关键词: 肝毛细线虫, 动物模型, 小鼠

Abstract:

Objective To establish a mouse infection model of Capillaria hepatica to facilitate the research on hepatic capillariasis. Methods The C. hepatica infected rats were collected from endemic area in Luoyang, Henan. The C. hepatica eggs were collected from the liver of an infected rat by 1% pepsin digestion. The collected eggs were divided into 3 groups with 1 000, 5 000 and 10 000 per well in duplicate, then cultured in vitro at 28 ℃ in an incubator. The eggs were observed under microscope for their embryonated rate at Day 18, 21, 23, 25, 29. The fully embryonated eggs were divided into two groups and incubated in distilled water and artificial gastric juice respectively under 37 ℃. The hatched larvae were observed. Three male Kunming mice with 4 weeks old in each group were inoculated orally with 5, 10, 20, 60, 100, 200, 500 and 800 C. hepatica embryonated eggs. The survival rate of each group was observed as evidence of successful infection. Another sixteen mice were infected orally with 60 C. hepatica embryonated eggs to systemically observe the infection and development of C. hepatica worms and eggs in livers at Day 14, 18, 21, 29, 35, 55, 90, 365 post infection by necropsying 2 mice at each time point. Results Seventeen rats were captured from endemic area Luoyang, Henan, and five identified to be infected with C. hepatica with infection ratio of 5/17. Rattus norvegicus rat was the only species infected with C. hepatica with infection rate of 5/8. Most of C. hepatica eggs observed in the livers of infected rats were at Ⅲ to Ⅳ cell divided stage. After being cultured in vitro, the cell in collected C. hepatica eggs started to develop and divide to multiple cells. The embryonated eggs could be seen on Day 16 of culture. The rates of embryonated eggs in the culture groups of 1 000, 5 000 and 10 000 eggs per well were 39.6% (67/169), 35.2% (45/128) and 21.4% (98/458) on Day 18; 74.0% (148/200), 75.1% (411/547) and 60.9% (340/558) on Day 23; 88.0% (125/142), 89.2% (140/157) and 79.3% (168/212) on Day 25; and 94.9% (131/138), 97.0% (254/262) and 88.6% (140/158) on Day 29; respectively, with statistical significance between each culture time points (P < 0.05). No larva was hatched from embryonated eggs either in distilled water or in artificial gastric juice. The Kunming mice were successfully infected with 200 or less embryonated eggs of C. hepatica and all survived during the infection. The mice could not survive with infection of more than 500 eggs. In the groups of mice infected with 60 C. hepatica embryonated eggs, the larvae of C. hepatica could be observed in the liver tissue on Day 14. The matured and pregnant female adult worms were observed on Day 18. The adult worms started to die and the unembryonated eggs were laid into liver tissue on Day 35. All worms degenerated and only eggs were observed in liver tissue on Day 90 and 365 post infection. Conclusion The embryonated eggs of C. hepatica can be acquired by in vitro culture of eggs collected from infected rat and C. hepatic infection model was successfully established in Kunming mice with less than 200 embryonated infective eggs.

Key words: Capillaria hepatica, Animal model, Mice