The Impact of Fiber Source on Digestive Function, Fecal Microbiota, and Immune Response in Adult Dogs.

This study evaluated the impact of different fiber sources on intestinal function, fecal microbiota, and overall health in dogs. Twelve dogs were used in a crossover design, involving three periods of 6 weeks and three diets: a low-fiber diet (CTR), a cereal-fiber and beet-pulp-supplemented diet (BRA), and a fruit-fiber-supplemented diet (FRU). Each period included a digestibility trial and fecal and blood sampling in the last week. Short-chain fatty acids (SCFAs) and microbiota taxonomy (16S rRNA Illumina-MiSeq) and functionality (Shotgun-NovaSeq 6000) were determined in the feces. General biochemistry, complete blood cells, and lymphocyte subsets were also analyzed. The fiber-supplemented diets showed lower digestibility without significant changes in the fecal consistency. The BRA diet showed higher total SCFA concentrations (p = 0.056), with increases in alpha diversity and particular beneficial genera, such as Lachnospira, Bifidobacterium, and Faecalibacterium. The BRA microbiota was also associated with an overabundance of genes related to carbohydrate and amino acid metabolism. The FRU diet had a distinct impact on the microbiota composition and functionality, leading to higher levels of CD8 lymphocytes. These findings emphasize the importance of selecting the right fiber source when formulating dog diets, as it can have a differential impact on gut microbiota and animal health.

Potential benefits of yeast Saccharomyces and their derivatives in dogs and cats: a review.

Yeast Saccharomyces and its derivatives have been largely used in livestock and poultry nutrition for their potential positive impact on growth, performance, and general health. Originally included in animal diets as a source of protein, yeasts can also offer a wide range of by-products with interesting bioactive compounds that would confer uses beyond nutrition. Although its supplementation in livestock, poultry and even in humans is well documented, the available body of literature on the use of yeast and its derivatives in companion animals' food, mainly dogs and cats' diets, is still developing. Despite this, gut microbiota modulation, immune system enhancement or decreasing of potentially pathogenic microorganisms have been reported in pets when using these products, highlighting their possible role as probiotics, prebiotics, and postbiotics. This review attempts to provide the reader with a comprehensive on the effects of Saccharomyces and its derivatives in pets and the possible mechanisms that confer their functional properties.

Looking for Possible Benefits of Combining Short-Chain Fructo-Oligosaccharides (scFOS) with Saccharomyces cerevisiae Sc 47 on Weaned Pigs Orally Challenged with Escherichia coli F4.

The objective of this work was to evaluate the effect of supplementing short-chain fructo-oligosaccharides (scFOS) combined or not with live yeast Saccharomyces cerevisiae Sc 47 on weanling pigs challenged with Escherichia coli F4+. We allocated ninety-six piglets to four experimental diets: control (CTR); supplemented with scFOS (5 g/kg Profeed® P95) (scFOS); S. cerevisiae Sc 47 (1 g/kg Actisaf® Sc 47 HR +) (YEA); or both (SYN). Parameters included: performance; E. coli F4+ detection; fermentation activity; inflammatory biomarkers; and ileal histomorphology. Our results showed that supplementing scFOS was able to reduce the incidence of diarrhea, and both supplements were able to lower counts of EHEC along the gut. Supplementing scFOS was mostly associated with changes in the gut ecosystem and increases in the lactobacilli population, while S. cerevisiae Sc 47 registered increases in the numbers of ileal intraepithelial lymphocytes. The synbiotic mixture showed the lowest diarrhea incidence and fecal scores, benefiting from complementary modes of action and possible synergistic effects due to a hypothesized yeast-LAB cross-feeding phenomenon in the foregut. In conclusion, our results evidence that supplementing scFOS or Saccharomyces cerevisiae Sc 47 is efficacious to fight post-weaning colibacillosis, and combining both could be beneficial in high-risk scenarios.

Potential effect of two Bacillus probiotic strains on performance and fecal microbiota of breeding sows and their piglets.

