苏太猪抗F18+大肠杆菌病基础群FUT1基因多态性及其与生长发育的关联分析

本研究采用PCR-RFLP方法对苏太猪F18+大肠杆菌抗病育种基础群α-(1,2)岩藻糖转移酶基因1（FUT1）M307位点多态性进行了分析,并探讨了其与部分生长发育性能的相关性。苏太猪F18⁺大肠杆菌抗病育种基础群FUT1基因M307位点经Hin6Ⅰ酶切后,产生AA、AG和GG 3种基因型,频率分别为0.235、0.609和0.156,群体极显著的偏离了Hardy-Weinberg平衡状态(P<0.01)。总体上AA基因型的个体生长发育优于AG和GG基因型的个体,其中AA基因型个体的60 kg体长显著高于AG和GG基因型的个体(P<0.05),断奶重显著高于GG基因型的个体(P<0.05),体高和后躯宽显著高于AG基因型的个体(P<0.05)。试验结果表明,苏太猪抗病育种基础群中FUT1基因第307位点的AA基因型不仅对仔猪断奶后水肿病和腹泻病具有抗性,而且具有较高的早期生长发育性能。     

苏太猪抗F18+大肠杆菌病基础群FUT1基因多态性及其与生长发育的关联分析

本研究采用PCR-RFLP方法对苏太猪F18+大肠杆菌抗病育种基础群α-(1,2)岩藻糖转移酶基因1（FUT1）M307位点多态性进行了分析,并探讨了其与部分生长发育性能的相关性。苏太猪F18⁺大肠杆菌抗病育种基础群FUT1基因M307位点经Hin6Ⅰ酶切后,产生AA、AG和GG 3种基因型,频率分别为0.235、0.609和0.156,群体极显著的偏离了Hardy-Weinberg平衡状态(P<0.01)。总体上AA基因型的个体生长发育优于AG和GG基因型的个体,其中AA基因型个体的60 kg体长显著高于AG和GG基因型的个体(P<0.05),断奶重显著高于GG基因型的个体(P<0.05),体高和后躯宽显著高于AG基因型的个体(P<0.05)。试验结果表明,苏太猪抗病育种基础群中FUT1基因第307位点的AA基因型不仅对仔猪断奶后水肿病和腹泻病具有抗性,而且具有较高的早期生长发育性能。     

苏太仔猪FUT1基因M307位点多态性与F18大肠杆菌抗病相关性的体外鉴定

采用PCR-RFLP方法检测了江苏苏太断奶仔猪FUT1基因M307位点等位基因多态性分布,在所检的49头仔猪中,GG基因型个体16头,AG基因型19头,AA基因型14头。在此基础上,制备上述不同基因型个体仔猪小肠上皮细胞,分别与表达F18ab菌毛的野生型大肠杆菌、表达F18ac菌毛含fed操纵子全基因的重组大肠杆菌和V型系统表面分泌表达F18abFedF亚单位的重组大肠杆菌进行体外黏附试验和黏附抑制试验。研究结果表明:FUT1基因M307位点中GG型和AG型仔猪小肠上皮细胞均能黏附上述3种大肠杆菌,而AA型个体小肠上皮细胞则不能黏附。将上述3种大肠杆菌分别与抗F18ab菌毛高免血清、F18ac菌毛高免血清及抗F18abFedF亚单位单因子血清作用后,则失去黏附仔猪肠上皮细胞能力。上述结果对苏太猪从体外试验上证明了FUT1基因M307位点多态性与断奶仔猪腹泻和水肿病存在着直接的相关性。     

苏太猪FUT1基因多态性及其与生产性能的关联分析

采用PCR-RFLP方法对苏太猪FUT1基因多态性进行分析。结果表明:苏太猪FUT1基因开放阅读框307位点经Hin6Ⅰ酶切后,产生GG型和AG型,不存在抗性纯合子AA型。分析这两种基因型与苏太猪生产性能间的相关性,结果表明:AG型个体平均生产性能均高于GG型个体,且AG型个体的1胎断奶窝重、2胎断奶窝重、3胎总产仔数和6胎断奶窝重显著(P<0.05)高于GG型个体,对一些生产性状而言,AG型是有益基因型。     

