The Relationship Between The Occurrence Of Skin Ulcer Syndrome In Cultured Apostichopus Japonicus And Heterotrophic Bacterial Flora

As Apostichopus japonicus cultivation began more and more popular and culturing scale increased gradually day by day,skin ulcer syndrome broke out cosmically during winter seed rearing and culturing periods,which resulted in high death rate and serious economic loss.Its dominating symptoms were mouth swelling,shaking head,eviscerating bowels,body shrinking,skin ulcering,body disjointing and death.As it was a acute disease with high infectivity and wide epidemic,it restricted health cultivation and continuable development of sea cucumber seriously.At present,the study on diseases in cultured sea cucumber are focus on epidemiology,aetiology and tissue pathology,but the mechanism of disease happening,especially microecology characteristics have not seen any reports yet.This study firstly reported the water quality status,the number and distribution characteristics of bacteria in cultural ponds and the relation between the preponderant bacteria that were isolated from ponds water,top layer bottom mud,intestinal tract and focus tissue of sea cucumber during low temperature culturing period and aestivation period on the base of long inspection of three sea cucumber farms in different areas nearby Qingdao,with the purpose of finding out the water quality status,opening out the relation between pathogenic bacteria and heterotrophic bacterial flora in cultural environment at the beginning of cultured sea cucumber suffering from skin ulcer syndrome and clarifying the essential causation of skin ulcer syndrome.In addition,this study also identified the pathogeny of skin ulcer syndrome.From January to May of year 2006,at the beginning of cultured Apostichopus japonicus suffered from skin ulcer syndrome in three sea cucumber farms in different areas nearby Qingdao,detection of water quality and the number and distribution of heterotrophic bacteria and vibrio bacteria of cultural ponds as well as total count of heterotrophic bacteria and vibrio bacteria in intestinal tract of sea cucumber in normal pond and the pond where sea cucumber were suffered from skin ulcer syndrome in early stage were analyzed with the methods of HPC and VPC.At the same time,this study compared and analyzed the relativity between the preponderant bacteria that were isolated from above abnormal ponds water,top layer bottom mud,intestinal tract and focus tissue of sea cucumber.Besides,artificial infection test was carried out with preponderant bacteria 060102A and 060330B that were isolated from focus tissue of sea cucumber and were verified to be the pathogen causing the skin ulcer syndrome,bacterial classification was carried out samely.The water detection included T,S,pH,DO,COD,NH4+-N and NO2--N.The results showed that as water temperature and dissolved oxygen both formed divided layers in cultural ponds water the bottom layer water began to worsen at some degree;total heterotrophic bacteria and vibrio bacteria number in bottom layer water were both higher than that in surface layer water in general,the whole water bacteria number was in low-level;total heterotrophic bacteria number in top layer bottom mud and intestinal tract were in high-level,whereas total vibrio bacteria number was in low-level relatively.The preponderant bacteria that were isolated from top layer bottom mud and intestinal tract were identical to that in focus tissue of sea cucumber,whereas almost had no relation with ponds water.Morphological observation,semi-automatic identification through API system,traditional physiological and biochemical methods as well as 16S rRNA sequence analysis were applied in the bacterial classification.The results showed that 060102A was Pseudoalteromonas sp.;060330B had same character as Pseudomonas genus bacteria,its characteristics were similar to Pseudomonas putida,with 99.5 % identity.Thus bacterium 060330B was identified as Pseudomonas putida,and regarded as one of the pathogens causing skin ulcer syndrome of cultured sea cucumber.In addition,epidemiology investigation and detections of several samples revealed that the ponds in which cultivation history reached two years or more and having thick black bottom mud with fishy smell were likely to result in the happening of skin ulcer syndrome of cultured sea cucumber at that time.The occurrence of skin ulcer syndrome in cultured sea cucumber was closely related with water deterioration caused by divided water layers and pathogenic bacteria number in ponds bottom mud.From July to October of year 2006,at the beginning of cultured Apostichopus japonicus suffered from skin ulcer syndrome in three sea cucumber farms in different areas nearby Qingdao,detection of water quality and the number and distribution of heterotrophic bacteria and vibrio bacteria of cultural ponds as well as total count of heterotrophic bacteria and vibrio bacteria in intestinal tract of sea cucumber were analyzed with the methods of HPC and VPC.At the same time,this study compared and analyzed the relativity between the preponderant bacteria that were isolated from ponds water,top layer bottom mud,intestinal tract and focus tissue of sea cucumber.Besides,artificial infection test was carried out with preponderant bacterium 060904E that was isolated from focus tissue of sea cucumber and was verified to be the pathogen causing the skin ulcer syndrome,bacterial classification was carried out samely.The water detection included T,S,pH,DO,COD,NH4+-N and NO2--N.The results showed that the whole cultural ponds water began to worsen at some degree,total heterotrophic bacteria and vibrio bacteria number were 3.3×104cfu/mL and 2.0×104cfu/mL respectively on average,which both the differences were not apparent in water layers.Total heterotrophic bacteria and vibrio bacteria number were 5.9×106cfu/g and 1.4×106cfu/g respectively on average of top layer bottom mud in ponds.Besides,total heterotrophic bacteria and vibrio bacteria number in intestinal tract of sea cucumber were 5.8×106cfu/g and 1.9×106cfu/g.The preponderant bacteria that were isolated from ponds water,top layer bottom mud and intestinal tract were identical to that in focus tissue of sea cucumber.Thus bacterium 060904E was identified as Vibrio harveyi,and regarded as the pathogen causing skin ulcer syndrome of cultured sea cucumber during aestivation period.The occurrence of skin ulcer syndrome in cultured sea cucumber was closely related with the water deterioration and number of pathogenic vibrio bacteria in water and bottom mud of cultural ponds.This study revealed that Pseudomonas putida and Vibrio harveyi were important pathogenies that causing skin ulcer syndrome of cultured sea cucumber in Qingdao cultivation area,which established theory basis on aetiology research of cultured sea cucumber in future.At the same time,this research opened out that the occurrence of skin ulcer syndrome in cultured sea cucumber was closely related with water deterioration and pathogenic bacteria number in bottom mud under the condition of pond cultivation,which provided supervising references on health management,improving cultivation technology and strengthening prevention and treatment of diseases in cultured sea cucumber.