Andrew G. Van Kessel

Association of a missense mutation in the bovine leptin gene with carcass fat content and leptin mRNA levels

Previously, we have shown that alleles of the BM1500 microsatellite, located 3.6 kb downstream of the leptin gene in cattle, were associated with carcass fat measures in a population of 154 unrelated beef bulls. Subsequently, a cytosine (C) to thymine (T) transition that encoded an amino acid change of an arginine to a cysteine was identified in exon 2 of the leptin gene. A PCR-RFLP was designed and allele frequencies in four beef breeds were correlated with levels of carcass fat. The T allele was associated with fatter carcasses and the C allele with leaner carcasses. The frequencies of the SNP alleles among breeds indicated that British breeds have a higher frequency of the T allele whereas the continental breeds have a higher occurrence of the C allele. A ribonuclease protection assay was developed to quantify leptin mRNA in a separate group of animals selected by genotype. Animals homozygous for thymine expressed higher levels of leptin mRNA. This may suggest that the T allele, which adds an extra cysteine to the protein, imparts a partial loss of biological function and hence could be the causative mutation.

Characterization of intestinal microbiota and response to dietary virginiamycin supplementation in the broiler chicken.

ABSTRACT The inclusion of antibiotic growth promoters, such as virginiamycin, at subtherapeutic levels in poultry feeds has a positive effect on health and growth characteristics, possibly due to beneficial effects on the host gastrointestinal microbiota. To improve our understanding of the chicken gastrointestinal microbiota and the effect of virginiamycin on its composition, we characterized the bacteria found in five different gastrointestinal tract locations (duodenal loop, mid-jejunum, proximal ileum, ileocecal junction, and cecum) in 47-day-old chickens that were fed diets excluding or including virginiamycin throughout the production cycle. Ten libraries (five gastrointestinal tract locations from two groups of birds) of approximately 555-bp chaperonin 60 PCR products were prepared, and 10,932 cloned sequences were analyzed. A total of 370 distinct cpn60 sequences were identified, which ranged in frequency of recovery from 1 to 2,872. The small intestinal libraries were dominated by sequences from the Lactobacillales (90% of sequences), while the cecum libraries were more diverse and included members of the Clostridiales (68%), Lactobacillales (25%), and Bacteroidetes (6%). To assess the effects of virginiamycin on the gastrointestinal microbiota, 15 bacterial targets were enumerated using quantitative, real-time PCR. Virginiamycin was associated with increased abundance of many of the targets in the proximal gastrointestinal tract (duodenal loop to proximal ileum), with fewer targets affected in the distal regions (ileocecal junction and cecum). These findings provide improved profiling of the composition of the chicken intestinal microbiota and indicate that microbial responses to virginiamycin are most significant in the proximal small intestine.

Immunomodulatory Activities of Oat β-Glucan In Vitro and In Vivo

Previous studies have shown that β‐glucans extracted from yeast or fungi potentiate immune responses. In the present study, the immunomodulatory activities of β‐(1→3, 1→4)‐glucan, derived from oats, were investigated. The ability of oat β‐glucan (OβG) to stimulate IL‐1 and TNF‐α release from murine peritoneal macrophages and the murine macrophage cell line P338D1, was assessed. In vitro stimulation of macrophages with OβG resulted in the production of IL‐1 in a dose and time‐dependent manner, whereas only small amounts of TNF‐α could be detected in the culture supernatants. OβG also induced the production of IL‐2, IFN‐γ and IL‐4 secretion in a dose‐dependent manner in cultured spleen cells. The intraperitoneal administration of OβG in mice resulted in the accumulation of leucocytes, predominantly macrophages, in the peritoneal cavity. Furthermore, OβG was tested for its ability to enhance non‐specific resistance to a bacterial challenge in mice. Survival of mice challenged with Staphylococcus aureus was enhanced by a single intraperitoneal administration of 500 μg of OβG 3 days prior to bacterial challenge. In conclusion, these studies demonstrated that OβG possesses immunomodulatory activities capable of stimulating immune functions both in vitro and in vivo.