杜洛克猪FUT1基因M229与M307位点多态性及连锁不平衡分析

研究表明,FUT1基因M229和M307位点皆为断奶仔猪抗F18大肠杆菌重要变异位点。本试验采用PCRSSCP和PCR-RFLP方法分别对171头杜洛克猪群体M229和M307位点多态性进行分析,结果表明:M229和M307位点均检测到3种基因型,其中M307位点AA、AG、GG基因型个体数分别为47、100、24头,基因型频率分别为0.275、0.585、0.140,等位基因A为优势基因;M229位点CC、CT、TT基因型个体数分别为90、74、7头;基因型频率分别为0.526、0.433、0.041,等位基因C为优势基因;两种基因型都呈现交叉分布,其中M307位点AA基因型个体中M229位点CC/CT基因型分布较多,TT基因型分布比例较低。通过Haploview软件分析M229和M307位点之间的连锁不平衡水平,结果发现FUT1基因M229和M307位点之间的r2值为0.46,呈现较低连锁不平衡水平。因此,今后在对杜洛克猪抗F18大肠杆菌育种工作中,可以尝试对FUT1基因M307和M229位点同时选育,以期为进一步确证这两个位点的遗传效应及利用其开展抗病育种选育提供一定的试验依据。     

军牧1号白猪FUT1基因M307位点多态性研究

肠毒素大肠杆菌F18是引起断奶仔猪腹泻和仔猪水肿病的主要病原菌,α-1岩藻糖转移酶基因FUT1是控制大肠杆菌F18侵染猪小肠的受体蛋白表达的候选基因。采用PCR—RFLP方法对军牧1号白猪FUTl基因M307核苷酸G／A突变位点的多态性进行分析。结果表明,军牧1号白猪FUT1基因M307位点存在3种基因型,分布趋势为AG〉AA〉GG,等位基因A的频率为0．6048,为优势等位基因。X^2适合性检验结果表明,军牧1号白猪FUT1基因在M307位点上呈Hardy—Weinberg平衡状态。研究结果揭示军牧1号白猪有抵抗肠毒素大肠杆菌F18的遗传基础,在军牧1号白猪群体中可以通过标记辅助的方法培育对大肠杆菌F18具有抗性的新品系。     

F18大肠杆菌抗性型与敏感型苏太断奶仔猪十二指肠组织比较转录组分析

旨在揭示培育品种—苏太猪(杜洛克×梅山猪)F18大肠杆菌抗性的调控通路和重要候选基因,同时进一步探究中外猪品种F18大肠杆菌抗性调控遗传基础的差异。本研究以课题组前期获得的苏太猪和梅山猪F18大肠杆菌抗性型与敏感型断奶仔猪全同胞个体为研究对象,通过转录组测序筛选苏太猪F18大肠杆菌抗性相关的调控通路以及重要候选基因,并在细胞水平,利用qPCR和Western blot分析F18大肠杆菌刺激小肠上皮细胞IPEC-J2后重要候选基因(蛋白)的表达变化,同时利用qPCR检测重要候选基因在苏太和梅山断奶仔猪F18大肠杆菌抗性型与敏感型个体十二指肠组织中的差异表达情况。结果显示:1)在苏太断奶仔猪F18大肠杆菌抗性型与敏感型个体中筛选出238个差异表达基因,主要参与Toll样受体信号通路(toll-like receptor signaling pathway)和糖脂类通路(glycosphingolipid biosynthesis-lacto and neolacto series),其中TLR5、IL-1β、FUT2为重要候选基因;2)不同血清型F18大肠杆菌(F18ac、F18ab)菌体分别刺激小肠上皮细胞IPEC-J2后,FUT2、IL-1β、TLR5基因mRNA表达水平显著上调(P0.05),并且其蛋白表达水平也表现为明显的上调,由此表明,TLR5、IL-1β和FUT2在断奶仔猪F18大肠杆菌感染过程中发挥重要的调控作用;3)组织差异表达分析显示,TLR5、IL-1β和FUT2在苏太猪抗性型与敏感型个体十二指肠组织中表达差异均达到显著水平(P0.05),而梅山猪中TLR5、IL-1β表达差异达到极显著水平(P0.01),FUT2表达水平差异不显著(P0.05)。结合课题组前期关于梅山猪及外来引进品种F18大肠杆菌抗性相关分子机制研究及报道,本研究进一步证明中外猪品种F18大肠杆菌抗性调控的遗传基础确实存在差异,Toll样受体信号通路及CD14、TLR5等基因可能在中国地方品种—梅山猪抵抗F18大肠杆菌感染过程中发挥着免疫调控作用,而鞘糖脂合成通路及FUT2等基因可能在外来猪品种F18大肠杆菌受体形成过程中起关键作用。     