The effect of long-term administration of two Bacillus strains was tested on 98 breeding sows and their litters allotted into three treatments: a control group (CON); supplemented with 5 × 108 cfu/kg B. subtilis - 541 (BSU); or with 5 × 108 cfu/kg B. amyloliquefaciens - 516 (BAM). Reproductive and performance variables were recorded over three cycles with 56 dams remaining through the third lactation. Blood and fecal samples were taken longitudinally from 12 sows per treatment on days 8 and 21 of the third lactation and milk samples were taken on day 21. Feces from one piglet per litter was sampled on days 21 and 33 and jejunal gene expression was assessed in two piglets on day 21. Changes in fecal microbiota were assessed by 16S rRNA gene sequencing (Illumina MiSeq) and gene expression by Open-Array technology. Metabolomic responses were analyzed in milk by NMR and Ig-G and Ig-A specific antibodies were determined by ELISA. No significant differences were observed on feed intake, body weight, or fat mobilization of the sows. However, a significant increase in the total number of piglets born was observed in supplemented sows. Although the increase was seen from the first cycle with BAM, improvements were not seen with BSU until the third cycle. BAM also increased the number of born-alive and weaned piglets. NMR analysis showed an impact of BAM on milk composition. No differences were found in milk or blood immunoglobulins. A different structure of the fecal microbiota was found in supplemented sows, with changes across phylum, family, and genus. These changes were greater at day 8, suggesting a relevant role of probiotics establishing a new intestinal balance after labor. Shifts in the microbiota were also seen in the piglets, with a clearer impact post-weaning than in suckling. In this regard, correlations between microbial groups of sows and piglets showed a higher link with weaned (d33) than with suckling pigs (d21), reinforcing the idea of an early maternal carry-over. No changes due to treatment in jejunal gene expression were detected; however, piglet size had a clear impact on different genes. In summary, the addition of both probiotics, and particularly Bacillus amyloliquefaciens, demonstrated potential benefits on the prolificacy of sows. Daily feeding of Bacillus amyloliquefaciens resulted in an increase in the number of weaned piglets. The high correlations between the compositions of the microbiota of sows and their piglets are evidence of maternal imprinting, with effects lasting beyond weaning.

The aim of the present study was to determine if the inclusion of probiotic microorganisms in the mother's diet during gestation and the lactation period is capable of modifying the performance of mothers and piglets and the possible effect on the intestinal health of piglets after separation from the mother. For this, 98 females were distributed in three experimental treatments: a control diet, or the same diet in which one of two probiotic strains to be tested (Bacillus subtilis or Bacillus amyloliquefaciens) were incorporated. The experimental diets were administered during pregnancy and the lactation phase for three consecutive productive cycles. Among the most striking results, it is worth highlighting the impact of probiotic treatments on the reproductive performance of sows. Both supplemented groups showed a higher number of total piglets per sow. Furthermore, sows that received the Bacillus amyloliquefaciens diet showed a significant increase in the number of live-born piglets. Probiotic supplementation also showed effects on the fecal microbiota composition of the mothers and their piglets. Changes in the composition of sow milk were also observed. In summary, results demonstrated the potential benefits of supplementing probiotics, and particularly a strain of Bacillus amyloliquefaciens, to improve prolificacy, modulate the intestinal microbial composition, and improve the performance of piglets during lactation.

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.

Evaluation of dietary supplementation of a novel microbial muramidase on gastrointestinal functionality and growth performance in broiler chickens.