Extensive Profiling of a Complex Microbial Community by High-Throughput Sequencing

ABSTRACT Complex microbial communities remain poorly characterized despite their ubiquity and importance to human and animal health, agriculture, and industry. Attempts to describe microbial communities by either traditional microbiological methods or molecular methods have been limited in both scale and precision. The availability of genomics technologies offers an unprecedented opportunity to conduct more comprehensive characterizations of microbial communities. Here we describe the application of an established molecular diagnostic method based on the chaperonin-60 sequence, in combination with high-throughput sequencing, to the profiling of a microbial community: the pig intestinal microbial community. Four libraries of cloned cpn60 sequences were generated by two genomic DNA extraction procedures in combination with two PCR protocols. A total of 1,125 cloned cpn60 sequences from the four libraries were sequenced. Among the 1,125 cloned cpn60 sequences, we identified 398 different nucleotide sequences encoding 280 unique peptide sequences. Pairwise comparisons of the 398 unique nucleotide sequences revealed a high degree of sequence diversity within the library. Identification of the likely taxonomic origins of cloned sequences ranged from imprecise, with clones assigned to a taxonomic subclass, to precise, for cloned sequences with 100% DNA sequence identity with a species in our reference database. The compositions of the four libraries were compared and differences related to library construction parameters were observed. Our results indicate that this method is an alternative to 16S rRNA sequence-based studies which can be scaled up for the purpose of performing a potentially comprehensive assessment of a given microbial community or for comparative studies.

Comparison of Ileum Microflora of Pigs Fed Corn-, Wheat-, or Barley-Based Diets by Chaperonin-60 Sequencing and Quantitative PCR

ABSTRACT We have combined the culture-independent methods of high-throughput sequencing of chaperonin-60 PCR product libraries and quantitative PCR to profile and quantify the small-intestinal microflora of pigs fed diets based on corn, wheat, or barley. A total of 2,751 chaperonin-60 PCR product clones produced from samples of ileum digesta were examined. The majority (81%) of these clones contained sequences independently recovered from all three libraries; 372 different nucleotide sequences were identified, but only 14% of the 372 different sequences were recovered from all three libraries. Taxonomic assignments of the library sequences were made by comparison to a reference database of chaperonin-60 sequences combined with phylogenetic analysis. The taxa identified are consistent with previous reports of pig ileum microflora. Frequencies of each sequence in each library were calculated to identify taxa that varied in frequency between the corn, barley, and wheat libraries. The chaperonin-60 sequence inventory was used as a basis for designing PCR primer sets for taxon-specific quantitative PCR. Results of quantitative PCR analysis of ileum digesta confirmed the relative abundances of targeted taxa identified with the library sequencing approach. The results of this study indicate that chaperonin-60 clone libraries can be valid profiles of complex microbial communities and can be used as the basis for producing quantitative PCR assays to measure the abundance of taxa of interest during experimentally induced or natural changes in a community.

β-Glucan, extracted from oat, enhances disease resistance against bacterial and parasitic infections

The effect of beta-glucan, extracted from oats, on the enhancement of resistance to infections caused by Staphylococcus aureus and Eimeria vermiformis was studied in mice. In vitro study using macrophages isolated from the peritoneal cavity showed that beta-glucan treatment significantly enhanced phagocytic activity. In vivo study further demonstrated that beta-glucan treatment induced a significant (P<0.05) protection against the challenge with 5 x 10(8) of S. aureus in mice. Fecal oocyst shedding in the C57BL/6 mice infected with E. vermiformis was diminished by beta-glucan treatment by 39.6% in intraperitoneal and 28.5% in intragastric group compared to non-treated control. Patency period was shorter and antigen (sporozoites and merozoites) specific antibodies were significantly (P<0.05-0.01) higher in beta-glucan-treated group compared to non-treated control group. There were an increasing number of splenic IFN-gamma-secreting cells in glucan-treated group via intraperitoneal route, which might be responsible for the enhancement of the disease resistance. Glucan treatment was able to effectively change the lymphocytes population (Thy 1.2(+), CD4(+) and CD8(+) cells) in the mesenteric lymph nodes and Peyers patches in mice infected with E. vermiformis. In conclusion, the oral or parenteral oat beta-glucan treatment enhanced the resistance to S. aureus or E. vermiformis infection in the mice.