杜洛克FUT1基因多态性及其与产仔性能的关联分析

采用PCR-RFLP方法对杜洛克FUT1基因多态性进行分析。结果发现:杜洛克FUT1基因开放阅读框307位点经Hin6Ⅰ酶切后,检测到AA、AG和GG3种基因型,AA基因型的频率为0.157,A基因的频率为0.353。分析这3种基因型与杜洛克产仔性能间的关联性。结果表明:抗性纯合子AA基因型的个体1～5胎每个胎次的产仔数均值均高于AG和GG基因型,且第1胎和第4胎的产仔数均值显著高于AG和GG基因型(P<0.05),AA基因型对产仔性能而言是有益基因型。     

FUT1基因在上海地区5个猪群体内的多态性分析

仔猪腹泻和水肿病主要是由肠毒素型大肠杆菌的特定抗原F18的不同血清型引起的。α-1岩藻糖转移酶基因(FUT1)可作为F18介导的黏附呈敏感性或抗性遗传性状的一个候选基因。试验采用PCR-RFLP技术分析FUT1基因多态性,并检测其在5个猪群体:杜洛克猪(77头)、长白猪(47头)、大白猪(144头)、申农猪(72头)、梅山猪(69头)的基因型和基因频率。结果表明,FUT1基因在杜洛克猪、大白猪、申农猪群体内检测出AA、AG和GG3种基因型。在杜洛克群体内的基因型频率为AA(0.16)、AG(0.40)、GG(0.44);在大白猪群体内基因型频率为AA(0.03)、AG(0.40)、GG(0.57);在申农猪群体内基因型频率为AA(0.01)、AG(0.25)、GG(0.74);在长白猪群体内只检测出AG(0.21)和GG(0.79),没有AA抗病型;在梅山猪群体中检测的所有样品为GG型。     

致病性F18大肠杆菌黏附素受体易感性仔猪的体外鉴定

在PCR-RFLP方法分析了不同猪个体FUT1基因M307位点等位基因多态性的基础上，制备M307位点为GG和AG2种类型仔猪小肠上皮细胞分别与表达F18ab菌毛的野生型大肠杆菌、表达F18ac菌毛含fed操纵子全基因的重组大肠杆菌及表面分泌表达F18ab菌毛FedF亚单位的重组大肠杆菌进行体外黏附和黏附抑制试验。结果表明，上述野生菌或重组菌对GG和AG2种基因型的30～35日龄断奶仔猪小肠上皮细胞均具有较好的黏附能力。上述3种大肠杆菌分别与抗F18ab纯菌毛血清、F18ac纯菌毛血清及抗F18ab菌毛FedF亚单位单因子血清作用后．则丢失黏附小肠上皮细胞能力。而GG基因型的3日龄仔猪小肠上皮细胞不能很好的黏附上述野生菌或重组菌．但是可以很好地黏附表达987P菌毛的大肠杆菌。     

莱芜黑猪FUTⅠ基因检测及其与生长性能的关系

本研究采用PCR RFLP法对莱芜黑猪FUTⅠ基因M307位点多态性及其与生长性能的关系进行分析。结果表明：莱芜黑猪FUTⅠ基因M307位点均存在丰富的多态性,而且在该突变位点显著偏离Hardy Weinberg平衡状态（P＜0.01）。3种基因型对莱芜黑猪的初生重、断奶重和70日龄保育重（和断奶仔猪腹泻和水肿病密切相关）等均无显著影响（P＞0.05）。本研究结果提示：肠毒素大肠杆菌 F18抗性基因有可能起源于西方猪种,对莱芜黑猪无显著影响,有必要对其中所具备的遗传抗性做更深入的研究,寻找、定位其相应的QTL和抗性基因。     

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.     

