양식 돌기해삼 , Apostichopus japonicus의 세균 감염증과 방어인자에 관한 연구

Abstract

Sea cucumber (Apostichopus japonicus) is an economically important species and recently its culture has become very popular in china. However, the disaccord of the development of intensive culture with the research of Apostichopus japonicus biology and culture technique has caused the emergence of significant diseases and severe economic loss. Cultured Apostichopus japonicus suffering from serious diseases with massive mortality. Clinical signs included mouth tumidity, shaking head, skin ulceration during the winter from 2004 to 2006 in Dalian, China. In order to make clear the cause of diseases, prevent and cure them, we carried out a series of research, including Apostichopus japonicus immunity, bacterial diversity in the internal bodily and external environment of healthy and diseased sea cucumbers, identification and biological characteristics of pathogenic bacteria infection, the relationship between bacterial infection and organism immunity. The results can be summarized as follows: 1. Bacteria diversity in the internal bodily and external environment of healthy and diseased sea cucumber (1) Indoors the number of heterotrophic bacteria and Vibrio spp. in intestine and sediment was highest, and that in skin, coelomic fluid, net and water was similar. The predominant bacteria belonged to Vibrio spp. (62.71 %), Enterobacteriaceae (8.47 %), Aeromonas spp. (6.48 %), Pseudomonas spp. (6.48 %), Moraxella spp. (5.08 %), Acinetobacter spp. (3.39 %), Alcaligenes sp. (1.69 %), Agrobacterium sp. (1.69 %), respectively. (2) Outdoors the number of bacteria in intestine and sediment was the highest. The predominant bacteria belonged to Vibrio spp. (46.15 %), Flavobacterium spp. (12.82 %), Enterobacteriaceae (10.26 %), Aeromonas spp. (10.26 %), Pseudomonas spp. (5.13 %), Agrobacterium spp. (5.13 %), Pseudoalteromonas sp. (2.56 %), Moraxella sp. (2.56 %), Acinetobacter sp. (2.56 %), Alcaligenes sp. (2.56 %), respectively. (3) The number of heterotrophic bacteria and Vibrio spp. isolated from skin and sediments in diseased Apostichopus japonicus was 3∼4 times more than in the healthy individuals. The predominant bacteria belonged to Vibrio spp. (64.51 %), Serratia spp. (12.9 %), Shewanella spp. (12.9 %), Pseudoalteromonas spp. (6.45 %) and Flavobacterium sp. (3.22 %), and the thirdly most predominant strains were V. splendidus (41.94 %), Serratia odorifera biogroup Ⅰ(12.9 %）and Shewanella spp. (12.9 %), respectively. Shewanella spp. was only isolated from diseased Apostichopus japonicus. 2. Determination of pathogenic bacteria and microbiological charateristics of Shewanella marisflavi (1) On the basis of challenge tests, 13 of 31 strains isolated were pathogenic to Apostichopus japonicus, which were 8 strains of V. splendidus, 3 strains of Shewanella spp. and 2 strains of Pseudoalteromonas tetraodonis, respectively. Interestingly, 8 of 13 strains of V. splendidus isolated were pathogenic and their frequency appeared was highest. (2) Artificial infection by all these pathogens causes disease and mortality in sea cucumber, swordtail fish (Xiphophorus helleri) and mice. Based on LD50, the virulence order of pathogens was: S. marisflavi ＞ V. splendidus ＞P. tetraodonis. and the LD50 for sea cucumber was higher than for swordtail fish and mice. (3) Environmental stressors of salinity, pH and temperature on virulence of pathogens for Apostichopus japonicus would shorten time of death and induce higher mortality. Apostichopus japonicus was more susceptive to higher temperature, lower salinity and lower pH. Environmental factors beyond normal limit seem to be responsible for the disease. (4) Colonies of S. marisflavi were light red, 1.2∼2mm sized rod with viscosity on marine agar (MA, Difco) medium after incubating