Nonstarch Polysaccharides Modulate Bacterial Microbiota, Pathways for Butyrate Production, and Abundance of Pathogenic Escherichia coli in the Pig Gastrointestinal Tract†

ABSTRACT The impact of nonstarch polysaccharides (NSP) differing in their functional properties on intestinal bacterial community composition, prevalence of butyrate production pathway genes, and occurrence of Escherichia coli virulence factors was studied for eight ileum-cannulated growing pigs by use of terminal restriction fragment length polymorphism (TRFLP) and quantitative PCR. A cornstarch- and casein-based diet was supplemented with low-viscosity, low-fermentability cellulose (CEL), with high-viscosity, low-fermentability carboxymethylcellulose (CMC), with low-viscosity, high-fermentability oat β-glucan (LG), and with high-viscosity, high-fermentability oat β-glucan (HG). Only minor effects of NSP fractions on the ileal bacterial community were observed, but NSP clearly changed the digestion in the small intestine. Compared to what was observed for CMC, more fermentable substrate was transferred into the large intestine with CEL, LG, and HG, resulting in higher levels of postileal dry-matter disappearance. Linear discriminant analysis of NSP and TRFLP profiles and 16S rRNA gene copy numbers for major bacterial groups revealed that CMC resulted in a distinctive bacterial community in comparison to the other NSP, which was characterized by higher gene copy numbers for total bacteria, Bacteroides-Prevotella-Porphyromonas, Clostridium cluster XIVa, and Enterobacteriaceae and increased prevalences of E. coli virulence factors in feces. The numbers of butyryl-coenzyme A (CoA) CoA transferase gene copies were higher than those of butyrate kinase gene copies in feces, and these quantities were affected by NSP. The present results suggest that the NSP fractions clearly and distinctly affected the taxonomic composition and metabolic features of the fecal microbiota. However, the effects were more linked to the individual NSP and to their effect on nutrient flow into the large intestine than to their shared functional properties.

Transcriptome profiling of the small intestinal epithelium in germfree versus conventional piglets

BackgroundTo gain insight into host-microbe interactions in a piglet model, a functional genomics approach was used to address the working hypothesis that transcriptionally regulated genes associated with promoting epithelial barrier function are activated as a defensive response to the intestinal microbiota. Cesarean-derived germfree (GF) newborn piglets were colonized with adult swine feces, and villus and crypt epithelial cell transcriptomes from colonized and GF neonatal piglets were compared using laser-capture microdissection and high-density porcine oligonucleotide microarray technology.ResultsConsistent with our hypothesis, resident microbiota induced the expression of genes contributing to intestinal epithelial cell turnover, mucus biosynthesis, and priming of the immune system. Furthermore, differential expression of genes associated with antigen presentation (pan SLA class I, B2M, TAP1 and TAPBP) demonstrated that microbiota induced immune responses using a distinct regulatory mechanism common for these genes. Specifically, gene network analysis revealed that microbial colonization activated both type I (IFNAR) and type II (IFNGR) interferon receptor mediated signaling cascades leading to enhanced expression of signal transducer and activator of transcription 1 (STAT1), STAT2 and IFN regulatory factor 7 (IRF7) transcription factors and the induction of IFN-inducible genes as a reflection of intestinal epithelial inflammation. In addition, activated RNA expression of NF-kappa-B inhibitor alpha (NFκBIA; a.k.a I-kappa-B-alpha, IKBα) and toll interacting protein (TOLLIP), both inhibitors of inflammation, along with downregulated expression of the immunoregulatory transcription factor GATA binding protein-1 (GATA1) is consistent with the maintenance of intestinal homeostasis.ConclusionThis study supports the concept that the intestinal epithelium has evolved to maintain a physiological state of inflammation with respect to continuous microbial exposure, which serves to sustain a tight intestinal barrier while preventing overt inflammatory responses that would compromise barrier function.

