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

Samantha O Sterndale,Mark O’Dea,John R Pluske,Danica J Evans,Sam Abraham,Jae Cheol Kim,David W Miller,Josephine P Mansfield,Julie Clarke,Shafi Sahibzada

doi:10.1186/s40104-019-0366-1

Samantha O Sterndale, Mark O’Dea + Show 8 more

Open Access

https://doi.org/10.1186/s40104-019-0366-1

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Abstract

BackgroundThis 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.ResultsA 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.ConclusionThese 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.

Highlights

This study investigated the validity of the DNA-marker based test to determine susceptibility to ETECF4 diarrhoea by comparing the results of two DNA sequencing techniques in weaner pigs following experimental infection with F4 enterotoxigenic Escherichia coli (ETEC-F4)
To further analyse the genotype of each pig, the results obtained by Sanger sequencing and next generation sequencing (NGS) were compared to the PCRRFLP method
The results confirmed that pigs with a C nucleotide by Sanger sequencing had more than 50% C nucleotides via NGS at the mucin 4 (MUC4) site, and were classified as resistant

Summary

Introduction

This study investigated the validity of the DNA-marker based test to determine susceptibility to ETECF4 diarrhoea by comparing the results of two DNA sequencing techniques in weaner pigs following experimental infection with F4 enterotoxigenic Escherichia coli (ETEC-F4). Not all pigs are susceptible to developing an ETEC-F4 infection as this depends, in part, on the presence and amount of F4 receptors found in the brush border membrane of the small intestine. Piglets identified as susceptible have a sufficient amount of F4 receptors in the small intestine (SI), allowing the ETEC to adhere and develop ETEC-F4 diarrhoea [4, 5]. In experiments that require pigs to be predisposed to developing ETEC-F4 diarrhoea, polymerase chain reaction-restriction fragment length polymorphism testing that targets the MUC4 gene has been used to determine which pigs are susceptible [4, 9]. Limited research has been completed analysing for susceptibility using reads of individual DNA strands within a sample (each strand theoretically representing a single cell), comparing the proportion of G to C alleles and comparing these results to the gel electrophoresis DNA-based marker test as described by [4]

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