Zhangyuan Pan

Microarray analysis of differential gene expression in sensitive and resistant pig to Escherichia coli F18

In this study, Agilent two-colour microarray-based gene expression profiling was used to detect differential gene expression in duodenal tissues collected from eight full-sib pairs of Sutai pigs differing in adhesion phenotype (sensitivity and resistance to Escherichia coli F18). Using a two-fold change minimum threshold, we found 18 genes that were differentially expressed (10 up-regulated and eight down-regulated) between the sensitive and resistant animal groups. Our gene ontology analysis revealed that these differentially expressed genes are involved in a variety of biological processes, including immune responses, extracellular modification (e.g. glycosylation), cell adhesion and signal transduction, all of which are related to the anabolic metabolism of glycolipids, as well as to inflammation- and immune-related pathways. Based on the genes identified in the screen and the pathway analysis results, real-time PCR was used to test the involvement of ST3GAL1 and A genes (of glycolipid-related pathways), SLA-1 and SLA-3 genes (of inflammation- and immune-related pathways), as well as the differential genes FUT1, TAP1 and SLA-DQA. Subsequently, real-time PCR was performed to validate seven differentially expressed genes screened out by the microarray approach, and sufficient consistency was observed between the two methods. The results support the conclusion that these genes are related to the E. coli F18 receptor and susceptibility to E. coli F18.

Investigation of the relationship between SLA-1 and SLA-3 gene expression and susceptibility to Escherichia coli F18 in post-weaning pigs.

Porcine post-weaning diarrhea and edema disease are principally caused by Escherichia coli strains that produce F18 adhesin. FUT1 genotyping and receptor binding studies divided piglets into E. coli F18-resistant and -sensitive groups, and the roles of SLA-1 and SLA-3 were investigated. SLA-1 and SLA-3 expression was detected in 11 pig tissues, with higher levels of SLA-1 in lung, immune tissues and gastrointestinal tract, and higher levels of SLA-3 also in lung and lymphoid tissues. Both genes were expressed higher in F18-resistant piglets, and their expression was positively correlated in different tissues; a negative correlation was observed in some tissues of F18-sensitive group, particularly in lung and lymphatic samples. Gene ontology and pathway analyses showed that SLA-1 and SLA-3 were involved in 37 biological processes, including nine pathways related to immune functions. These observations help to elucidate the relationship between SLA class I genes and E. coli F18-related porcine gastrointestinal tract diseases.

Study on the age-dependent tissue expression of FUT1 gene in porcine and its relationship to E. coli F18 receptor

Escherichia coli (E. coli) that produces adhesin F18 is the main pathogen responsible for porcine post-weaning diarrhea and edema disease. The receptor for E. coli F18 has not been described in pigs, however the alpha (1,2)-fucosyltransferase (FUT1) gene on chromosome 6 has been proposed as a candidate. The objective of this study, therefore, was to investigate the relationship between FUT1 gene expression and E. coli F18 receptor in Sutai pigs of different ages (8-, 18-, 30- and 35-day-old). FUT1 gene expression was detected in 11 pig tissues with the highest level in lung, and expressed consistently at the four time points. In most tissues, FUT1 gene expression levels decreased from days 8 to 18, then continually increased on days 30 and 35, with expression around weaning time higher than that on day 8. Gene ontology and pathway analysis showed that FUT1 was involved in 32 biological processes, mainly those integral to the membrane, or involved in glycosylation, as well as regulation of binding, interestingly participating in three pathways related to glycosphingolipid biosynthesis. From this analysis and the high linkage disequilibrium between the FUT1 gene and the E. coli F18 receptor locus, we can speculate that higher expression of the FUT1 gene in small intestine is beneficial to the formation of receptors to the E. coli F18 strain and is related to the sensitivity to the pathogen.

Beneficial genotype of swine FUT1 gene governing resistance to E. coli F18 is associated with important economic traits.

1Key Laboratory for Animal Genetics, Breeding, Reproduction and Molecular Design of Jiangsu Province, College of Animal Science and Technology, and 2College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, People’s Republic of China 3Suzhou Taihu Pig Breeding Centre, Jiangsu Province, Suzhou, Jiangsu 215000, People’s Republic of China 4Jiangsu Engineering Research Centre for Molecular Breeding of Pig, Changzhou, Jiangsu 213149, People’s Republic of China

The effect of mutation at M307 in FUT1 gene on susceptibility of Escherichia coli F18 and gene expression in Sutai piglets

Escherichia coli F18 (ECF18) is a common porcine enteric pathogen. The pathogenicity of ECF18 bacteria depends on the existence of ECF18 receptor in the brush border membranes of piglet’s small intestinal mucosa. Alpha (1) fucosyltransferase gene (FUT1) has been identified as the candidate gene controlling the adhesion to ECF18 receptor. The genetic variations in the position of M307 nucleotide in open reading frame of FUT1 have been proposed as a marker for selecting resistant pigs. The piglets were divided into three groups, AA, AG and GG, according to the genotypes present at M307 of FUT1. Small intestinal epithelium cells of piglets with AA, AG and GG genotypes were selected to test the adhesion capability of the wild type E.coli expressing F18ab fimbriae, the recombinant E. coli expressing F18ac fimbriae or the recombinant E. coli secreting and surface-displaying the FedF subunit of F18ab fimbriae, respectively. Here, we examined the distribution and expression of porcine FUT1 mRNA in different tissues in Sutai pigs using real-time PCR. The results showed that piglets with AA genotype show resistance, whereas piglets with GG or AG genotypes are sensitive to the pathogenic E. coli F18 in Sutai piglets. FUT1 was expressed in all the tissues that were examined, and the gene’s expression was highest in the lungs. There was no significant difference in expression level among the three genotypes in the liver, lung, stomach and duodenum, where the gene expression was relatively high. The present analysis suggested that mutation at M307 in FUT1 gene determines susceptibility of small intestinal epithelium to E. coli F18 adhesion in Sutai piglet and the expression of FUT1 gene may be regulated by other factors or the mutation was likely to be in linkage disequilibrium with some cis-regulatory variants.

