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Effect of mucin 4 allele on susceptibility to experimental infection with enterotoxigenic F4 Escherichia coli in pigs fed experimental diets.

ABSTRACT: Background:This study investigated the validity of the DNA-marker based test to determine susceptibility to ETEC-F4 diarrhoea by comparing the results of two DNA sequencing techniques in weaner pigs following experimental infection with F4 enterotoxigenic Escherichia coli (ETEC-F4). The effects of diet and genetic susceptibility were assessed by measuring the incidence of piglet post-weaning diarrhoea (PWD), faecal E. coli shedding and the diarrhoea index. Results:A DNA marker-based test targeting the mucin 4 gene (MUC4) that encodes F4 fimbria receptor identified pigs as either fully susceptible (SS), partially or mildly susceptible (SR), and resistant (RR) to developing ETEC-F4 diarrhoea. To further analyse this, DNA sequencing was undertaken, and a significantly higher proportion of C nucleotides was observed for RR and SR at the XbaI cleavage site genotypes when compared to SS. However, no significant difference was found between SR and RR genotypes. Therefore, results obtained from Sanger sequencing retrospectively allocated pigs into a resistant genotype (MUC4-), in the case of a C nucleotide, and a susceptible genotype (MUC4+), in the case of a G nucleotide, at the single nucleotide polymorphism site. A total of 72 weaner pigs (age ~?21?days), weighing 6.1?±?1.2?kg (mean?±?SEM), were fed 3 different diets: (i) positive control (PC) group supplemented with 3?g/kg zinc oxide (ZnO), (ii) negative control (NC) group (no ZnO or HAMSA), and (iii) a diet containing a 50?g/kg high-amylose maize starch product (HAMSA) esterified with acetate. At days five and six after weaning, all pigs were orally infected with ETEC (serotype O149:F4; toxins LT1, ST1, ST2 and EAST). The percentage of pigs that developed diarrhoea following infection was higher (P?=?0.05) in MUC4+ pigs compared to MUC4- pigs (50% vs. 26.8%, respectively). Furthermore, pigs fed ZnO had less ETEC-F4 diarrhoea (P?= 0.009) than pigs fed other diets, however faecal shedding of ETEC was similar (P?>?0.05) between diets. Conclusion:These results confirm that MUC4+ pigs have a higher prevalence of ETEC-F4 diarrhoea following exposure, and that pigs fed ZnO, irrespective of MUC4 status, have reduced ETEC-F4 diarrhoea. Additionally, sequencing or quantifying the single nucleotide polymorphism distribution at the XbaI cleavage site may be more reliable in identifying genotypic susceptibility when compared to traditional methods.

SUBMITTER: Sterndale SO

PROVIDER: S-EPMC6636048 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Effect of mucin 4 allele on susceptibility to experimental infection with enterotoxigenic F4 <i>Escherichia coli</i> in pigs fed experimental diets.

Sterndale Samantha O SO Evans Danica J DJ Mansfield Josephine P JP Clarke Julie J Sahibzada Shafi S Abraham Sam S O'Dea Mark M Miller David W DW Kim Jae Cheol JC Pluske John R JR

Journal of animal science and biotechnology 20190717

<h4>Background</h4>This study investigated the validity of the DNA-marker based test to determine susceptibility to ETEC-F4 diarrhoea by comparing the results of two DNA sequencing techniques in weaner pigs following experimental infection with F4 enterotoxigenic <i>Escherichia coli</i> (ETEC-F4). The effects of diet and genetic susceptibility were assessed by measuring the incidence of piglet post-weaning diarrhoea (PWD), faecal <i>E. coli</i> shedding and the diarrhoea index.<h4>Results</h4>A ...[more]

PMID: 31346463

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Project description:This study aimed to characterize in silico enterotoxigenic Escherichia coli F4- and F18-positive isolates (n = 90) causing swine postweaning diarrhea, including pathogenic potential, phylogenetic relationship, antimicrobial and biocide resistance, prophage content, and metal tolerance rates. F4 strains belonged mostly to the O149 and O6 serogroups and ST100 and ST48 sequence types (STs). F18 strains were mainly assigned to the O8 and O147 serogroups and ST10, ST23, and ST42. The highest rates of antimicrobial resistance were found against streptomycin, sulfamethoxazole, tetracycline, trimethoprim, and ampicillin. No resistance was found toward ciprofloxacin, cefotaxime, ceftiofur, and colistin. Genes conferring tolerance to copper (showing the highest diversity), cadmium, silver, and zinc were predicted in all genomes. Enterotoxin genes (ltcA, 100% F4, 62% F18; astA, 100% F4, 38.1% F18; sta, 18.8% F4, 38.1% F18; stb, 100% F4, 76.2% F18) and fimbria-encoding genes typed as F4ac and F18ac were detected in all strains, in addition to up to 16 other virulence genes in individual strains. Phage analysis predicted between 7 and 20 different prophage regions in each strain. A highly diverse variety of plasmids was found; IncFII, IncFIB, and IncFIC were prevalent among F4 isolates, while IncI1 and IncX1 were dominant among F18 strains. Interestingly, F4 isolates from the early 1990s belonged to the same clonal group detected for most of the F4 strains from 2018 to 2019 (ONT:H10-A-ST100-CH27-0). The small number of single-nucleotide polymorphism differences between the oldest and recent F4 ST100 isolates suggests a relatively stable genome. Overall, the isolates analyzed in this study showed remarkably different genetic traits depending on the fimbria type.IMPORTANCE Diarrhea in the postweaning period due to enterotoxigenic E. coli (ETEC) is an economically relevant disease in pig production worldwide. In Denmark, prevention is mainly achieved by zinc oxide administration (to be discontinued by 2022). In addition, a breeding program has been implemented that aims to reduce the prevalence of this illness. Treatment with antimicrobials contributes to the problem of antimicrobial resistance (AMR) development. As a novelty, this study aims to deeply understand the genetic population structure and variation among diarrhea-associated isolates by whole-genome sequencing characterization. ST100-F4ac is the dominant clonal group circulating in Danish herds and showed high similarity to ETEC ST100 isolates from China, the United States, and Spain. High rates of AMR and high diversity of virulence genes were detected. The characterization of diarrhea-related ETEC is important for understanding the disease epidemiology and pathogenesis and for implementation of new strategies aiming to reduce the impact of the disease in pig production.