This study was conducted to assess the effect of dietary supplementation of Muramidase 007 to broiler chickens on gastrointestinal functionality, evaluating growth performance, apparent ileal digestibility, intestinal histomorphology, vitamin A in plasma and cecal microbiota. A total of 480 one-day male chicks (Ross 308) were distributed in 16 pens allocated in 2 experimental diets: the control diet (CTR) without feed enzymes, coccidiostat or growth promoters, and the experimental diet (MUR): CTR supplemented with 35,000 units (LSU(F))/kg of the Muramidase 007. Digesta and tissue samples were obtained on days 9 and 36 of the study. A lower feed conversion ratio was observed in the MUR treatment. Apparent ileal digestibility of DM, organic matter and energy were improved by Muramidase 007. It was also observed that MUR improved digestibility of total fatty acids, mono-unsaturated fatty acids and poly-unsaturated fatty acids, and content of vitamin A in plasma at day 9 (P < 0.05). Histomorphological analysis of jejunum samples revealed no differences in the villus height or crypt depth; but a higher number of goblet cells and intraepithelial lymphocytes at day 36 with MUR. No differences were observed in plate counts of enterobacteria or Lactobacillus along the gastrointestinal tract, neither on the cecal short-chain fatty acids. An statistical trend was observed for reduction of cecal clostridia at day 9 for MUR. Analysis of cecal microbiota structure by 16S rRNA gene sequencing revealed relevant changes correlated to age. At day 9, broilers receiving MUR showed decreased alpha diversity compared to CTR that was not detected at day 36. Changes in specific taxonomic groups with an increase in Lactobacillus genus were identified. In conclusion, evaluation of the variables in this study indicates that dietary Muramidase 007 contributes to improve feed conversation ratio and gastrointestinal function in broiler chickens. Effects could have been mediated by slight shifts observed in the intestinal microbiota. More studies are guaranteed to fully understand the mechanisms involved.

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.

Response of gastrointestinal fermentative activity and colonic microbiota to protected sodium butyrate and protected sodium heptanoate in weaned piglets challenged with ETEC F4.

This study aimed to evaluate the potential of two new fat-protected butyrate or heptanoate salts to improve gut health and control post-weaning colibacillosis in weaning piglets challenged with enterotoxigenic Escherichia coli (ETEC) F4+, particularly focusing on their impact on intestinal microbiota and fermentative activity along the gastrointestinal tract (GIT). Seventy-two 21-d-old pigs were fed a plain diet (CTR) or supplemented with sodium butyrate (BUT) or sodium heptanoate (HPT), both at 0.3%. After a week of adaptation, animals were orally challenged at days 8 and 9 with 5.8 · 109 and 6.6 · 1010 cfu, respectively, and were euthanised on d 4 and d 8 post-inoculation (PI) (n = 8) to collect blood, digesta and tissue samples and characterise microbial groups, pathogen loads (qPCR), fermentation, ileal histomorphometry and immune markers. Colonic microbiota was analysed by 16S rRNA gene MiSeq sequencing. Supplementing both acid salts did not compensate clinical challenge effects nor performance impairments and neither histomorphometry nor serum biomarkers. Changes in the gastric fermentative activity were registered, BUT reducing lactic acid concentrations (day 8 PI), and with HPT fewer animals presenting detectable concentrations of propionic, butyric and valeric acids. At ileum BUT increased acetic acid concentration (day 8 PI), and both additives reduced short-chain fatty acids (SCFA) in the colon. Increases in enterobacteria and coliforms counts in ileal digesta (day 4 PI, p < 0.10) and mucosa scrapes (p < 0.05) were registered although E. coli F4 gene copies were unaffected. Regarding changes in the colonic microbiota (day 4 PI), Prevotellaceae and Prevotella were promoted with BUT supplementation whereas only minor groups were modified in HPT-treated animals. Summarising, although the pathogen loads or inflammatory mediators remained unresponsive, butyrate and heptanoate showed a significant impact on microbial fermentation along the whole GIT, being able to modify different bacterial groups at the colon. It could be hypothesised that these effects might be mediated by a carry-over effect of the changes observed in gastric fermentation, but possibly also to a better nutrient digestion in the foregut as a result of the reduced colonic SCFA concentrations.

I-FABP, Pig-MAP and TNF-α as biomarkers for monitoring gut-wall integrity in front of Salmonella Typhimurium and ETEC K88 infection in a weaned piglet model.

