G. Q. Zhu

Genetic variation at the alpha-1-fucosyltransferase (FUT1) gene in Asian wild boar and Chinese and Western commercial pig breeds.

Escherichia coli F18 bacteria producing enterotoxins and/or shigatoxin (ETEC/STEC) are main pathogens that cause oedema disease and postweaning diarrhoea in piglets, and alpha-1-fucosyltransferase (FUT1) gene has been identified as a candidate gene for controlling the expression of ETEC F18 receptor. The genetic variations at nucleotide position 307 in open reading frame of FUT1 gene in one wild boar breed and 20 western commercial and Chinese native pig breeds were investigated by polymerase chain reaction-restriction fragment length polymorphism. The results showed that the genetic polymorphisms of the FUT1 locus were only detected in western pig breeds and the Chinese Taihu (including Meishan pig, Fengjing pig and Erhualian pig), Huai and Lingao pig breeds; only Duroc and Pietrain possessed the resistant AA genotype, while the wild boar and other Chinese pig breeds only presented the susceptible genotype GG. The results indicated that Chinese native pig breeds lack genetic factors providing resistance to ETEC F18 bacteria. The resistant allele to ETEC F18 might originate from European wild boar. It was inferred that oedema and postweaning diarrhoea caused by ETEC F18 have close relationship with the growth rate, which can explain why on the contrary Chinese native pig breeds have stronger resistance to oedema and postweaning diarrhoea in piglets compared with western pig breeds.

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.

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.

Differential expression of genes BPI , TAP1 , SLA-1 and SLA-3 in Escherichia coli F18-resistant and sensitive Meishan post-weaning piglets

The genetic basis and mechanism of sensitivity or resistance to the significant pathogen Escherichia coli F18 is not clear in native Chinese pig breeds and may differ from that in non-native breeds. Our previous research showed that the genes BPI, TAP1, SLA-1 and SLA-3 may play important roles in resistance to E. coli F18 in post-weaning piglets. This study was based on success in selecting and identifying full sib Chinese native Meishan breed post-weaning E. coli F18-resistant and sensitive piglets. Real-time PCR was used to detect expression levels of the genes BPI, TAP1, SLA-1 and SLA-3 in liver, spleen, thymus, lymph node, duodenum, and jejunum tissues between E. coli F18-resistant and -sensitive Meishan piglets. Only the BPI gene was obviously tissue specific; it was highly expressed in the duodenum and jejunum, but expressed at a low level in other tissues. The other three genes were expressed in all the studied tissues, and particularly highly in immune organs and intestinal tissues. The expression level of BPI in the duodenum of the resistant group was significantly higher than that in the sensitive group (P < 0.01). The SLA-3 expression level in the thymus of the resistant group was significantly higher than that in the sensitive group (P < 0.05). TAP1 and SLA-1 gene expression levels were generally higher in the resistant group than the sensitive group, but there were no significant differences. Genes BPI and SLA-3 play an important role in the processes of resistance to E. coli F18 in Meishan weaned piglets. We speculate that BPI protein may have a direct killing effect on Gram-negative bacteria such as E. coli strain F18 in the intestine; the resistance of Meishan weaned piglets to E. coli F18 is likely to be related to the upregulation of intestinal BPI. The upregulation of SLA-3 may increase the resistance of weaned piglets to E. coli F18 by regulation of the immune response.

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.’’

Genetic Diversity, Genetic Distance and Phylogeny of Functional ApoVLDL-II and Lipoprotein Lipase Genes

Abstract Musa, H.H., Bao, W.B., Wang, K.H., Chen, G.H. and Zhu, G.Q. 2008. Genetic diversity, genetic distance and phylogeny of functional apoVLDL-II and lipoprotein lipase genes. J. Appl. Anim. Res., 34: 143–147. Five chicken populations including Anka, Rugao, Wenchang, Silkies and Red jungle fowl were used to study the genetic diversity of functional apoVLDL-II and lipoprotein lipase genes. Mutation was detected by PCR-RFLP and PCR-SSCP, thereafter, SNP was screened by direct gene sequence. The genetic diversity and distance were analyzed by POPGENE and the phylogeny tree was constructed by PHYLIP. Anka showed the highest genetic diversity, effective number of allele and Shannons information index among populations studied. Anka and Silkies observed the highest genetic distance. In addition, Red jungle fowl was more closely and genetically related to Wenchang than other populations, followed by Silkies, Rugao and Anka. From this study we concluded that the phylogenic tree developed from the molecular marker provided enough information to predict the likely originator and the genetic distances existing among the five chicken populations.