Relationship between the expression level of SLA-DQA and Escherichia coli F18 infection in piglets.

The expression of SLA-DQA was assayed by Real-time PCR to analyze the differential expression between ETEC F18-resistant and -sensitive post-weaning piglets, and then to compare the expression levels of SLA-DQA in 11 different tissues from 8-, 18-, 30- and 35-day-old ETEC F18-resistant piglets, which aimed at discussing the role of SLA-DQA in resistance to ETEC F18. The results showed that SLA-DQA is broadly expressed in 11 tissues with the highest expression level in lymph nodes, and a relatively higher expression level in lung, spleen, jejunum, and duodenum. In tissues of lymph node, lung, spleen, jejunum, and duodenum, the mRNA expression of SLA-DQA in resistant individuals was significantly higher than that in sensitive ones (P<0.05). In most tissues, the expression of SLA-DQA increased from 8 to 18 and 30 days (weaning day), and increased persistently to 35 days of post-weaning. Expression levels of SLA-DQA on 35 days in most tissues were significant higher than that on 8, 18 and 30 days (P<0.05). The results demonstrated that the resistance to ETEC F18 in post-weaning piglets is related to up-regulation of mRNA expression of SLA-DQA to a certain extent. The analysis suggested that SLA-DQA may be not the direct immune factor that resisted the Escherichia coli F18, but perhaps enhanced humoral immunity and cell immunity to reduce the transmembrane signal transduction of ETEC F18 bacterial LPS and then led to the resistance to ETEC F18 in piglets.

Erratum to: The effect of mutation at M307 in FUT1

In our paper that was published online 21 June 2011 in Molecular Biology Reports (The effect of mutation at M307 in FUT1 gene on susceptibility of Escherichia coli F18 and gene expression in Sutai piglets) there is an error in two primers we quoted for detection and quantification of FUT1 gene expression in various pig tissues by qPCR. In the section ‘‘Design and synthesis of the real-time PCR primer’’ the correct text should be ‘‘The length of the amplified FUT1 fragment was 126 bp, with the forward primer being 50-TTTTAAGCCCCCAAACTGCC-30 and the reverse primer being 50-TAAATCGACCCCATCAG CCTC-30.’’

Isolation of new alleles of the swine TLR4 gene and analysis of its genetic variation: Isolation of new alleles of the swine TLR4 gene and analysis of its genetic variation

Using the PCR-SSCP method, the genetic variation in exon 1 of the TLR4 gene was detected among 893 animals, including Asian wild boars, 3 imported commercial and 10 Chinese indigenous swine breeds. This was conducted to analyze the polymorphisms of exon 1 of TLR4 gene in native and foreign pig breeds and aimed at providing a theoretical foundation for further research on the role that TLR4 gene played in immune and defense system. New alleles were isolated for exon 1 of the swine TLR4 gene for the first time, There were 6 genotypes and 3 alleles, in which the Duroc appeared AA, BB, CC, AB, AC and BC genotypes; Sutai pig, which has Duroc pig origin, were detected to be BB, CC, and BC genotypes; Yorkshire and Landrace were detected to be CC and BC genotypes. Wild boar and all 10 Chinese native pig breeds appeared highly conserved in exon 1 of TLR4 gene, with only CC genotype. Among the 3 homozygous genotypes, the CC genotype matches the sequence in GenBank, while a G93C synonymous mutation and a G194A nonsense mutation were found in the BB and AA genotypes, respectively. The correlation between these two mutation points of TLR4 gene with resistance to stress and disease is worthy of further study.Using the PCR-SSCP method, the genetic variation in exon 1 of the TLR4 gene was detected among 893 animals, including Asian wild boars, 3 imported commercial and 10 Chinese indigenous swine breeds. This was conducted to analyze the polymorphisms of exon 1 of TLR4 gene in native and foreign pig breeds and aimed at providing a theoretical foundation for further research on the role that TLR4 gene played in immune and defense system. New alleles were isolated for exon 1 of the swine TLR4 gene for the first time, There were 6 genotypes and 3 alleles, in which the Duroc appeared AA, BB, CC, AB, AC and BC genotypes; Sutai pig, which has Duroc pig origin, were detected to be BB, CC, and BC genotypes; Yorkshire and Landrace were detected to be CC and BC genotypes. Wild boar and all 10 Chinese native pig breeds appeared highly conserved in exon 1 of TLR4 gene, with only CC genotype. Among the 3 homozygous genotypes, the CC genotype matches the sequence in GenBank, while a G93C synonymous mutation and a G194A nonsense mutation were found in the BB and AA genotypes, respectively. The correlation between these two mutation points of TLR4 gene with resistance to stress and disease is worthy of further study.