犬贾第虫携病毒株体外纯培养的建立

Establishment of In vitro Cultivation of Giardia canis Trophozoites Infected with Giardia canis Virus

目的培养一携带犬贾第虫病毒的犬贾第虫(Giardiacanis)细胞株。方法用蔗糖密度梯度离心-G1耐酸漏斗过滤法纯化犬贾第虫包囊,经口接种5日龄长爪沙鼠(Merionesunguiculata),8d后于其十二指肠无菌收集犬贾第虫滋养体,置改良的TYI-S-33培养基中培养,待滋养体在培养管壁上形成细胞单层后进行传代。同时进行冻存和复苏实验,以及纯度、稳定性、细胞生物学特性、微生物污染等4项指标检测。滋养体经液氮冻融3次后3000×g离心15min,取上清,用磷钨酸负染,透射电镜观察病毒粒子。结果犬贾第虫滋养体接种14d后虫体逐渐适应了培养环境,在培养管壁上形成细胞单层,经上述4项指标检测,证明形成了稳定的犬贾第虫细胞株。电镜观察,见滋养体内有外观球形呈20面体结构、直径约为36nm的病毒样粒子。结论建立了携带GCV的犬贾第虫细胞株的体外纯培养。

Objective To cultivate a Giardia canis isolate with G.canis virus （GCV）. Methods Five-day-old Meriones unguiculatus was infected with the cysts of G. canis isolated from dogs in Changchun and purified by sucrose density gradient centrifugation-G1 acid funnel filtration method. Trophozoites were isolated aseptically from the duodenum of the infected rodent after 8 days, then transferred to modified TYI-S-33 medium and cultivated at 37 ℃. The trophozoites were centrifuged with 3 000 × g, 15 min after liquid nitrogen freeze-thawing three times and the supernatant stained negatively by phosphotungstic acid was observed with transmission electron microscope. Results G. canis trophozoites which adapted gradually to the environment and grew a cellular monolayer after 14 days were examined by freezing and thawing experiment, purity quotient, stability, biology characteristics and microbial contamination detection. The results demonstrated that a stable G.canis trophozoite cell isolate was established. G. canis virus with icosahedron spherical shape and 36 nm in diameter was observed by electron microscope. Conclusion In vitro cultivation of G. can/s trophozoites with GCV is established.