关键词: 细粒棘球蚴, 多房棘球蚴, 微囊, 原头节, 感染, 免疫

Abstract: Objective To investigate the effect of intraperitoneal inoculation of Echinococcus granulosus (Eg) or E. multilocularis (Em) micorcysts on hepatic infection and pathogenesis of Em in mice liver. Methods The protoscoleces (PSCs) of Eg and Em were collected and cultured in vitro for 6 and 8 weeks to encyst, respectively. Sixty C57 female mice were randomly assigned to 4 groups, which were Em/Em infection, Em/Eg infection, pure Em infection and sham surgery groups, with 15 mice in each group. The mice of designated infection groups were intraperitoneally injected with 50 Em microcysts (diluted in 1 ml 1 × PBS), the mice in the Eg/Em infection group were intraperitoneally inoculated with 50 Eg microcysts, and 1 ml of normal saline, respectively. After 1 month, the mice in all three groups underwent laparotomy and were injected with 2 000 Em PSCs (200 µl) through hepatic portal vein (HPV). The mice in the sham operation group were injected with the same amount of normal saline into HPV in abdominal cavity. Mice blood was collected from the tail vein before intraperitoneal inoculation and at 1, 3 and 6 months after inoculation. In the collected blood samples, serum antibodies against hydatid cyst fluid antigen were detected by ELISA. At 1, 3 and 6 months after HPV infection, 5 mice in each group were dissected to inspect the liver lesions of infection with naked eyes and conduct quantitative scoring. The big lobe of liver was collected from mice to prepare paraffin section, and stain with HE to observe the changes in liver lesion tissue. SPSS 21.0 was used for statistical analysis, and an independent sample t-test was used to compare the mean between the two groups. Results The diameter of Eg microcysts cultured for 6 weeks in vitro is about 300 to 500 µm, and the diameter of Em microcysts cultured for 8 weeks is about 150 to 300 µm. ELISA results showed that after 1, 3, and 6 months of intraperitoneal infection with echinococcus microcapsules, the levels of anti-cyst fluid protein antibodies (A₄₀₅ values) in the serum of mice infected with Em/Em and Eg/Em elevated and maintained at high level. One month after intraperitoneal inoculation of microcysts, the serum anti cyst fluid protein antibody levels (A₄₀₅ values) in mice infected with Em/Em and Eg/Em groups were 2.77 ± 0.62 and 2.35 ± 0.23, respectively, which were more than 10 times higher than 0.15 ± 0.02 in the sham operation group. After 1, 3, and 6 months of HPV infection, the autopsy was performed. Eg cysts were seen in the abdominal cavity of the Eg/Em infected group, while no cysts were seen in the Em/Em group, the Em liver infected group and the sham group. Liver infection at different stages was the most severe in the Em liver infection group, where the liver surface was mostly covered with punctate or vesicular lesions of varying sizes. In the Eg/Em infection group, the infected lesions were weak or normal, and there were 1-3 small lesions on the liver surface that tend to become normal as the infection cycle extends. No infectious lesions were found in the Em/Em group and the sham group at different stages. The results of the lesion severity score showed that the scores of each stage in the group with Em liver infection (4.8 ± 1.3, 5.0 ± 1.5, 4.5 ± 1.4) were higher than those in the group with Eg/Em infection (1.6 ± 0.9, 1.2 ± 1.1, 1.6 ± 0.9) (t = 4.92, 4.81, 5.24, all P < 0.01). The results of HE staining showed that 1 month after HPV infection with Em PSCs, there was a large number of inflammatory cells infiltrating the lesions in the Em liver infection group, mainly eosinophils; In the Eg/Em infected group, there was less infiltration of inflammatory cells in the lesion. At 3 months after infection, the inflammatory cells in the focus of the simple Em liver infection group decreased relative to the prophase, with neutrophils predominating, and a collagen fibre layer formed around the focus; In the Eg/Em infection group, the focus was mainly collagen fibre layer; At 6 months after infection, the lesions in both the Em liver infection group and the Eg/Em infection group showed a trend of recovery, mainly with connective tissue hyperplasia. Conclusion Em microcysts infection via abdominal cavity can completely block Em reinfection in mice liver, and Eg infection via abdominal cavity can suppress Em infection in mice liver to a greater extent.

Key words: Echinococcus granulosus, E. multilocularis, Microcysts, Protoscoleces, Infection, Immunity