A spiroplasma associated with tremor disease in the Chinese mitten crab (Eriocheir sinensis).

An epidemic of tremor disease has been a serious problem in Chinese mitten crabs, Eriocheir sinensis, in China in recent years. The disease-causing agent was previously considered to be a rickettsia-like organism. Here, analysis of the 16S rRNA gene sequence, light and electron microscopy and cultivation in vitro were used to identify the agent. Sequence analysis of the 16S rRNA gene found it to have 98 % identity with that of Spiroplasma mirum. The agent was able to be passed through membrane filters with pores 220 nm in diameter and could be cultivated by inoculating the yolk sac of embryonated chicken eggs and M1D medium. Rotary motion and flexional movement were seen by light microscopy, and electron microscopy showed that the organism had a helical morphology and lacked a cell wall. The organism produced small colonies with a diameter of 40-50 microm after 17-25 days of incubation on solid M1D medium. The agent was found in blood cells, muscles, nerves and connective tissues of crabs inoculated with a filtrate of yolk sacs or with cultures grown in M1D medium, and it was similar in structure to those grown in eggs and cultivation broth. Disease was reproduced by experimental infection with the cultivated organisms. This study has demonstrated that the causative agent of tremor disease in the Chinese mitten crab is a member of the genus Spiroplasma. This is believed to be the first time a spiroplasma has been found in a crustacean. These findings are not only significant for studies on pathogenic spiroplasmas, but also have implications for studies of freshwater ecology.

Morphology of spiroplasmas in the Chinese mitten crab Eriocheir sinensis associated with tremor disease.

The structure of spiroplasmas in the Chinese mitten crab associated with tremor disease was studied by transmission electron microscopy techniques, including both negative staining and ultrasectioning. A spiral structure, which is a typical form of spiroplasmas, could be detected and the observations showed for the first time the presence of spiroplasmas in the cells of the crab. Propagation of spiroplasmas within the cells presented various forms that could be sorted into three morphological types: rounded, regular helical, and pleiomorphic or intermediate forms. The spiroplasmas appeared as round bodies during the fallow stage of development or under poor conditions. They showed various shapes such as helices, saccate, branched, tadpole-like and tortoise-like structures while growing, and became long and congregated in late stages of development. When spiroplasmas were isolated from chicken eggs and cultured in M1D medium they appeared to undergo similar morphological changes to those in the crab. The spiroplasmas contained chromatin filaments and peripheral ribosome-like granules and were delimited by distinct unitary membranes. Average diameters were calculated at 0.1 to 0.35 microm for rounded forms and 0.1-0.2 microm for helical or long forms, and they varied in length from 3-12 microm.