Study On Etiology,histopathology Of Dark-body Diseases In Cultured Spotted Knifejaw(Oplegnathus Punctatus) And Variance Of Microbial Community Structure Under Antibiotic Intervention

In recent years,dark-body disease occurred in spotted knifejaw(Oplegnathus punctatus)during the culture period.The main symptoms were dark body,weak vitalit y,anorexia and becoming thinner.After dissecting,firstly massive colourless or light yellow ascites was found in abdominal cavity;secondly,the intestinal showed transparent and atrophic;thirdly,the liver was congestive and fragile;fourthly,the gill filaments was hemorrhagic and adhesive.The cumulative mortality was up to 50~90%,which brought a great economic losses and also have a serious impact on production and healthy development of spotted knifejaw.In this research,etiology,histopathology and intestina l microecology of the dark-body disease have been studied systematically in order to find out the disease pathogens and pathogenesis.By using the traditional method of pathogen isolation,a bacterial strain named OD1 was isolated from intestinal,liver,gill,renal and other tissue of the diseased fish.The artificial infection test showed that the bacteria was the pathogen of dark-body disease.The morphological analysis showed that the pathogen has one peritrichous and was a short rod-shaped gram-negative bacteria,with size 1.8~2?m× 0.8~1?m.Using physiological and biochemical identification combined with 16 S rDNA gene sequence identification,it was found that the pathogen has a high similarity with vibrio harveyi,which is up to 99%.The drug susceptibility test showed that the bacteria was high sensitive to tetracycline,novobiocin,doxycycline and cefotaxime,and moderate sensitive to cefradine and clarithromycin,and resistant to penicillin and erythrocin.The histopathological study of spotted knifejaw natural y infected with OD1 was carried out through microscope and electron microscope.The results showed that,the pathological changes were found in most tissues and organs of diseased fish,of which the digestive system was most seriously affected.The intestinal mucosal epithelial cell was necrosis and shedding,the epithelial cell nucleus was dissociated and there was almost no complete cell structure.The infiltration of blood cell was found in hepar,the hepatocyte was atrophy and rupture,the hepatocyte nucleus was swel ed,and the intracellular organelle decreased.The outer membrane of gallbladder was dissolved and its smooth muscle degenerated into a network structure.In addition to the digestive system,different degrees of pathological changes also occurred in other major organs.The gill cartilage was disappeared and the epithelial cel s was hypertrophied and integrated.The results of ultra-pathological showed that the gill epithelial cell membrane was dissolved,the nuclear membrane was disappeared and the nuclear cytoplasm was concentrated.The renal tubular cavity was closed,atrophied and nearly disappeared and the glomerulus showed hyperemia and swel.The spleen tissues became loosen and the blood cel s were necrosis and accumulated.The myocardial fibers was twisted and cell swel ed and fractured.In order to resolve the pathogen and pathogenesis of dark-body disease in spotted knifejaw and explore the changes of the bacterial community structure in the intestine of individuals with dark-body disease and after medical treatment,the Il umina HiSeq PE250 high-throughput technique was used to analyze the bacterial community diversit y and structure of the fish intestine from healthy,sub-healthy,typical dark-body disease infected and oral florfenicol intervened individuals,as well as the culture water and pellet feed.The results showed that the dominant bacterial communities in intestine were Proteobacteria,Firmicutes and Tenericutes,under the conditions at different health levels and florfenicol intervention,and the proportions of corresponding OTU number into all the OTU in the samples were above 85%.It could affect the ranking order of the top 20 dominant bacterial species in intestine when the dark-body disease occurred,with the relative abundance of vibrios in Proteobacteria increasing significantly.In addition,the dark-body disease symptoms aggravated gradually as the increase of vibrios' abundance.Feeding with florfenicol could reduce the vibrios' abundance from 60.33% to 1.29% in the intestine of diseased fish,and also changed its structure of bacterial community in a large degree,which proved that florfenicol was effective in treating dark-body disease.It also found that the culture water and pellet feed had a certain influence in intestine bacterial community,and the effect of the culture water was higher than the pellet feed.The study reported etiology and pathology of spotted knifejaw dark-body disease.The present results would provide a reference basis and technical support for the health culture and disease control in spotted knifejaw.