Having sensitive serum biomarkers able to determine the structural changes of the small intestine suffering from bacterial digestive diseases could be a valuable tool particularly in piglets at weaning, when intestinal infections are highly prevalent. We evaluated the usefulness of three inflammatory and gut-wall-integrity biomarkers to assess the degree of intestinal histo-morphological damage in piglets. Piglets were orally challenged with Salmonella Typhimurium or enterotoxigenic Escherichia coli (ETEC) to get a variable range of response according to individual variability. Forty-eight piglets were challenged with Salmonella Typhimurium and seventy-two with enterotoxigenic Escherichia coli K88. Clinical signs and faecal score were recorded. At Days 4 and 8 post-inoculation, blood was sampled, animals euthanised and distal ileum dissected. Morphological measures were obtained from the gut tissue, and serum tumour necrosis factor-alpha (TNF-α), pig major acute-phase protein (Pig-MAP) and intestinal fatty acid-binding protein (I-FABP) were determined. Animals developed mild-to-severe diarrhoea after the challenge. When analysing the complete set of analytical results, a high correlation was found among the three serum biomarkers. The most representative morphological indicator was the villus:crypt ratio (V:C), which showed a strong negative correlation with all three biomarkers. Regression analyses between faecal score and the previous variable showed linear relations. When the range of V:C was analysed, based on the quartile distribution of each serum variable, a marked increase in their concentration was observed with greater villus damage. Summarising, the combination of I-FABP, Pig-MAP and TNF-α may be useful for determining the intestinal injury degree and barrier integrity in recently weaned pigs.

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.

Probiotics have been demonstrated to be useful to enhance gut health and prevent gastrointestinal infections in humans. Additionally, some multi-strain probiotic combinations have been suggested to have greater efficacy than single strains. The objective of this study is to demonstrate the potential of a combination of the probiotic strains: Bifidobacterium longum subsp. infantis CECT 7210 (brand name B. infantis IM1®) and B. animalis subsp. lactis BPL6 to enhance gut health and to ameliorate the outcome of a Salmonella challenge using a weaning piglet model. Seventy-two 28-day-old weanling piglets, 7.7 (±0.28) kg of body-weight, were distributed in a 2 × 2 factorial design; treated or not with the probiotic combination and challenged or not with the pathogen. Animals were orally challenged after an adaptation period (Day 8) with a single dose (5 × 108 cfu) of Salmonella Typhimurium. One animal per pen was euthanized on Day 12 (Day 4 post-inoculation [PI]) and Day 16 (Day 8 PI). All parameters responded to the challenge and 4 deaths were registered, indicating a severe but self-limiting challenge. Improvements registered in the challenged animals due to the probiotic were: increased voluntary feed-intake (P probiotic × challenge = 0.078), reduced fecal excretion of Salmonella (P = 0.028 at Day 1 PI and P < 0.10 at Days 3 and 5 PI), decreased rectal temperature (P probiotic × day = 0.048) and improvements in the villous:crypt ratio (P probiotic × challenge < 0.001). Moreover, general probiotic benefits were observed in both challenged and non-challenged groups: decreased diarrhea scores of the PI period (P = 0.014), improved fermentation profiles on Day 8 PI (increased ileal acetic acid [P = 0.008] and a tendency to lower colonic ammonia concentrations [P = 0.078]), stimulation of intestinal immune response by increasing villous intraepithelial lymphocytes (P = 0.015 on Day 8 PI) and an improved villous:crypt ratio (P = 0.011). In conclusion, the multi-strain probiotic had a positive effect on reducing pathogen loads and alleviating animals in a Salmonella challenge. In addition, enhanced gut health and immunity was recorded in all animals receiving the probiotic, indicating an improvement in the post-weaning outcome.

Evaluation of the Probiotic Strain Bifidobacterium longum subsp. Infantis CECT 7210 Capacities to Improve Health Status and Fight Digestive Pathogens in a Piglet Model.

