Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China.
Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, Nanjing 210023, China.
Vet Microbiol. 2014 Jun 25;171(1-2):215-20. doi: 10.1016/j.vetmic.2014.03.016. Epub 2014 Mar 25.
Spiroplasma is a novel pathogen of commercially exploited crustaceans. To more full clarify its pathogenic mechanism at the molecular level in vitro, a spiroplasma-infected hemocytes model was developed as described here, using a modified method of hemocytes culture from the Chinese mitten crab Eriocheir sinensis. It has been standardized by employing L-15 growth medium supplemented with 15% fetal bovine serum along with 0.15% glucose, 0.75% NaCl, antibiotics (100 U ml(-1) penicillin, 100 U ml(-1) streptomycin) and a suitable pH of 7.20-7.40, incubated at 28°C without the requirement for 5% carbon dioxide. Cytopathic effects of Spiroplasma eriocheiris in the cultures, including cell debris and cellular exudates, were observed as early as 36 h post-inoculation. The green fluorescent dye Alex-488 was used as an immunofluorescence marker of S. eriocheiris to study its adhesion to, and infection of, the host cell. At about 24-h post-inoculation, a large number of spiroplasmas were observed infecting the host hemocytes cells, which became slug-like and widely distributed in the culture. Intracellular inclusion bodies containing spiroplasmas, and a large number of free spiroplasmas released from the ruptured cells, were observed using transmission electron microscopy. All the results show that S. eriocheiris can invade E. sinensis hemocytes in vitro, and this can provide a significant step forward toward further study of the relationship between the novel pathogen spiroplasma and its hosts in laboratory experimental studies.
螺原体是一种新型的商业开发甲壳类动物病原体。为了更全面地阐明其在体外的分子水平致病机制，开发了一种螺原体感染血细胞模型，该模型是根据中华绒螯蟹（Eriocheir sinensis）血细胞培养的改良方法建立的。通过采用 L-15 生长培养基，补充 15%胎牛血清、0.15%葡萄糖、0.75%氯化钠、抗生素（100 U ml(-1)青霉素、100 U ml(-1)链霉素）和适宜的 pH 值（7.20-7.40），在 28°C 下培养，无需 5%二氧化碳，使该模型得以标准化。在接种后 36 小时，即可观察到 Spiroplasma eriocheiris 在培养物中的细胞病变效应，包括细胞碎片和细胞渗出物。Alex-488 绿色荧光染料被用作 S. eriocheiris 的免疫荧光标记物，以研究其对宿主细胞的黏附和感染。在接种后约 24 小时，大量的螺原体被观察到感染宿主血细胞，这些血细胞变成类似鼻涕虫的形状，并在培养物中广泛分布。用透射电子显微镜观察到含有螺原体的细胞内包涵体，以及大量从破裂细胞中释放的游离螺原体。所有结果表明，S. eriocheiris 可以在体外入侵中华绒螯蟹的血细胞，这为进一步研究新型病原体螺原体与宿主之间的关系提供了重要的一步，特别是在实验室实验研究中。
