Incubation of Isolated Wheat Starch with Proteolytic or Lipolytic Enzymes and Different Extraction Media Reveals a Tight Interaction Between Puroindolines and Lipids at Its Granule Surface

Differences in hardness of wheat cultivars have been related to differences in interactions between the starch granule surface and the gluten protein matrix that are mediated by the proteins puroindoline (PIN) A and B. We examined whether or not PINs and (polar) lipids are associated at the starch granule surface, and, if so, how they interact with the starch granule surface itself. Starch was isolated from a soft wheat cultivar containing both wild‐type PINs and incubated with peptidases or lipases, or in extraction media (typically used for defatting). Protein, PIN, and lipid levels revealed that PINs and lipids are tightly associated together at the starch granule surface. Our results imply that PINs need lipids for binding to the granule surface but not vice versa.     

Development of a cytometric bead array screening tool for the simultaneous detection of pro-inflammatory cytokines in porcine plasma

Lipopolysaccharide (LPS) has been widely used as a model of immune challenge in pigs as it induces the immediate synthesis of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6, which trigger the production of the acute phase proteins (APPs) C-reactive protein (CRP), haptoglobin (Hp) and pig-Major Acute Phase Protein (pig-MAP). To measure secreted proteins in porcine plasma, specific and sensitive Enzyme-Linked Immuno Sorbent Assays (ELISAs) are well-suited to perform single parameter analysis, yet this approach is time-consuming and expensive for multi-parameter analyses. During the last decade, multiplex bead-based flow cytometry has been increasingly applied as it offers the opportunity to estimate protein ratios in a small sample volume. Cytometric bead array (CBA) is a flow cytometric application using a diversity of beads with unique fluorescence intensities, covalently coupled to a capture antibody for each protein of interest. Detection antibodies, either directly or indirectly conjugated to a fluorochrome, are added to accomplish the desired sandwich format. The aim of the present study was to develop a CBA 3-plex assay for the major pro-inflammatory cytokines TNF-α, IL-1β and IL-6, and an additional CBA 2-plex assay for the major APPs, CRP and pig-MAP, in porcine plasma. Results were compared to commercial ELISA kits. For the CBA 3-plex assay, the limits of detection (LODs) varied between 0.005 and 0.363 ng/mL, the intra- and inter-assay coefficients of variation were <10% and <16%, respectively. For TNF-α, IL-1β, IL-6 and pig-MAP, CBA time-concentration profiles similar to those obtained with commercial ELISAs were observed. In conclusion, the novel validated CBA 3-plex assay provides a fast and economical screening tool for determination of pro-inflammatory cytokine profiles in limited porcine plasma volumes. This tool will be applied to study the immunomodulatory properties of drugs in a porcine LPS inflammation model. This study also demonstrated the applicability of CBA for measurement of APPs in pigs, although a different combination than pig-MAP with CRP is recommended.     

The search for the gene mutations underlying enterotoxigenic Escherichia coli F4ab/ac susceptibility in pigs: a review

Diarrhoea due to enterotoxigenic Escherichia coli with fimbriae F4 (ETEC-F4) is an important problem in neonatal and just weaned piglets and hence for the pig farming industry. There is substantial evidence for a genetic basis for susceptibility to ETEC-F4 since not all piglets suffer from diarrhoea after an ETEC-F4 infection. It is assumed that the wild boar was originally ETEC-F4 resistant and that susceptibility towards ETEC arose after domestication. There are different phenotypes in the pig determined by which of the three existing F4 variants (F4ab, F4ac or F4ad) they are susceptible or resistant for. This suggests that several F4 receptors exist, expressed individually or in combination with each other on the brush border of the piglet’s small intestine. As such, the mucin-type glycoproteins (IMTGP) are described as F4ab/ac receptors, while the intestinal neutral glycospingolipid (IGLad) is proposed as an F4ad receptor. GP74 is a putative F4ab receptor. However, the specific genes that encode for the susceptibility are not yet known. In the past decades, linkage analyses revealed that the loci encoding for the receptor(s) for the two most frequent variants F4ab and F4ac were mapped to the 13^th chromosome of the pig (Sus scrofa 13, SSC13). After fine mapping, the region of interest was mapped between two microsatellite markers, Sw207 and S0075, and interesting candidate genes surfaced. Numerous SNP analyses and a few expression studies on the three MUC-genes (MUC4, MUC13 and MUC20) and the transferrin receptor gene (TFRC) as well as on some other positional candidate genes have been performed in order to find the causative mutation for the ETEC-F4ab/ac receptor(s). However, until today, the exact mutation causing susceptibility to ETEC-F4 remains unknown.     