at 25 ℃ for at least 48h and green on thiosulfate-citrate-bile-salts-sucrose (TCBS) medium. In transmission electron microscopy, S. marisflavi had polar flagella and visible secretion around. (5) S. marisflavi was Gram-negative, oxidase- and catalase-positive and not sensitive to O/129. It caused clear hemolysis on sheep blood agar (β- hemolysis) and produced H2S. The condition of 4∼35 ℃, pH 6.0∼9.2 and 0∼8 % NaCl was needed for growth of S. marisflavi. (6) The phylogenetic tree based on 16S rDNA genes sequence revealed that the pathogen had the closest phylogenetic affiliations to the S. marisflavi KCCM 41822 [Accession number：AY485224], the similarity and bootstrap value between them were 99.86 % and 100 %, respectively. (4) ECPs of S. marisflavi showed positive values for caseinolytic, amylase, lipase and phospholipase activity. The proteinase activity decreased below 40 ℃, ECP's optimal condition were under 2.0∼4.0 % NaCl, 28 ℃, pH 7.5, BHIA and incubating 35 hours. 3. Apostichopus japonicus cellular immunity and the relationship between bacterial infection and organism immunity (1) Through light and electron microscopy, coelomocytes of Apostichopus japonicus can be divided into six types: lymphoid cells, spherulocytes, ameboid phagocytes, hyaline cells (or hyalinocytes), fusiform cells and crystal cells. Furthermore, spherulocytes can be divided into typeⅠ,Ⅱ and Ⅲ. The annual average of total coelomocytes count (TCC) was (3.85±0.21) × 106 cells/㎖, the count of lymphoid cells, spherulocytes and ameboid phagocytes was (1.58±0.11) × 106cells/㎖, (41±1.47) %; (1.16±0.13) × 106 cells/㎖, (30±0.89) % and (0.99±0.07) × 106 cells/㎖, (25±0.98) % of THC, respectively. (2) Spherulocytes and amebocytes had the ability to phagocytize carmine and yeast, and phagocytic percent and phagocytic index were correlated to the size and the kind of foreign bodies. Furthermore, phagocytic ability was correlated to temperature, the peak phagocytic percent and index appeared at 18 ℃, followed by at 12 ℃, and then at 4 ℃ and 25 ℃. In addition, coelomocytes had the ability to agglutinate in vitro and the agglutination was correlated to the stimulation by temperature and foreign bodies. (3) Antibacterial activity (Ua), lysozyme activity (Ul), acid phosphatase (ACP), alkalphosphatase (ALP), catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) except for phenoloxidase (PO) were determined in different tissues (tentacle, body wall, intestine, respiratory tree, muscle, coelomic fluid, extraction of coelomocytes and body surface mucus) of Apostichopus japonicus. Additionally, the discrepancy of immune enzymes activity in tissues existed among different seasons. Two peak values of all the enzymes appeared on May and November, the least appeared on August. (4) Enzyme cytochemistry showed that the activity of ALP, ACP and POD was determined in lymphoid cells and spherulocytes. Ameboid phagocytes was ALP positive and all coelomocytes were PO negative. The stratum corneum and epithelium layer of tentacle, intestine, respiratory tree, muscle and body wall were ALP negative. The epithelium layer and back layer of tentacle, mid-gut, respiratory tree, muscle and body wall were ACP negative. The connective tissue of tentacle, intestinal mucosa, outer connective tissue of respiratory tree and corium cutis of body wall were POD negative. All the tissues above were PO negative. (5) The number of THC in Apostichopus japonicus after infected by pathogenic bacteria showed a repeated trend: reduces first and then increases. The variety of spherulocytes and lymphoid cells occurred synchronously. (6) Both pathogenic and non-pathogenic bacteria induced similar degree of immune related enzymes activity. The effect of higher bacterial cell concentration had more obvious than that of the lower concentration.