不同宿主源弯曲菌黏附侵袭肠上皮细胞及在鸡肠道内定殖的比较

Comparison on adhesion and invasion to intestinal epithelial cells and chicken intestine colonization of Campylobacter strains from different hosts

摘要:

【背景】弯曲菌(Campylobacter)是重要的人畜共患病原菌，可在多种动物肠道定殖，但不同宿主源弯曲菌对肠上皮细胞的黏附侵袭特征及在鸡肠道内的定殖能力并不明确。【目的】探究不同宿主源弯曲菌对不同宿主肠上皮细胞黏附侵袭及在鸡肠道内定殖能力的差异性。【方法】利用 5株来自不同宿主源弯曲菌，包括人源、鸡源、鸭源和牛源空肠弯曲菌(Campylobacter jejuni)及猪源结肠弯曲菌(Campylobacter coli)，在对菌株PCR鉴定、运动力及生物膜形成能力测定的基础上，分别测定各菌株对人源肠上皮细胞Caco-2、猪源肠上皮细胞IPEC-J2和大鼠源肠上皮细胞IEC-6的黏附能力，通过庆大霉素保护试验测定菌株对肠上皮细胞的侵袭能力，比较黏附量和侵袭量的差异；将5株弯曲菌分别口服攻毒鸡，于攻毒后不同日龄(different days post inoculation，DPI)采集肠道样品测定弯曲菌的菌落数，比较不同弯曲菌在鸡肠道内定殖的差异。【结果】人源弯曲菌运动力显著高于其他4株动物源弯曲菌，而牛源和猪源弯曲菌生物膜形成能力显著高于其他菌株。黏附侵袭测定结果显示，人源弯曲菌对Caco-2细胞的黏附能力显著高于动物源弯曲菌，但侵袭能力显著低于动物源弯曲菌；鸭源和牛源弯曲菌对IPEC-J2细胞的黏附能力显著低于其他菌株，而且鸭源弯曲菌的侵袭能力显著低于其他菌株；不同菌株对IEC-6细胞的黏附能力无显著差异，但鸡源弯曲菌侵袭能力显著低于其他菌株。不同弯曲菌口服攻毒鸡后1、3和6d动物源弯曲菌定殖水平显著高于人源，在攻毒后10d和15d仅牛源弯曲菌显著高于人源，于攻毒后15d所有菌株达到约8-10Log₁₀(CFU/g)的稳定定殖水平。【结论】来源于不同宿主的弯曲菌对不同宿主肠上皮细胞均具有黏附侵袭能力，同时可在鸡肠道内稳定定殖，提示弯曲菌在不同动物间传播和适应性定殖的特征，对开展弯曲菌针对性防控措施具有一定的借鉴意义。

关键词:弯曲菌;宿主;肠上皮细胞;黏附;侵袭;定殖

Abstract:

[Background] Campylobacter is a major zoonotic bacterial pathogen which can colonize the intestinal tract of a variety of animals. However, adhesion and invasion to intestinal epithelial cells and chicken intestine colonization of Campylobacter strains from different hosts are unclear. [Objective] To compare the adhesion and invasion to intestinal epithelial cells and the chicken intestine colonization of Campylobacter strains from different hosts. [Methods] Five Campylobacter strains isolated from different hosts, including human-, chicken-, duck-, and bovine-derived Campylobacter jejuni strains and a porcine-derived C.coli strain, were studied. The strains were identified by multiplex PCR and tested for motility and biofilm formation ability. The adhesion of each strain to human intestinal epithelial cell line Caco-2, porcine intestinal epithelial cell line IPEC-J2, and rat intestinal epithelial cell line IEC-6 was determined respectively. The invasion of each strain to intestinal epithelial cells was determined by gentamicin protection test. The differences in adhesion and invasion were statistically analyzed. Further, chickens were challenged orally with the five strains respectively. We collected the cecal samples on different days post inoculation (DPI) to determine the chicken intestine colonization ability of Campylobacter. [Results] The motility of human-derived C.jejuni was significantly higher than that of other four animal-derived Campylobacter strains, and the biofilm formation ability of bovine- and porcine-derived strains was significantly higher than that of other strains. The human-derived C.jejuni showcased stronger adhesion and weaker invasion to Caco-2 cells than animal-derived strains. The adhesion ability of duck- and bovine-derived strains to IPEC-J2 cells was significantly lower than that of other strains, and the invasion ability of the duck-derived strain was significantly lower than that of other strains. There was no significant difference in the adhesion ability of the five strains to IEC-6 cells, while the invasion ability of chicken-derived strain was significantly lower than that of other strains. The animal-derived strains showed higher colonization levels than the human-derived strain at 1, 3 and 6 DPI, while only the bovine-derived strain had significantly higher colonization level than the human-derived strain at 10 and 15 DPI. All the strains reached a stable colonization level of 8-10 Log₁₀ (CFU/g) at 15 DPI. [Conclusion] Campylobacter strains from different hosts can adhere to and invade the intestinal epithelial cells of various animal origins and colonize chicken intestine, which suggest that Campylobacter can transmit among and colonize different animals. The results are helpful for developing targeted prevention and control measures against Campylobacter.

Key words:Campylobacter;host;intestinal epithelial cells;adhesion;invasion;colonization