamnosus HN001) with or without galacto-oligosaccharides against enterotoxigenic Escherichia coli (ETEC) F4 infection in post-weaning pigs. Ninety-six piglets were distributed into 32 pens assigned to five treatments: one non-challenged (CTR+) and four challenged: control diet (CTR-), with probiotics (>3 × 1010 CFU/kg body weight each, PRO), prebiotic (5%, PRE), or their combination (SYN). After 1 week, animals were orally inoculated with ETEC F4. Feed intake, weight, and clinical signs were recorded. On days 4 and 8 post-inoculation (PI), one animal per pen was euthanized and samples from blood, digesta, and tissues collected. Microbiological counts, ETEC F4 real-time PCR (qPCR) quantification, fermentation products, serum biomarkers, ileal histomorphometry, and genotype for mucin 4 (MUC4) polymorphism were determined. Animals in the PRO group had similar enterobacteria and coliform numbers to the CTR+ group, and the ETEC F4 prevalence, the number of mitotic cells at day 4 PI, and villus height at day 8 PI were between that observed in the CTR+ and CTR- groups. The PRO group exhibited reduced pig major acute-phase protein (Pig-MAP) levels on day 4 PI. The PRE diet group presented similar reductions in ETEC F4 and Pig-MAP, but there was no effect on microbial groups. The SYN group showed reduced fecal enterobacteria and coliform counts after the adaptation week but, after the inoculation, the SYN group showed lower performance and more animals with high ETEC F4 counts at day 8 PI. SYN treatment modified the colonic fermentation differently depending on the MUC4 polymorphism. These results confirm the potential of the probiotic strains and the prebiotic to fight ETEC F4, but do not show any synergy when administered together, at least in this animal model.

Effects of Bifidobacterium longum Subsp. infantis CECT 7210 and Lactobacillus rhamnosus HN001, Combined or Not With Oligofructose-Enriched Inulin, on Weaned Pigs Orally Challenged With Salmonella Typhimurium.

Salmonella is a common causative agent of enteric disease and is developing mechanisms of resistance to antimicrobials. Probiotics, such as bifidobacteria and lactobacilli, and prebiotic fibers are a potential alternative to counteract this pathogen as they have demonstrated effectiveness in preventing its adhesion, reducing intestinal damage, and enhancing the host immune system. Furthermore, the benefits are expected to be potentiated when these compounds are administered together. A trial was performed to evaluate the efficacy of two probiotic strains (Bifidobacterium longum subsp. infantis CECT 7210 (Laboratorios Ordesa S.L.) and Lactobacillus rhamnosus HN001, combined or not with a prebiotic containing oligofructose-enriched inulin, against Salmonella Typhimurium. Ninety-six piglets (28 days old) were distributed into 32 pens assigned to 5 treatments: one non-challenged (control diet, CTR+) and four challenged: control diet (CTR-) or supplemented with probiotics (>3 × 1010 cfu/kg each strain, PRO), prebiotic (5%, PRE), or their combination (SYN). After 1 week of adaptation, animals were orally challenged with Salmonella Typhimurium. Feed intake, weight, and clinical signs were recorded. On days 4 and 8 post-inoculation (PI), one animal per pen was euthanized, and samples from blood, digestive content, and ileal tissues were collected to determine Salmonella counts, fermentation products, ileal histomorphology, and serum TNF-α and Pig-MAP concentrations. The effect of the oral challenge was evidenced by animal performance, fecal consistency, and intestinal architecture. Regarding the experimental treatments, animals belonging to the PRO group experienced a faster clearance of the pathogen, with more pigs being negative to its excretion at the end of the study and recovering the impaired ileal villi/crypt ratio more rapidly. Animals receiving the PRE diet showed a lower intestinal colonization by Salmonella, with no countable levels (<3 cfu/g) in any of the analyzed samples, and an augmented immune response suggested by serum Pig-MAP concentrations. Treatments including the prebiotic (PRE and SYN) showed similar changes in the fermentation pattern, with an increase in the molar percentage of valeric acid concentration in the colon. The SYN group, however, did not show any of the outcomes registered for PRO and PRE in Salmonella colonization or in immunity markers, suggesting the lack of synbiotic action in this animal model. Further research is needed to better understand the complex mechanisms behind these effects.

Impact of in-feed sodium butyrate or sodium heptanoate protected with medium-chain fatty acids on gut health in weaned piglets challenged with Escherichia coli F4.

Short and medium-chain fatty acids (SCFA and MCFA, respectively) are commonly used as feed additives in piglets to promote health and prevent post-weaning diarrhoea. Considering that the mechanism and site of action of these fatty acids can differ, a combined supplementation could result in a synergistic action. Considering this, it was aimed to assess the potential of two new in-feed additives based on butyrate or heptanoate, protected with sodium salts of MCFA from coconut distillates, against enterotoxigenic Escherichia coli (ETEC) F4+ using an experimental disease model. Two independent trials were performed in 48 early-weaned piglets fed a control diet (CTR) or a diet supplemented with MCFA-protected sodium butyrate (BUT+; Trial 1) or sodium heptanoate (HPT+; Trial 2). After 1 week of adaptation, piglets were challenged with a single oral inoculum of ETEC F4+ (minimum 1.4 · 109 cfu). One animal per pen was euthanised on days 4 and 8 post-inoculation (PI) and the following variables assessed: growth performance, clinical signs, gut fermentation, intestinal morphology, inflammatory mediators, pathogen excretion and colon microbiota. None of the additives recovered growth performance or reduced diarrhoea when compared to the respective negative controls. However, both elicited different responses against ETEC F4+. The BUT+ additive did not lead to reduce E. coli F4 colonisation but enterobacterial counts and goblet cell numbers in the ileum were increased on day 8 PI and this followed higher serum TNF-α concentrations on day 4 PI. The Firmicutes:Bacteroidetes ratio was nevertheless increased. Findings in the HPT+ treatment trial included fewer animals featuring E. coli F4 in the colon and reduced Enterobacteriaceae (determined by 16S RNA sequencing) on day 4 PI. In addition, while goblet cell numbers were lower on day 8 PI, total SCFA levels were reduced in the colon. Results indicate the efficacy of MCFA-protected heptanoate against ETEC F4+ and emphasise the potential trophic effect of MCFA-protected butyrate on the intestinal epithelium likely reinforcing the gut barrier.

Efficacy of medium-chain fatty acid salts distilled from coconut oil against two enteric pathogen challenges in weanling piglets.

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.