Probiotics have been demonstrated to be useful to enhance gut health and prevent gastrointestinal infections. The objective of this study is to demonstrate the potential of the probiotic strain Bifidobacterium longum subsp. infantis CECT 7210 (B. infantis IM1) to prevent and fight intestinal disease by using a Salmonella Typhimurium (Trial 1) or an enterotoxigenic Escherichia coli K88 (Trial 2) oral challenge in a weaning piglet model. Seventy-two piglets were used in each trial. After an adaptation period, animals were orally challenged. One animal per pen was euthanized at Days 4 and 8/9 (Trial 1/Trial 2) post-inoculation (PI). Animal performance, clinical signs, pathogen excretion, fermentation, immune response, and intestinal morphology were evaluated. In Trial 1, most parameters responded to the challenge, whereas, in Trial 2, effects were much milder. Consistent effects of the probiotic were detected in both experiments: Reduction of pathogen excretion (P = 0.043 on Day 3 PI, Trial 1) or ileal colonization (33% reduction of animals with countable coliforms; P = 0.077, Trial 2); increases in intraepithelial lymphocytes (P = 0.002 on Day 8 PI in Trial 1, P = 0.091 on Day 4 PI in Trial 2), and improvement of the fermentation profile by increasing butyric acid in non-challenged animals [P challenge × probiotic (interaction) = 0.092 in Trial 1 and P = 0.056 in Trial 2] concomitant with an enhancement of the villus:crypt ratio on Day 8/9 PI (P interaction = 0.091 for Trial 1 and P = 0.006 for Trial 2). Challenged animals treated with the probiotic showed reduced feed intakes (P interaction = 0.019 in Trial 1 and P = 0.020 in Trial 2) and had lower short-chain fatty acid concentrations in the colon (P interaction = 0.008 in Trial 1 and P = 0.082 in Trial 2). In conclusion, this probiotic demonstrated potential to reduce the intestinal colonization by pathogens and to stimulate local immune response. However, effects on feed intake, microbial fermentation, and intestinal architecture showed a differential pattern between challenged and non-challenged animals. Effects of the probiotic intervention were dependent on the structure of the ecosystem in which it was applied.

New properties of wheat bran: anti-biofilm activity and interference with bacteria quorum-sensing systems.

Some plant extracts, have been demonstrated to interfere with the microbial metabolism of several pathogenic bacteria. Within this antimicrobial properties it has been described the potential to inhibit or destroy biofilms or to interfere in quorum-sensing (QS) systems. However, to our knowledge, no study exploring this potential of wheat-bran (WB) has been published. The purpose of the present study is to evaluate the anti-biofilm activity of WB against a cow mastitis strain of Staphylococcus aureus and also its possible interference with bacterial QS systems. The potential of inhibition and destruction of the biofilm was studied by different in vitro assays. Also, we tested the ability of WB to interfere in bacterial QS by degrading acyl-homoserine lactones (AHL) as one of the most studied QS signal molecules for Gram-negative bacteria. The soluble extract of WB at 0.5% showed anti-biofilm activity, inhibiting biofilm formation and also destroying it. Similarly, the > 300 kDa fraction from WB had significant anti-biofilm activity in both in vitro assays. The WB also showed a potential to interfere with bacterial QS systems, as it was demonstrated to contain certain lactonase activity able to reduce AHL concentration in the medium. The present study reveals two additional beneficial properties of WB extract never explored before, which may be related to the presence of defence compounds in the plant extract able to interfere with microbial biofilms and also QS systems.

Casein modified gold nanoparticles for future theranostic applications.

The synthesis and characterization of gold nanoparticles (AuNPs 20 nm sized) modified with k-casein derived peptides in order to monitor the peptide effect as bacterial adhesion inhibitor, thanks to the carrier/concentrator effect of the AuNPs is here presented. Some aspects related to the stability of AuNP/peptide conjugates for a potential application in the design of an electrochemical biosensor for pathogen bacteria detection are also discussed. This peptide based nanoparticle assay takes advantage of the dual character of the AuNPs: as carrier of the biorecognition molecule and also as electrocatalytic label, allowing the evaluation of the pathogen bacteria-peptide interaction in a simple and rapid way through the chronoamperometric monitoring of the hydrogen evolution reaction on screen-printed carbon electrodes. The developed proof of concept theranostic system may open the way to therapeutic and biosensing applications with interest for various fields.

Influence of dietary ingredients on in vitro inflammatory response of intestinal porcine epithelial cells challenged by an enterotoxigenic Escherichia coli (K88).

