江西地区腹泻仔猪肠道致病菌谱的分析及梭杆菌的分离鉴定

Analysis of the pathobionts of intestinal microflora in diarrhea piglets in Jiangxi Province and the isolation and identification of its Fusobacterium

为研究与仔猪腹泻相关的肠道菌群的组成,本试验利用细菌16S rDNA高通量测序分析对比腹泻与健康仔猪肠道菌群的组成差异,并建立荧光定量PCR计数方法测定仔猪肠道梭杆菌数,结合变性梯度凝胶电泳(PCR-DGGE)图谱技术对腹泻仔猪肠道梭杆菌属进行分析;依据菌群分析结果,对腹泻仔猪肠道梭杆菌进行分离鉴定。相比健康仔猪肠道菌群组成,腹泻组(D组)仔猪肠道梭杆菌属(Fusobacterium)丰度显著高于健康组(H组),而厚壁菌门(Firmicutes)、乳杆菌属(Lactobacillus)相对丰度显著低于H组(P<0.05);在种水平上,D组死亡梭杆菌(Fusobacterium mortiferum)显著高于H组(P<0.05),而食淀粉乳杆菌(Lactobacillus amylovorus)则显著低于H组;荧光定量PCR检测结果也表明腹泻新生仔猪肠道中梭杆菌数量显著高于健康仔猪(P<0.05);仔猪肠道梭杆菌属PCR-DGGE图谱结果显示,具核梭杆菌(Fusobacterium nucleatum)在健康与腹泻仔猪中均有被检测到,但仅在D组中检测到的梭杆菌为uncultured Fusobacterium sp.。依据菌群分析结果,采用哥伦比亚血琼脂梭杆菌分离培养基对腹泻仔猪粪便样品进行梭杆菌分离。综上,江西地区腹泻仔猪肠道死亡梭杆菌相对丰度升高,而食淀粉乳杆菌等乳酸杆菌相对丰度偏低,梭杆菌可能是引起仔猪腹泻的致病共栖菌谱(pathobionts)中的重要成员;并从腹泻仔猪肠道成功分离、鉴定出具核梭杆菌和死亡梭杆菌,该结果为诊断及预防仔猪腹泻提供试验依据。

In order to study the composition of intestinal microflora associated with piglets’ diarrhea,the comparison of intestinal microflora between diarrhea and healthy piglets was compared by using 16 S rDNA high-throughput sequencing and denaturing gradient gel electrophoresis(DGGE) techniques.The quantitative real-time PCR(q-PCR) method was established to determine the number of Fusobacterium in feces of piglets.According to the results of molecular analysis,Fusobacterium were isolated and identified.Compared to the intestinal microflor composition of healthy piglets,the relative abundance of Fusobacterium in the diarrhea group(D group) was significantly higher than that in the healthy group(H group) at phylum and genus level;but the relative abundance of Firmicutes,Lactobacillus was significantly lower(P <0.05).The relative abundance of Fusobacterium mortiferum of D group was significantly higher than that of H group at species level,while Lactobacillus amylovorus was significantly lower than that of H group(P <0.05).The results of real-time PCR also showed that the number of Fusobacterium in intestinal tract of diarrhea piglets was significantly higher than that of healthy piglets(P<0.05).The results of PCR-DGGE pattern of Fusobacterium in piglet’ s intestinal tract showed that the Fusobacterium nucleatum was detected in the intestines of healthy and diarrhea piglets;but just only uncultured Fusobacterium sp.in D group.Then Fusobacterium were isolated from fecal samples of diarrhea piglets by Columbia blood agar base.The conclusion showed that in the inlestinal tract of diarrhea piglets in Jiangxi Province,the relative abundance of Fusobacterium mortiferum was increased,while the relative abundance of Lactobacillus such as Lactobacillus amylovorus was declined.So Fusobacterium may be an important member of the pathobionts in diarrhea piglets.Fusobacterium nucleatum and Fusobacterium mortiferum were successfully isolated and identified from the intestinal tract of diarrhea piglets.The results would provide a experimental evidence for the diagnosis and prevention of diarrhea in piglets.