Broiler chicken health, welfare and fluctuating asymmetry in organic versus conventional production systems

The aim of this study was to test three predictions: (1) that a combination of animal-based parameters indicates better health and welfare in broilers from organic versus conventional farms, (2) that broilers from organic farms show less fluctuating asymmetry (FA) than broilers from conventional farms, and (3) that, at the level of the individual bird, the relationship between FA and welfare is negative and strongest in conventional broilers. On 140 slaughter-age birds randomly selected from seven organic and seven conventional flocks in Belgium we measured standardised FA and five conventional animal-based welfare indicators (tonic immobility duration, latency-to-lie, and condition of the foot pad, hock, and breast). The caeca from the birds from four organic and four conventional flocks were removed for assessing the presence of two bacterial pathogens (Salmonella and Campylobacter) and the concentration of the health-promoting lactic acid bacteria. Finally, the blood serum concentration of the acute phase protein alpha-1-acid glycoprotein (AGP), a non-specific indicator of immunological stress, was determined. Salmonella was found in the caeca content of two (2.5%) birds only (both from the same conventional farm), whereas 44 (55.7%) birds were infected with Campylobacter. The prevalence of Campylobacter, the concentration of lactic acid bacteria, the duration of tonic immobility, and the condition of the breast and foot pad did not differ between both production systems. Apart from a higher concentration of AGP, organic birds had better scores for hock condition and a longer latency-to-lie indicating better leg health. In addition, organic birds scored better on the aggregated welfare index (i.e. the average of the five standardised welfare indicators). As both production systems differed in many aspects (e.g. slower-growing genotypes, slaughter age, feed, stocking density, group size) it is impossible to assign differences in welfare/health indicators to a single factor. Whatever the causes may be our findings suggest that, despite the potentially elevated risk of immunological challenge, broiler chicken welfare is generally superior in organic farms as compared with conventional farms in Belgium. Regarding the validity of FA as welfare indicator, the prediction of lower FA in the population with highest aggregated welfare score was confirmed but, at the level of the individual, no associations between FA and the aggregated welfare index were found irrespective of whether data from organic and conventional broilers were analysed separately or combined.     

Soil properties influencing the denitrification potential of Flemish agricultural soils

The denitrification potential of the soil horizons between 0- and 90-cm depth of 20 agricultural fields, representative of the most frequent combinations of agricultural crops and soil textures in Flanders (Belgium), and the factors affecting the denitrification potential were studied in the laboratory under controlled conditions. The denitrification potential in the presence of an added soluble C and N source was measured at 15°C after saturation of air-dried soil samples with water. The denitrification potential of the lower horizons was generally negligible compared to the upper horizons. The lower denitrification potential of the deeper horizons could partially be explained by their limited C availability. The denitrification potential of the upper horizons strongly depended on texture. Based on this parameter the soils could be divided into three groups: soils with a high clay content (>30% clay) were characterised by a high denitrification potential (>8.33 µg N g^-1 dry soil day^-1); soils with medium texture had a medium denitrification potential, between 0.41 and 7.25 µg N g^-1 dry soil day^-1; and soils with a high sand content (>80% sand) had a low denitrification potential (<2.58 µg N g^-1 dry soil day^-1). In most cases, extending the saturation period during pre-incubation increased the denitrification potential. Comparison of the denitrification potential of the upper horizons with and without addition of a soluble C source showed that the denitrification potential of the upper horizons of these soils was limited by their percentage of endogenous C. The measured denitrification potentials indicate that denitrification losses in soils high in clay content can be important when NO₃ ^- concentrations are high.     

Supplementing salinomycin to diets for growing-finishing pigs

Feeding experiments with growing-finishing pigs were carried out to study the effect of different dietary salinomycin concentrations upon animal performance. Besides a dietary treatment without antibiotic, three treatments consisted of 20/10 mg/kg, 40/20 mg/kg and 60/30 mg/kg salinomycin (the first and second number refer to the salinomycin content in grower and finisher diet, respectively). Compared to the control group, feeding diets with 20 or 40 mg/kg salinomycin resulted in significantly higher weight gain in the grower period (25-45 kg body weight), while also feed conversion was lower (although not statistically significant). In the finisher period (45-100 kg body weight) salinomycin had no significant effect upon weight gain or feed conversion efficiency, but the 20 and 30 mg/kg treatments yielded the best results. Considering the grower and finisher period together the dietary treatments 40/20 mg/kg and 60/30 mg/kg showed highest growth rate and most favourable feed conversion, although not statistically significant. Carcass characteristics (dressing percentage, carcass length, backfat thickness, percent meat) were not affected by dietary salinomycin supplementation.     

Impact of Puroindolines on Semisweet Biscuit Quality: A Fractionation–Reconstitution Approach

Puroindoline (PIN) proteins are a factor determining wheat kernel endosperm texture. Biscuits are preferably made from flour from soft wheat (Triticum aestivum L.). Such wheat contains relatively high levels of wild‐type PINs, the impact of which on biscuit quality is unclear. We here studied the impact of PINs on biscuit texture using model flour samples reconstituted from starch and gluten fractions with varying PIN levels. These were obtained by fractionating flour from soft or durum wheat containing either wild‐type or no PINs, respectively. This approach allowed largely retaining the interaction between PINs and either starch or gluten, such as it exists in flour. High PIN levels enhanced air incorporation during dough preparation, increased dough (lateral) expansion, and yielded larger biscuits with higher porosity, which was mainly because of the larger pores. Biscuit fracture stress negatively correlated with PIN level. Porosity contributed to biscuit mechanical properties, but PINs also affected biscuit matrix strength, which in turn affected fracture stress. PINs seem to exert their softening effect when present above a threshold level, and then they have a stronger impact on biscuit fracture stress than the wheat endogenous lipids.