Enterotoxigenic Escherichia coli (ETEC) K88 is the main bacterial cause of diarrhea in piglets around weaning and the adhesion of ETEC to the intestinal mucosa is a prerequisite step for its colonization. In this study, the adhesion of a fimbriated ETEC and a non-fimbriated E. coli (NFEC) to the intestinal cells and the activation of the innate immune system were evaluated using a porcine intestinal epithelial cell line (IPEC-J2). The impact of several feedstuffs (wheat bran (WB); casein glycomacropeptide (CGMP); mannan-oligosaccharides (MOS); locust bean extract (LB) and Aspergillus oryzae fermentation extract (AO)) on ETEC attachment and the inflammatory response were also studied. The gene expression of TLR-4; TLR-5; IL-1ß; IL-8; IL-10 and TNF-α were quantified using Cyclophilin-A, as a reference gene, and related to a non-challenged treatment. The fimbriated strain was markedly better than the non-fimbriated strain at adherence to intestinal cells and inducing an inflammatory response. All the feedstuffs studied were able to reduce the adhesion of ETEC, with the greatest decrease with CGMP or MOS at highest concentration. Regarding the inflammatory response, the highest dose of WB promoted the lowest relative expression of cytokines and chemokines. All tested feedstuffs were able to reduce the adhesion of ETEC to IPEC-J2 and interfere on the innate inflammatory response; however WB should be further studied according to the beneficial results on the intestinal inflammatory process evidenced in this study.

Effect of a microencapsulated feed additive of lactic and formic acid on the prevalence of Salmonella in pigs arriving at the abattoir.

The main objective of this work was to investigate the potential of a microencapsulated mixture of organic acids (formic and lactic acid) added to the feed of fattening pigs to reduce the prevalence and shedding of Salmonella when arriving to the abattoir. Two trials were performed. In Trial 1, 24 fattening pigs received one of three diets: a control diet, the same diet including lactic and formic acid (0.4% each) (non-protected blend; NPB), or a lipid microencapsulated blend (0.14% each acid) (protected blend; PB). After 10 days, digesta samples from various parts of gastrointestinal tract were taken. No changes were detected either in pH, total short chain fatty acids (SCFA), lactic/formic acid concentrations in any of the sections studied or in caecal lactic acid bacteria or enterobacteria. In Trial 2, 261 pigs from a commercial farm were distributed between the three previous diets in the five weeks before slaughtering. At the abattoir, no change was detected in caecal pH, but increased concentrations (p < 0.05) of formic and lactic acid were found with the NPB and PB treatments. The total SCFA concentration was higher with the PB compared to the control diet (p = 0.002) with a lower percentage of branched chain fatty acids. Both acidified diets decreased enterobacteria in the caecum but did not modify lactobacilli. NPB treatment decreased Salmonella seroprevalence (p < 0.001). A significant (p < 0.05) increase in Salmonella faecal shedding was found related to the stress previous to slaughter with the control group and PB, but not in the NPB group. More studies will be needed to confirm the usefulness of protected acids to prevent Salmonella prevalence and shedding at the abattoir.

Evaluation of the inclusion of a mixture of organic acids or lactulose into the feed of pigs experimentally challenged with Salmonella Typhimurium.

A mixture of organic acids and lactulose for preventing or reducing colonization of the gut by Salmonella Typhimurium was evaluated in pigs. A total of 63 4-week-old commercial piglets were randomly distributed into three different experimental dietary groups: a plain diet without additives (PD) and the same diet supplemented with either 0.4% (w/v) formic acid and 0.4% lactic acid (w/v) (AC) or 1% (w/v) lactulose (LC). After 7 days of adaptation, two-thirds of the pigs (14 from each diet) were challenged with a 2-mL oral dose of 10(8)CFU/mL of Salmonella Typhimurium, leaving the remaining animals unchallenged (UC). After 4 and 10 days post-challenge, pigs were euthanized and the ileum and caecum content were aseptically sampled to (a) quantify lactic, formic, and short-chain fatty acids (SCFA), (b) quantify bacterial populations and Salmonella by fluorescence in situ hybridization and (c) qualitatively analyse bacterial populations through denaturing gradient gel electrophoresis (DGGE). Modification of fermentation products and counts of some of the bacterial groups analysed in the challenged pigs receiving the treatments AC and LC were minimal. Treatments only influenced the bacterial diversity after 10 days post-challenge, with AC generating a lower number of DGGE bands than UC (P<0.05). Neither the inclusion of a mixture of 0.4% (w/v) formic and 0.4% (w/v) lactic acids nor of 1% (w/v) lactulose in the feed influenced numbers of Salmonella in the ileum and caecum of experimentally challenged pigs.

