Study On The Pathogen Detection Of Reddish Body Syndrome Turbots (Scophthalmus Maximus)

Turbot, Scophthalmus maximus, is an important aquaculture fish species in ourcountry. Recently, the frequent outbreak of the turbot reddish body disease hasaffected the healthy aquaculture of the turbot seriously. Turbot reddish body diseaserefers to the occurrence of local haemorrhages in skin along the fin base and vertebraon the blind side of the farmed turbot. But it was reported that the causative pathogensof turbot “reddish body syndrome”(RBS) are diverse including bacteria, virus andparasite, which makes it difficult for the diagnosis and treatment of the RBS turbot intime. Therefore, it is very necessary to using a reliable and effective method ofpathogen detection for correct diagnosis and treatment.Five batches of sick turbots with suspected red body syndrome were sampledform4turbot farms located in the Jiaodong coastal area, Shandong Province, inMarch, September, October and December2011and in January2012, respectively.Three kinds of RBS cases have been observed among the sampled sick turbots afterpathological symptoms examination and pathogen detection, designated asbacteria-infected RBS turbot, virus-infected turbot and vibrio-virus co-infection RBSturbot, respectively.Results for the pathological symptom observation revealed that, the symptoms ofthe sick turbots collected in January2012are not the RBS disease, but the other fourbatches of sick turbots showed obvious RBS with local haemorrhage in the skin. Itwas found that the RBS turbots with different pathogens indicated differentsyndromes: the gill filaments of vibrio-infected RBS turbots are usually brightly redwhile the gill filaments of virus-infected RBS turbots are usually anemia and grey.Thus whether or not the gill filaments of RBS turbots are anemia and grey in color isthe most direct and effective index to discriminate the bacterial infection from virus infection. In addition, when the vibrio-infected RBS turbots became seriously sick,their skins would start to ulcerate and bleed.Results for the parasites detection by light microscopy indicated that all the RBSturbots tested were not infected by parasites. However, the pathogenic vibrio wasdetected in three batches of RBS turbots collected in September, October andDecember2011, evidenced by the occurrence of the yellow vibrio colonies in TCBSselection medium, in which only vibrio can grow. And virus particles were detected inthe sick turbots collected in March and December2011by transmission electronmicroscopic (TEM) examination (both negative staining and microtome section). Insummary, the diagnostic results for pathogen detection showed3cases ofbacterial-infected RBS turbot (September and October), virus-infected RBS turbot(March) and vibrio-virus co-infection RBS turbot (December).TEM examination showed that the virus particles were present in both thevirus-infected and vibrio-virus co-infected RBS turbots. In negative staining,spherical virions with or without envelope were found. They were about120~130nmin diameter. Similar size of virion was also observed in the microtome sections of skintissues. And the virus particles in the cytoplasm of the infected cells were sphericalwith a diameter of about130nm, too. It was also observed in the pathological cellsthat the mitochondria swell up and the cristae were partially disintegrated.Results for the cytopathic effect (CPE) examination suggested that the isolatedvirus can proliferate in the flounder gill (FG) cells. Obvious CPE, showing that theinfected cells rounded and detached from the substrate, was observed in the FG cellsat the3rd day after exposure to the virus crude extracts, followed by piling up of theFG cells and more and more cells detached at the9th day after virus inoculation. And11days later, most cells disintegrated and died. Although the typical virus particleswere not found in the infected FG cells by TEM, the virus inclusion bodies-likestructure really existed in the cytoplasm of the infected FG cells, and also the numberof lysosomes and secondary lysosomes increased, and the cytoplasm became morehomogeneous and vacuolated in the infected FG cells. It was also found that thevirus-infected FG cells became hypertrophic and about twice of the size of uninfected normal cells. The CPE was also observed in the FG cells infected by the FGcells-derived virus concentrate, but the occurrence delayed4days in comparison withthe tissue-derived virus infection. This indicated that the newly amplified virus in FGcells still contained the capability of virus infectivity but the activity slightlydecreased.In a word, in the present study various pathogen detection methods includinglight microscopic examination, selective cultivation of vibrio, TEM observation (bothnegative staining and microtome section), cytopathic effect (CPE) examination andthe specific amplification of PCR were examined for their suitability for rapidpathogen detection of RBS turbots by analyzing five cases of RBS turbots. All thedata obtained will contribute to the reliable and effective diagnostics and treatment ofthe RBS turbots as well as the healthy aquaculture of turbots.