| Macrobrachium rosenbergii is an important freshwater prawn in Chinese aquaculture. But with the intensive development of freshwater aquaculture and the aquatic environment deterioration, more serious diseases have appeared, especially bacterial disease, which have high incidence and caused massive mortalities. In August2010, a breeding pond of M.rosenbergii has occurred sudden disease in Gaoyou city, Jiangsu province, PR. China Clinical signs of the diseased M.rosenbergii included weakness and their aggregation at the side of the ponds, and rapid death when seining. The outbreaks of the disease epizootics have caused drastic decreases in production and catastrophic economic losses. After the preliminary analysis and research, we identified the pathogen, and it is a spiroplasma, strain MR-1008.Spiroplasma was found as a kind of microbe in70th last century and extensively parasitized in insects and plants. Some are agents which were severely pathogenic. By now,38species of spiroplasma have been named. There are five main characters of a spiroplasma including filterability, helicity, rotary motility, the wall-free membrane, and growth in medium. Light microscopy (LM), transmission electron microscopy (TEM), molecular biological methods (16S rRNA gene analytic method as a gold analytic standard widely accepted of the species pedigree in microbes) and in vitro culture were used to identify the pathogen. It was found that16S rDNA of MR-1008had98%identity with that of Sproplasma mirum. After the process above, the spiroplasma was identified by morphologic, microbiological method and Koch’s Rule. Except Eriocheir sinensis, Procambarus clarkii and Penaeus. vannamei, we found spiroplasma in crustacean again, and it was the first time isolated from M.rosenbergii. Therefor it is necessary to do further research on its pathogenesis mechanism and biological characteristics.Spiroplasma eriocheiris was the first spiroplasma (GenBank Accession No: AY920929) that has been found in a freshwater crustacean, it can cause tremor disease of Chinese mitten crabs and lead to mortality rate as high as100%in15days. Experimental infection with MR-1008can cause crab paroxysmal tremor and100%of mortality, but with a longer time. Meanwhile, infected M.rosenbergii with S.eriocheiris artificially can only cause54.2%of mortality in30days. But infected M.rosenbergii with MR-1008, the mortality rate was up to83.3%. These results suggested that spiroplasma isolated from different hosts had different infective intensities or the hosts respond differently to spiroplasmainfection, and the two spiroplasmas exhibited some differences. E.eriocheris is more sensitive to spiroplasma than M. rosenbergii. In addition, there were no significant differences between these two spiroplasmas from different hosts with ELISA and Western blotting with polyclonal antibody of MR-1008and monoclonal antibody7C8of S.eriocheiris.The spiroplasma was isolated from diseased M. rosenbergii and cultured through triply cloning in R2medium for the use of biological characteristics studies. The results were as follows:MR-1008showed optimal growth temperature at30℃. Under the optimal conditions, MR-1008reached the log-phase for24hr. The optimal growth pH of MR-1008was at pH7.2-7.6and optimal growth pH salinity was at0‰～1‰.The result of drug sensitivity test showed that Ofloxacin, Clindamycin, Clarithromycin, Nitrofurantoin, Minocycline, Norfloxacin, Levofloxacin, Tetracycline, Midecamycin can highly inhibited MR-1008.With those results above, it revealed that the causative agent of diseased M. rosenbergii from Gaoyou is a spiroplasma. But there were different virulence and infectivity of aquatic spiroplasma strains which from different hosts, and the polyclonal antibodies were also different, that may caused by variation among different hosts and host selection pressures. |
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