Gemma González-Ortiz

Lactulose and Lactobacillus plantarum, a Potential Complementary Synbiotic To Control Postweaning Colibacillosis in Piglets

ABSTRACT The potential of a prebiotic oligosaccharide lactulose, a probiotic strain of Lactobacillus plantarum, or their synbiotic combination to control postweaning colibacillosis in pigs was evaluated using an enterotoxigenic Escherichia coli (ETEC) K88 oral challenge. Seventy-two weanlings were fed four diets: a control diet (CTR), that diet supplemented with L. plantarum (2 × 1010 CFU · day−1) (LPN), that diet supplemented with 10 g · kg−1 lactulose (LAC), or a combination of the two treatments (SYN). After 7 days, the pigs were orally challenged. Six pigs per treatment were euthanized on days 6 and 10 postchallenge (PC). Inclusion of lactulose improved the average daily gain (ADG) (P < 0.05) and increased lactobacilli (P < 0.05) and the percentage of butyric acid (P < 0.02) in the colon. An increase in the ileum villous height (P < 0.05) and a reduction of the pig major acute-phase protein (Pig-MAP) in serum (P < 0.01) were observed also. The inclusion of the probiotic increased numbers of L. plantarum bacteria in the ileum and colon (P < 0.05) and in the total lactobacilli in the colon and showed a trend to reduce diarrhea (P = 0.09). The concentrations of ammonia in ileal and colonic digesta were decreased (P < 0.05), and the villous height (P < 0.01) and number of ileal goblet cells (P < 0.05) increased, at day 10 PC. A decrease in plasmatic tumor necrosis factor alpha (TNF-α) (P < 0.01) was also seen. The positive effects of the two additives were combined in the SYN treatment, resulting in a complementary synbiotic with potential to be used to control postweaning colibacillosis.

Screening of extracts from natural feed ingredients for their ability to reduce enterotoxigenic Escherichia coli (ETEC) K88 adhesion to porcine intestinal epithelial cell-line IPEC-J2.

Enterotoxigenic Escherichia coli (ETEC) K88 is the most prevalent enteropathogen in weaned piglets, with the ability to express fimbria F4 and specifically attach to intestinal receptors in the young piglet. The prevention of ETEC K88 adhesion to the epithelium by interfering in this fimbria-receptor recognition provides an alternative approach to prevent the initial stage of disease. The aim of this study is to screen, among different feed ingredients (FI), their ability to reduce ETEC K88 attachment to the porcine intestinal epithelial cell-line (IPEC-J2). The selected FI consisted of products of a vegetable or dairy origin, and microbial by-products, which could be suitable to be included in piglets diet. Incubation of a mixture of each FI extract with the bacteria on IPEC-J2 monolayer was allowed. After washing with PBS to remove the non-adhered bacteria, the culture medium was added to grow the adhered bacteria and, simultaneously, to keep the cells alive. Then, the bacterial growth was monitored in a spectrophotometer reader for 12h. Casein glycomacropeptide (CGMP), locust bean (LB), exopolysaccharide (EPS) and wheat bran (WB) reduced the number of attached ETEC K88 to IPEC-J2, but no anti-adhesive effect was found for soybean hulls, sugar-beet pulp, locust gum, fructooligosaccharides, inulin, mushroom, mannanoligosaccharides or the fermented product from Aspergillus oryzae. The lineal analysis of dose responses demonstrated lineal activity (P<0.0001) for CGMP, LB, EPS and WB. These in vitro results suggest CGMP, LB, EPS and WB as good candidates to be included in piglets diet with supported functional activity against colibacillosis.

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.

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.

Effects of dietary supplementation of Bacillus amyloliquefaciens CECT 5940 and Enterococcus faecium CECT 4515 in adult healthy dogs

The aim of this study was to evaluate the effect of a probiotic preparation consisting of two probiotic strains, Bacillus amyloliquefaciens CECT 5940 and Enterococcus faecium CECT 4515 (each 5 · 108 CFU/g feed), on faecal consistency, faecal microbiology and nutrient digestibility in adult healthy dogs. Sixteen beagles (eight males and eight females) were divided into two groups: the Control group (CON), which was fed the basal diet, and the probiotic group (PRO), which received the basal diet supplemented daily with 1 · 108 CFU for 39 consecutive days. Faecal score was assessed before (BS) and throughout the supplementation period (SP). Fresh faecal samples were collected before supplementation, before finishing the supplementation period and after 6 days of withdrawal for microbial enumeration and pH measurement. During the supplementation period, a digestibility trial was performed. There were no differences in faecal scores or the digestibility coefficients between groups. Between groups no statistical differences were found in most microbiota analysed or in faecal pH. However, during the supplementation period, pathogenic clostridia dropped significantly in Group PRO (5.64 vs. 2.94 ± 0.53 CFU/g faeces; p < 0.001), when compared with the period BS. The use of the probiotic preparation had no impact on nutrient digestibility by adult healthy dogs; however, it could stabilise faecal microbiota by decreasing pathogenic clostridia.

