| The culture area around the Daqin island,located in Changdao County,Shandong province,had become the largest destination of large-scale deep-water cage in northern China,and the main cultured species was black rockfish(Sebastes schlegeli).After more than 20 years development,with the growing scale of the aquaculture,the occurrence of disease had becoming more and more frequent,which has become the key factor affecting the survival rate of the aquaculture.Especially in recent years,the outbreak of the disease had been characterized by rapid infection rate and high mortality,which had seriously affected the normal production.In this study,cage-cultured black rockfish was taken for study object.Epidemiology,etiology and pathology were studied systematically.The contents and results were as follows:1.Epidemiological investigationFrom 2016 to 2017,epidemiology and etiology on the main diseases of the cagecultured of the black rockfish were carried out.And Daqin Island as the survey site,the major water quality indexes,including temperature,salinity,p H,dissolved oxygen(DO),total heterotrophic bacteria and vibrios were detected,along with the bait and net fixture.The results of epidemiological investigation showed that the death of the black rockfish was mainly between June and November,and the main diseases were lymphatic cysts,skin ulcers and drums and eye rotting.Lymphatic cysts was viral disease,and the symptoms will gradually alleviated or relieved with the rising of water temperature,which will not cause massive mortality,so the skin ulcer became the main symptoms of sick black rockfish.The basic water quality indicators such as temperature,salinity,dissolved oxygen and p H were relative stable,which were suitable for aquaculture.But the amount of bacteria and Vibrio in water,net clothing and bait was higher in summer.The highest content of bacteria and vibrio in water body was 6.75 and 5.60 respectively.The highest value of bacteria and vibrio on the attachment of net clothing was up to 5.94 and 4.79,and the maximum value of bacteria and vibrio in bait could reach 6.66 and 5.57.2.Etiology and drug study on main disease of Sebastes schlegeliThe pathogenic bacteria was isolated and identified by morphological observation,routine physiological and biochemical tests,the sequencing of gyr B and 16 S r RNA gyr B gene sequence alignment.The results showed that the skin ulcer was mainly caused by two different bacteria-Photobacterium damselae subsp.damselae and Vibrio rotiferianus,and the symptom was completely different.The symptom infected by the Photobacterium damselae subsp.damselae was haemorrhage and redness on base of fins,and the surface of the skin and tail handle became gray,while more obverious traumatic ulcer appeared on the body surface of rockfish.The drug resistance test was carried out on two strains.The results showed that the the Photobacterium damselae were highly sensitive to the 10 drugs,such as the antibiotics,neomycin,florfenicol and naproxic acid,moderately sensitive to 6 drugs such as cefadine and cefazolin,and resistance to penicillin and pipperic acid.The Vibrio rotiferianus are highly sensitive to tetracycline,quinolones,coumarins and peptidyltransferases,but are moderately sensitive or insensitive to macrolides,peptides,sulfonamides,beta lactam and aminoglycosides.Put the berberine as option medicine,double dilution method was used to determine minimal inhibitory concentration(MIC)and mininal bactericidal concentration(MBC)of berberine on Photobacterium damselae subsp.damselae and Vibrio rotiferianus.The results showed that the MIC on two strains were both 128 mg/L,the MBC on Photobacterium damselae subsp.damselae was 512 mg/L,another was 256 mg/L.In the stability test,berberine solution with concentration of 512 mg/L were treated respectively at 20℃,40℃,60℃,80℃,100℃ and 121℃,its sterilization rate on indicator bacteria(P.damselae)could maintain above 99%.The berberine solution after 20℃,60℃ and 100℃ treatment were stored in photophobic place under room temperature for 35 d,its sterilization rate on P.damselae was still higher than 99%.These demonstrated that berberine had good drug stability.In addition,sterilization effect of berberine solution with different metal ion treatment,such as Na+,Mg2+,Ca2+,K+,almost unchanged.3.Pathological study on main disease of Sebastes schlegeliHistopathology and ultrahistopathological studies showed that the intestine,liver,kidney and gills appeared different degrees of changes infected by Photobacterium damselae subsp.damselae and Vibrio rotiferianus.After infected by Photobacterium damselae subsp.damselae,the epithelial cells of the intestinal villi were fester,the necrosis of the basement membrane was fused,the structure of the muscle layer was loose,the liver blood cell infiltration,hepatic sinusoid expand,cell vacuolization seriously,glomerular atrophy disappeared,renal tubule structure broken,infected cell vacuoles like degeneration,a large number of granuloma lesions were appeared,epithelial cells swelled,degenerated and abscissed.Compared with the former,it has similar symptoms after infected by Vibrio rotiferianus while a lesser.4.Correlation between intestinal flora and environmental bacteriaThe Illumina Hiseq 2500 platform high throughput sequencing technology was used to analyze the bacterial flora of the intestinal,water,bait and the net fixture.The results showed that the bacteria in the intestinal and the bait were mainly Proteobacteria and Firmicutes,and the sum of relative abundance was more than 95%,but in water and net fixture,the bacterial flora was mainly Proteobacteria,the second was Bacteroidetes and Firmicutes,then Planctomycetes,Actinobacteria,Verrucomicrobia and Cyanobacteria were higher than intestinal and bait.At the level of the genus,the relative abundance of Citrobacter,Acinetobacter and Pseudomonas had high dominance in all samples.In addition,the relative abundance of photolobacterium in intestinal and bait increased in July and November 2016.And the proportion of Vibrio increased significantly in August 2016,September 2016 and August 2017.This may have a great relationship with the disease outbreak in July to November.At the same time,the PCo A analysis showed the Intestinal flora had the greater relevance with bait flora,but little correlation with the flora of water and net fixture. |
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