Effect of barley and oat cultivars with different carbohydrate compositions on the intestinal bacterial communities in weaned piglets.

This experiment was aimed at comparing the intestinal microbial community composition in pigs fed hulled common barley supplemented with isolated barley mixed-linked beta-glucan, four hulless barley varieties and breeding lines with mixed-linked beta-glucan contents ranging from 41 to 84 g kg(-1) and different amylose/amylopectin ratios as well as two oat varieties. Seventy-two weaned piglets were allocated to one of nine diets composed of 81.5% cereal, 6% whey, 9% soy protein isolate and 3.5% minerals. After 15 days, pigs were sacrificed and ileum and colon contents were collected for quantitative real-time PCR (qPCR) and denaturing gradient gel electrophoresis to evaluate microbial communities. Shifts in intestinal microbial communities were observed with the hulless barley cultivars with a normal to high beta-glucan content and from normal starch toward either high-amylopectin or high-amylose starch. These hulless barleys had the lowest (P<0.05) microbial diversity, whereas oats had intermediate diversity compared with low-beta-glucan hulless cultivars and hulled varieties. Furthermore, hulless varieties favoured xylan- and beta-glucan-degrading bacteria whereas mixed-linked beta-glucan-supplemented hulled barley favoured lactobacilli. Numbers of lactobacilli decreased in the ileum of pigs fed hulless/high mixed-linked beta-glucan barley-based diets. Thus, cultivar differences in both the form and the quantity of carbohydrates affect gut microbiota in pigs, which provides information for future feeding strategies.

Fermentable Fiber Ameliorates Fermentable Protein-Induced Changes in Microbial Ecology, but Not the Mucosal Response, in the Colon of Piglets

Dietary inclusion of fermentable carbohydrates (fCHO) is reported to reduce large intestinal formation of putatively toxic metabolites derived from fermentable proteins (fCP). However, the influence of diets high in fCP concentration on epithelial response and interaction with fCHO is still unclear. Thirty-two weaned piglets were fed 4 diets in a 2 × 2 factorial design with low fCP/low fCHO [14.5% crude protein (CP)/14.5% total dietary fiber (TDF)]; low fCP/high fCHO (14.8% CP/16.6% TDF); high fCP low fCHO (19.8% CP/14.5% TDF); and high fCP/high fCHO (20.1% CP/18.0% TDF) as dietary treatments. After 21-23 d, pigs were killed and colon digesta and tissue samples analyzed for indices of microbial ecology, tissue expression of genes for cell turnover, cytokines, mucus genes (MUC), and oxidative stress indices. Pig performance was unaffected by diet. fCP increased (P < 0.05) cell counts of clostridia in the Clostridium leptum group and total short and branched chain fatty acids, ammonia, putrescine, histamine, and spermidine concentrations, whereas high fCHO increased (P < 0.05) cell counts of clostridia in the C. leptum and C. coccoides groups, shifted the acetate to propionate ratio toward acetate (P < 0.05), and reduced ammonia and putrescine (P < 0.05). High dietary fCP increased (P < 0.05) expression of PCNA, IL1β, IL10, TGFβ, MUC1, MUC2, and MUC20, irrespective of fCHO concentration. The ratio of glutathione:glutathione disulfide was reduced (P < 0.05) by fCP and the expression of glutathione transferase was reduced by fCHO (P < 0.05). In conclusion, fermentable fiber ameliorates fermentable protein-induced changes in most measures of luminal microbial ecology but not the mucosal response in the large intestine of pigs.