Different feed withdrawal times before slaughter influence caecal fermentation and faecal Salmonella shedding in pigs.

The effects of different pre-slaughter feed withdrawal times (FWT) on the gastrointestinal tract (GIT) weight and the gut environment of pigs and Salmonella shedding were investigated. Trial 1 evaluated the effects under experimental conditions (feed withdrawal for 18, 30 and 36 h) and trial 2 under commercial conditions (15 and 30 h). In trial 1, the GIT weight tended to decrease (P=0.07), the caecal pH increased (P<0.0001), short-chain fatty acids (SCFA) decreased (P<0.001) and percentage of branched-chain fatty acids (BCFA) increased as FWT increased. Similar results were observed in trial 2, but Enterobacteriaceae numbers and Salmonella positive pigs tended to increase whereas lactobacilli decreased (P<0.0005) as FWT increased. The increase in FWT involved changes in the gut microbial ecosystem that could be associated with the trend of increased caecal Enterobacteriaceae and Salmonella in faeces, and may represent a higher risk of carcass contamination in cases of laceration of viscera.

In vitro fermentation of carbohydrates by porcine faecal inocula and their influence on Salmonella Typhimurium growth in batch culture systems.

The aim of this study was to evaluate in vitro the influence of fermentable carbohydrates on the activity of porcine microbiota and survival of Salmonella Typhimurium in a batch culture system simulating the porcine hindgut. The carbohydrates tested were xylooligosaccharides, a mixture of fructooligosaccharides/inulin (FIN), fructooligosaccharides (FOS), gentiooligosaccharides (GEO) and lactulose (LAC). These ingredients stimulated the growth of selected Bifidobacterium and Lactobacillus species in pure cultures. In batch cultures, the carbohydrates influenced some fermentation parameters. For example, GEO and FIN significantly increased lactic acids compared with the control (no added carbohydrate). With the exception of LAC, the test carbohydrates increased the production of short-chain fatty acid (SCFA) and modified SCFA profiles. Quantitative analysis of bacterial populations by FISH revealed increased counts of the Bifidobacterium group compared with control and, with exception of FOS, increased Lactobacillus, Leuconostoc and Weissella spp. counts. Salmonella numbers were the lowest during the fermentation of LAC. This work has looked at carbohydrate metabolism by porcine microbiota in a pH-controlled batch fermentation system. It provides an initial model to analyse interactions with pathogens.

Changes in caecal microbiota and mucosal morphology of weaned pigs.

An experiment was designed to monitor the changes in caecal microbiota associated with early weaning. Twelve piglets (20+/-2 days) from six different litters were selected from a commercial source. For the two experimental groups, one animal from each litter was weaned onto a post-weaning diet (W) and the other remained with the sow (S). After 1 week, animals were sacrificed and caecal samples taken. Microbial counts for total bacteria, enterobacteria and lactobacilli populations were determined by quantitative PCR using SYBR Green dye. Microbial profiles were assessed by terminal restriction fragment length polymorphism (t-RFLP). Weaning promoted an increase in the enterobacteria:lactobacilli ratio (0.27 versus 1.67 log/log 16S rRNA gene copy number, P=0.05). Total bacteria and richness of the caecal microbial ecosystem (number of peaks) were similar in both experimental groups (49.3 for S and 53.4 for W, respectively, P=0.22), although the band patterns were clearly grouped in two different clusters by dendogram analysis. Weaning was also associated with a decrease in crypt density, an increase in mytotic index and a decrease in the number of goblet cells. A reduced immunological response was also observed and was manifested by an increase in intraepithelial lymphocytes and lymphocyte density in the lamina propria. Weaning appears to be critical in the establishment of the caecal microbiota in pigs with important changes, particularly in microbial groups and in caecal mucosal architecture.