A Proteinaceous Fraction of Wheat Bran May Interfere in the Attachment of Enterotoxigenic E. Coli K88 (F4+) to Porcine Epithelial Cells

Wheat bran (WB) from Triticum aestivum has many beneficial effects on human health. To the best of our knowledge, very little has been published about its ability to prevent pathogenic bacterial adhesion in the intestine. Here, a WB extract was fractionated using different strategies, and the obtained fractions were tested in different in vitro methodologies to evaluate their interference in the attachment of enterotoxigenic Escherichia coli (ETEC) K88 to intestinal porcine epithelial cells (IPEC-J2) with the aim of identifying the putative anti-adhesive molecules. It was found that a proteinaceous compound in the >300-kDa fraction mediates the recognition of ETEC K88 to IPEC-J2. Further fractionation of the >300-kDa sample by size-exclusion chromatography showed several proteins below 90 kDa, suggesting that the target protein belongs to a high-molecular-weight (MW) multi-component protein complex. The identification of some relevant excised bands was performed by mass spectrometry (MS) and mostly revealed the presence of various protease inhibitors (PIs) of low MW: Serpin-Z2B, Class II chitinase, endogenous alpha-amylase/subtilisin inhibitor and alpha-amylase/trypsin inhibitor CM3. Furthermore, an incubation of the WB extract with ETEC K88 allowed for the identification of a 7S storage protein globulin of wheat, Globulin 3 of 66 kDa, which may be one of the most firmly attached WB proteins to ETEC K88 cells. Further studies should be performed to gain an understanding of the molecular recognition of the blocking process that takes place. All gathered information can eventually pave the way for the development of novel anti-adhesion therapeutic agents to prevent bacterial pathogenesis.

Response of broiler chickens fed wheat-based diets to xylanase supplementation

&NA; The objective of this study was to evaluate the effect of xylanase supplementation on performance, footpad score (FPS), nutrient digestibility, and intestinal morphometry in broiler chickens. Two‐hundred‐eighty‐eight Ross 308 broiler chicks (one d old) were placed in one of 3 experimental treatments: positive control (PC), negative control (NC) (−150 kcal/kg), and XYL (NC supplemented with xylanase). Each treatment had 8 replicates with 12 animals each. Starter (zero to 21 d) and grower (21 to 42 d) diets, based on wheat and soy‐bean meal, were available ad libitum. Body weight gain (BWG) and feed intake were measured, and mortality corrected feed conversion ratio (FCR) was calculated. The relative weights of the empty gastrointestinal tract compartments and FPS were recorded on d 21 and d 42. On d 42, ileal contents were collected for nutrient digestibility determination. Statistical comparisons were performed using one‐way ANOVA (JMP Pro 12). The reduction of energy resulted in lighter birds at the end of the study (PC: 2,710 vs. NC: 2,546 g; P = 0.030) whereas xylanase supplementation numerically increased BWG by 84 g (P = 0.229) and improved FCR by 12 points (P = 0.145) compared with the NC. Significant differences in FPS were observed on d 21. Score 0 (no lessions) was predominant in PC and XYL treatments, while score 1 (mild lesions) had a higher frequency in NC birds. Xylanase supplementation numerically increased organic matter (5.9%) and energy (4.7%) utilization with values above those observed with the NC. No treatment effects were observed in any of the morphometric measurements, with the exception of the gizzard (P = 0.036) and the ileum (P = 0.088) on d 42. Xylanase resulted in higher relative gizzard weights compared to NC (P = 0.102). Supplementation of broiler diets with xylanase tended to influence performance, which may be due to a better utilization of nutrients. The increase in gizzard and ileum weights in birds 42 d but not 21 d of age suggests an adaptive response that takes time to develop.

Preliminary study: voluntary food intake in dogs during tryptophan supplementation.

Tryptophan, a precursor of important molecules such as serotonin, melatonin and niacin, is an essential amino acid for dogs. In pigs, tryptophan supplementation has been shown to induce a significant increase in food intake. The aim of the present study was to assess whether long-term tryptophan supplementation increases voluntary food intake in dogs and to observe whether this was accompanied by a change in serum ghrelin. In the present study, sixteen adult Beagle dogs were used, with four male and four female dogs fed diets supplemented with tryptophan (1 g/dog per d) during 81 d (Trp) and four male and four female dogs that were not supplemented (control). A voluntary food intake test was performed during 5 d following the supplementation period. The Trp group tended to show a higher food intake during the voluntary food intake test (58.0 (SE 5.37) v. 77.5 (SE 3.65) g/kg metabolic weight per d; P = 0.074). No significant differences were found for serum ghrelin concentrations.