SLA-DQA基因在F18大肠杆菌抗性和敏感性断奶仔猪间的差异表达分析

运用荧光定量PCR方法分析SLA-DQA基因在苏太猪F18大肠杆菌病抗性和敏感性资源群体中各个组织的分布和表达水平,以及在抗性组和敏感组中的表达差异,以探讨SLA-DQA基因在断奶仔猪抗大肠杆菌F18菌株感染中发挥的作用。试验结果显示,SLA-DQA基因在所检测的11个组织中均有表达,并呈现相似的表达规律,在肺、脾和淋巴结中的表达量较高,在空肠、十二指肠和胸腺中也有中度的表达。SLA-DQA基因在抗性组个体各个组织中的表达量普遍高于敏感性个体,而且在肺、脾、淋巴结、空肠和十二指肠5个组织中,SLA-DQA基因在抗性组个体中的表达量显著高于敏感性个体(P<0.05)。由此可见,当断奶仔猪受到大肠杆菌毒素侵扰后,SLA-DQA基因较高的表达量有利于SLA-II类抗原分子的合成,SLA-DQA基因虽然不是针对由大肠杆菌F18菌株造成的断奶仔猪腹泻和水肿病的直接免疫因子,但是在断奶仔猪受大肠杆菌F18菌株病原菌侵袭后引起的一系列生理变化和应答过程中发挥着重要的作用。     

The age-dependent expression of the F18+ E. coli receptor on porcine gut epithelial cells is positively correlated with the presence of histo-blood group antigens

F18(+)Escherichia coli have the ability to colonize the gut and cause oedema disease or post-weaning diarrhoea by adhering to specific F18 receptors (F18R) on the porcine epithelium. Although it is well established that a DNA polymorphism on base pair 307 of the FUT1 gene, encoding an alpha(1,2)fucosyltransferase, accounts for the F18R phenotype, the F18R nature is not elucidated yet. The aim of the present study was to investigate the correlation between the presence of H-2 histo-blood group antigens (HBGAs) or its derivative A-2 HBGAs on the porcine gut epithelium and F18(+)E. coli adherence. A significant positive correlation was found between expression of both the H-2 (r=0.586, P<0.01) and A-2 (r=0.775, P<0.01) HBGAs and F18(+)E. coli adherence after examination of 74 pigs aged from 0 to 23 weeks. The majority of the genetically resistant pigs (FUT1M307(A/A)) showed no HBGA expression (91.7%) and no F18(+)E. coli adherence (83.3%). In addition, it was found that F18R expression levels rise with increasing age during the first 3 weeks after birth and that F18R expression is maintained in older pigs (3-23 weeks old). Taken together, these data suggest that, apart from H-2 HBGAs, A-2 HBGAs might be involved in F18(+)E. coli adherence.     

Host genotype and amoxicillin administration affect the incidence of diarrhoea and faecal microbiota of weaned piglets during a natural multiresistant ETEC infection

Enterotoxigenic Escherichia coli (ETEC) is the aetiological agent of postweaning diarrhoea (PWD) in piglets. The SNPs located on the Mucine 4 (MUC4) and Fucosyltransferase 1 (FUT1) genes have been associated with the susceptibility to ETEC F4 and ETEC F18, respectively. The interplay between the MUC4 and FUT1 genotypes to ETEC infection and the use of amoxicillin in modifying the intestinal microbiota during a natural infection by multiresistant ETEC strains have never been investigated. The aim of this study was to evaluate the effects of the MUC4 and FUT1 genotypes and the administration of amoxicillin through different routes on the presence of diarrhoea and the faecal microbiota composition in piglets naturally infected with ETEC. Seventy-one piglets were divided into three groups: two groups differing by amoxicillin administration routes—parenteral (P) or oral (O) and a control group without antibiotics (C). Faecal scores, body weight, presence of ETEC F4 and F18 were investigated 4 days after the arrival in the facility (T0), at the end of the amoxicillin administration (T1) and after the withdrawal period (T2). The faecal bacteria composition was assessed by sequencing the 16S rRNA gene. We described that MUC4 and FUT1 genotypes were associated with the presence of ETEC F4 and ETEC F18. The faecal microbiota was influenced by the MUC4 genotypes at T0. We found the oral administration to be associated with the presence of diarrhoea at T1 and T2. Furthermore, the exposure to amoxicillin resulted in significant alterations of the faecal microbiota. Overall, MUC4 and FUT1 were confirmed as genetic markers for the susceptibility to ETEC infections in pigs. Moreover, our data highlight that group amoxicillin treatment may produce adverse outcomes on pig health in course of multiresistant ETEC infection. Therefore, alternative control measures able to maintain a healthy faecal microbiota in weaners are recommended.