Microscopic And Pathogenic Research Of Reared Stichopus Japonica Suffers From A Kind Of Skin Ulceration Disease

Stichopus japonica belongs to Echinodermata, Holothuroidea, Aspidochirota, Stichopodidae, Apostichopus. It usually inhabit in the shallow sea of north Pacific, Japan, Korea and Russia, and in china, it is mainly found in Liaoning, Hebei, Shandong and Jiangsu. Stichopus japonica is well know for its rich in Chondroitin sulfate, acidic mucopolysaccharides and collagen, and effect of enhancing human's immune system. For these advantages, Stichopus japonica is required more, and consequently, rearing industry of Stichopus japonica is booming.However, reared Stichopus japonica in China suffers from a kind of skin ulceration disease, which has caused severe economic loss in recent years. The course of this disease is often very short, usually several days. The infection involves a head shaking and viscera expelling at early time, and is followed with mouth and cloacal opening tumidity, then the body of the diseased individuals will be shrinking and titanic together with white or blue lesion spots on the body wall. In the later period, these small lesions grow more and bigger to large ulceration and finally causes the death of individuals. This disease is highly infectious, and can often cause all individuals reared in the same pool die in a very short time. And the pathogens are supposed to be bacterial by most researchers.Microscopic (Hematoxylin and Eosin Stain, Ecanning and Eransmission Electron Microscopies) methods, traditional bacteria research methods (Gram stain, Biochemistry test, Antibiotic sensitivity, and Electronic microscope negalive piofure) and biomolecular techniques (phylogenesis of 16S rRNA gene analysis of bacterium) have been introduced in this research to describe the lesions and to investigate and indentify the infecting microbial communities. The causative pathogen is isolated, identified and characterized in a classic way according to Koch's postulates. The lesions consist in a zone where the epidermis is totally destroyed and where collagen fibres and ossicles are exposed to the external medium. Under the light microscopic observation, the connection and structure of collagen fibers are irregular and destroyed compared to the samples of healthy individuals; the muscle fiber in the diseased individuals is connect much closer than the healthy ones, and lacks regular ear like structure as the healthy one's; and a kind of bulb like cells are observed only in the healthy individuals. Under the SEM observation, different kinds of bacterium are investigated to present predominately between the collagen fibers as well as ossicles, and most of the ossicles are not whole in structure. Lesions include ovoid-shaped and round bacterial morphotypes various in size. Under the TEM observation, a kind of cell that includes plenty of inclusions in healthy individuals is found to release the inclusions in the diseased individuals, and it is also shows that most cells in diseased ones are lack integrity in structure.Totally 9 strains of bacteria are isolated and purification, and they are numbered C6, TB, 4, BP1 to BP6, respectively. And only strain C6 could repeat symptom similar to the nature way in two weeks with an initial infection concentration of 108 unit mL-1. Strain C6 is a yellow, G-, long-bacillus in shape and 2μm×0.3μm in size. Among the 21 tested antibiotics, it is mainly sensitive to Chloramphenicol, Lincomycin and 6 other kinds. Among the 16 biochemistry items, only Oxidase and Ornithine decarboxylase are positive while others are shows negtive results. According to phenotype, biochemistry assay results and 16s rRNA gene analysis, strain C6 is identified to be Tenacibaculum sp. Other culturable bacterium colonies present predominantly in the lesions are isolated and purified, and totally 8 are isolated except for strain C6, and numbered, TB, 4, and BP1 to BP6. 16R rRNA gene phylogenesis of them indicates that they belong CFB group,γ- proteobacteria (Alteromonadales and Vibrionales). Strain TB shows similarity greater than 97% with Vibro sp.; strain 4 shows similarity greater than 99% with Shewanella sp.; strain BP1 shows similarity greater than 95% with Formosa sp., and Cytophaga sp.; strain BP2, BP3, BP4 and BP6 shows similarity greater than 99% with Pseudoalteromonas sp.; strain BP5 shows similarity greater than 99% with Alteromonas sp.. And bacteria of Vibro and Pseudoalteromonas are happened to be reported as causative pathogens of skin ulceration disease of Stichopus japonica.