Project description:Enterotoxigenic F4-fimbriated Escherichia coli is associated with diarrheal disease in neonatal and postweaning pigs. The F4 fimbriae mediate attachment of the bacteria to the pig intestinal epithelium, enabling an efficient delivery of diarrhea-inducing enterotoxins to the target epithelial cells. There are three variants of F4 fimbriae designated F4ab, F4ac and F4ad, respectively, having different antigenic and adhesive properties. In the present study, the binding of isolated F4ab, F4ac and F4ad fimbriae, and F4ab/ac/ad-fimbriated E. coli, to glycosphingolipids from erythrocytes and from porcine small intestinal epithelium was examined, in order to get a comprehensive view of the F4-binding glycosphingolipids involved in F4-mediated hemagglutination and adhesion to the epithelial cells of porcine intestine. Specific interactions between the F4ab, F4ac and F4ad fimbriae and both acid and non-acid glycosphingolipids were obtained, and after isolation of binding-active glycosphingolipids and characterization by mass spectrometry and proton NMR, distinct carbohydrate binding patterns were defined for each fimbrial subtype. Two novel glycosphingolipids were isolated from chicken erythrocytes, and characterized as GalNAc?3GalNAcß3Galß4Glcß1Cer and GalNAc?3GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer. These two compounds, and lactosylceramide (Galß4Glcß1Cer) with phytosphingosine and hydroxy fatty acid, were recognized by all three variants of F4 fimbriae. No binding of the F4ad fimbriae or F4ad-fimbriated E. coli to the porcine intestinal glycosphingolipids occurred. However, for F4ab and F4ac two distinct binding patterns were observed. The F4ac fimbriae and the F4ac-expressing E. coli selectively bound to galactosylceramide (Galß1Cer) with sphingosine and hydroxy 24:0 fatty acid, while the porcine intestinal glycosphingolipids recognized by F4ab fimbriae and the F4ab-fimbriated bacteria were characterized as galactosylceramide, sulfatide (SO(3)-3Galß1Cer), sulf-lactosylceramide (SO(3)-3Galß4Glcß1Cer), and globotriaosylceramide (Gal?4Galß4Glcß1Cer) with phytosphingosine and hydroxy 24:0 fatty acid. Finally, the F4ad fimbriae and the F4ad-fimbriated E. coli, but not the F4ab or F4ac subtypes, bound to reference gangliotriaosylceramide (GalNAcß4Galß4Glcß1Cer), gangliotetraosylceramide (Galß3GalNAcß4Galß4Glcß1Cer), isoglobotriaosylceramide (Gal?3Galß4Glcß1Cer), and neolactotetraosylceramide (Galß4GlcNAcß3Galß4Glcß1Cer).

Project description:BackgroundExcessive fat accumulation of pigs is undesirable, as it severely affects economic returns in the modern pig industry. Studies in humans and mice have examined the role of the gut microbiome in host energy metabolism. Commercial Duroc pigs are often fed formula diets with high energy and protein contents. Whether and how the gut microbiome under this type of diet regulates swine fat accumulation is largely unknown.ResultsIn the present study, we systematically investigated the correlation of gut microbiome with pig lean meat percentage (LMP) in 698 commercial Duroc pigs and found that Prevotella copri was significantly associated with fat accumulation of pigs. Fat pigs had significantly higher abundance of P. copri in the gut. High abundance of P. copri was correlated with increased concentrations of serum metabolites associated with obesity, e.g., lipopolysaccharides, branched chain amino acids, aromatic amino acids, and the metabolites of arachidonic acid. Host intestinal barrier permeability and chronic inflammation response were increased. A gavage experiment using germ-free mice confirmed that the P. copri isolated from experimental pigs was a causal species increasing host fat accumulation and altering serum metabolites. Colon, adipose tissue, and muscle transcriptomes in P. copri-gavaged mice indicated that P. copri colonization activated host chronic inflammatory responses through the TLR4 and mTOR signaling pathways and significantly upregulated the expression of the genes related to lipogenesis and fat accumulation, but attenuated the genes associated with lipolysis, lipid transport, and muscle growth.ConclusionsTaken together, the results proposed that P. copri in the gut microbial communities of pigs fed with commercial formula diets activates host chronic inflammatory responses by the metabolites through the TLR4 and mTOR signaling pathways, and increases host fat deposition significantly. The results provide fundamental knowledge for reducing fat accumulation in pigs through regulating the gut microbial composition. Video Abstract.

Dataset Information

Effect of mucin 4 allele on susceptibility to experimental infection with enterotoxigenic F4 Escherichia coli in pigs fed experimental diets.

Publications

Effect of mucin 4 allele on susceptibility to experimental infection with enterotoxigenic F4 <i>Escherichia coli</i> in pigs fed experimental diets.

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