Histopathology And Etiology Of Golden Cuttlefish(Sepia Esculenta) Vibrionic Necrosis Disease And Drugs Screening For Prevention And Treatment

Golden cuttlefish(Sepia esculenta)is the most economical squid in China and important fishing target.Since the 1980 s,due to the pollution of the marine environment and excessive fishing,its resources have declined significantly,and production has dropped dramatically.Currently,the golden cuttlefish has been extinct in many sea areas.In order to meet the demand for proliferation and resource recovery,since the beginning of 21 st century,China began to study the breeding techniques of the golden cuttlefish and achieved an important breakthrough in 2007.The golden cuttlefish breeding industry has developed rapidly.A disease with necrosis symptom in golden cuttlefish larvae had break in a hatchery in Huangdao District,Qingdao.In this study,the clinical symptoms and histopathology were investigated.The pathogen was isolated and identified,the route of transmission and drug screening were conducted.Aimed to provide basic data and theoretical reference for the prevention and control of the disease and the healthy development of the breeding industry in China.The results were as follows: 1.Histopathology and etiology of golden cuttlefish Vibrionic Necrosis DiseaseThe symptoms including: the dark body,anorexia,slow growth,while the skin,muscle,oral arm,tentacular arm and the internal organs were extensively ulcerated,and the mortality reached as high as 90%.Histopathological analysis showed that the chromatophore was disintegration,the myofilament was broken,the gill filament structure disintegrated,a large number of hepatic lobule epithelial cells were necrotic and the microvilli of intestinal epithelial cells were shed off.Ultra-pathological analysis showed that different degrees of lesions were existed in the liver,intestine,and gill of diseased golden cuttlefish,such as swelling of the nuclei,abnormal cells,dissolution of some cells structures,etc.Three dominant bacteria(SE-A,SE-B,SE-C)were isolated from the diseased golden cuttlefish larvae.Artificial infection experiments confirmed that the strains of SE-B and SE-C were the causative pathogens.Based on the result of morphology observation,physiological and biochemical,and molecular methods(16S rDNA)analysis,SE-B was identified as Vibrio alginolyticus,SE-C was identified as Vibrio tubiashii.According to the symptoms and the pathogens,the disease was named as "cuttlefish vibrionic necrosis disease"(CVND).2.Analysis of pathogenic bacteria and detection of virulence genesBy analyzing the bacterial distribution,dominance and bacterial characteristics of the strains among the diseased golden cuttlefish larvae,source water,breeding water and the live food-mysis,the route of pathogen transmission could be found out.Bacterial culture results showed that there were a lot of bacteria in the mysis and three dominant bacteria(KX-A,KX-B and KX-C)were isolated from the sample.One dominant strain(W1-A)was isolated from the water of the water inlet and another dominant strain(W2-A)was obtained from that of the breeding pool.According to the morphology observation,physiological and biochemical,and 16 S rDNA analysis,KXA was identified as Vibrio shilonii,KX-B?W1-A ? W2-A was identified as Vibrio alginolyticus,KX-C was identified as Vibrio tubiashii.Combined with the management,the quantity comparison of bacteria and Vibrio and the identification of dominant strains in breeding system,it was demonstrated that the two pathogens of the "CVND" in breeding system were mainly introduced by mysis.The results of virulence gene detection of Vibrio alginolyticus and Vibrio tubiashii showed that,within the eleven virulence genes that carried by Vibrio alginolyticus,tlh and tdh were related to hemolytic toxin and ompW,FlaA,and Collagenase were associated with virulence contribution.VthA associated with the hemolysin virulence gene was detected in Vibrio tubiashii.The relevant test results will provide basic data for the analysis of pathogenic pathogenic mechanisms.3.In vitro inhibitory effect of antibacterial drugs and Chinese herbal medicine on pathogenic bacteriaTwo pathogens of the "CVND" were investigated and antimicrobial drugs and Chinese herbs were screened.The chemotherapeutant sensitivity tests illustrated that SE-B was sensitive to doxycycline and florfenicol,etc.Comparatively it was insensitive to penicillin,streptomycin and gentamicin,etc.SE-C was sensitive to florfenicol and lomefloxacin,etc.However,insensitive to neomycin,streptomycin and clarithromycin,etc.Vitro antibacterial experiment demonstrated,the bacteriostatic effect of florfenicol was the best,and the minimum inhibitory concentrations to Vibrio alginolyticus and Vibrio tubiashii were 1.56 ?g/mL and 3.13 ?g/mL,the minimum bactericidal concentrations were 3.13 ?g/mL and 6.25 ?g/mL,separately.Using the Oxford cup and double dilution method,the in vitro antibacterial effect of 15 Chinese herbal targeted on pathogenic bacterias of the "CVND" was detected.The results showed that the four Chinese herbs of Fructus mume,Galla chinensis,Coptis chinensis Franch,and Rheum palmatum had the best antibacterial effect against two pathogenic bacterias.Four kinds of Chinese herbal medicines with different proportions were mixed and sixteen kinds of compounds were obtained.In vitro antibacterial experiments illustrated that,the antibacterial effect of Chinese herbal compounds were obviously better than that of Chinese herbal medicine,the maximum average inhibitory diameter was 23.79 mm,the minimum inhibitory concentrations was reduced to 1.17 mg/mL.By the vitro antibacterial experiment,two kinds of Chinese herbal compounds was selected,the results showed that the mixture of Fructus mume,Coptis chinensis,Franch Rheum palmatum and Galla chinensis with the propoetion of 4:1:4:6 had the best MIC was 0.59 mg/mL on the two pathogenic bacterias,while the mixture of Fructus mume,Coptis chinensis,Franch Rheum palmatum and Galla chinensis with the propoetion of 3:3:2:2 had the best MBC was 0.63mg/mL on the two pathogenic bacterias.In this study,we reported the pathogenic pathology,the analysis of infection pathways and the screening of antibiotics and Chinese herbal medicine of "CVND".This research would provid basic data and theoretical references for the disease prevention and control,and healthy development of the golden cuttlefish breeding industry.At the same time,a special low-toxicity,high-efficiency Chinese herbal compounds for the pathogens of the disease was screened,which laid the foundation for the development of Chinese herbal medicines and the protection of the safety of green foods.