养殖斑石鲷黑身病的病原病理学及药物干预下肠道菌群结构分析

发布时间：2018-11-26 20:11

【摘要】：近年来,斑石鲷(Oplegnathus punctatus)在养殖过程中发生一种黑身病,患病鱼体色变黑、活力差、摄食减少、身体消瘦,解剖发现腹腔内有大量无色或淡黄色积水,肠道透明萎缩,肝脏充血易碎,鳃丝出血黏连,累计死亡率达50~90%,给斑石鲷养殖业带来重大的经济损失,严重威胁了斑石鲷养殖产业的健康发展。本研究针对斑石鲷黑身病,从病原学、病理学和肠道菌群结构变化等方面进行了系统研究,以明确该病的发病原因,并为临床防控提供理论依据。利用传统细菌分离方法,从自然发病斑石鲷的肠、肝、鳃、肾等器官中分离得到一株优势细菌OD1,经人工感染试验表明该菌为斑石鲷黑身病的致病菌。对致病菌进行形态学分析发现该菌是革兰氏阴性杆菌,电镜下发现该菌的具有一条端生鞭毛,大小为1.8~2×0.8~1μm(长×宽)。生理生化特征及16S rDNA基因序列鉴定显示该病原菌与哈维氏弧菌(Vibrio harveyi)相似性高达99%。药敏试验结果表明,该菌对四环素、新生霉素、强力霉素和氟苯尼考等药物高度敏感,对头孢拉定、克拉霉素等药物中度敏感,对青霉素、红霉素等药物不敏感。对自然发病的斑石鲷进行病理学观察,结果发现,患病斑石鲷大部分组织器官都发生了不同程度的病理变化。其中,消化系统的病变最为严重,肠道黏膜上皮细胞坏死、脱落,上皮细胞核游离,几乎无完整细胞结构;肝脏内血细胞浸润,肝细胞萎缩、破裂,肝细胞核膨胀,胞内细胞器减少;胆囊外膜溶解消失,平滑肌变性成网状结构。除消化系统外,其他主要器官也发生了不同程度的病变,鳃丝间细胞溶解消失,上皮细胞增生相融,超微病理显示鳃丝上皮细胞膜溶解,核膜消失,核质浓缩;肾脏肾小管腔闭合、萎缩几近消失,肾小球充血肿胀;脾脏组织疏松,血细胞坏死堆积;心肌纤维扭曲,细胞肿胀断裂等。同时为了深入研究斑石鲷黑身病的病原与发病原因,探究患黑身病及药物治疗后斑石鲷肠道菌群的结构变化,本研究采用Ilumina HiSeq PE250高通量测序的方法对健康、亚健康、典型黑身病和口服氟苯尼考条件下的斑石鲷肠道以及养殖水体和颗粒饵料中的细菌多样性及群落结构进行了解析和相关性分析。结果显示,不同健康程度及氟苯尼考干预下斑石鲷肠道中细菌均以变形菌门、厚壁菌门和软壁菌门为主,且对应的OTU占样品全部OTU的比例均达到85%以上。黑身病的发生可影响斑石鲷肠道中丰度最高的前20种优势细菌种类的排名次序,其中变形菌门中的弧菌属的相对丰度显著增加,且随着弧菌属丰度的增加,斑石鲷的黑身病症状也逐渐加重。饵料中添加氟苯尼考投喂斑石鲷能使患病鱼肠道弧菌属的丰度从60.33%下降到1.29%,较大程度的改变了肠道的菌群结构,并证实氟苯尼考能有效防治黑身病。同时,养殖水体和颗粒饵料对斑石鲷肠道菌群也有一定影响,且养殖水体对其的影响高于颗粒饵料。本研究分析报道了斑石鲷黑身病的病原学、组织病理学和肠道菌群结构,其研究结果为今后斑石鲷的健康养殖和疾病防控提供了参考依据和技术支撑。
[Abstract]:In recent years, the Olegatus punctatus has a black body disease in the process of culture, the color of the diseased fish is black, the vitality is poor, the food consumption is reduced, the body is emaciated, the abdominal cavity is found to have a large amount of colorless or light yellow accumulated water, the intestinal tract is transparent and atrophic, the liver is hyperemia and fragile, The accumulated mortality rate is 50-90%, which brings great economic loss to the mariculture industry, which is a serious threat to the healthy development of the mariculture industry. In this study, a systematic study was carried out to identify the cause of the disease and to provide a theoretical basis for clinical prevention and control. A dominant bacterium, OD1, was isolated from the organs such as the intestine, the liver, the liver, the kidney and the like of the natural-onset grouper, using the traditional method of bacteria separation. The results of the artificial infection show that the bacteria are the pathogenic bacteria of the black body disease of the grouper. A morphological analysis of the pathogenic bacteria found that the bacterium was a gram-negative bacterium, and the bacterium was found to have an end-producing flagellum with a size of 1.8-2-0.8-1. m u.m (long-width-width) under the electron microscope. The physiological and biochemical characteristics and the 16S rDNA sequence identification showed that the similarity of the pathogenic bacteria and Vibrio harveyi was up to 99%. The results of the drug sensitivity test show that the bacteria are highly sensitive to the drugs such as tetracycline, neonatal mycin, doxycycline and florfenicol, and are sensitive to the drugs such as cefradine and clarithromycin, and are not sensitive to the drugs such as penicillin and erythromycin. The results showed that most of the tissues and organs of the grouper had different degree of pathological change. wherein, the pathological changes of the digestive system are the most serious, the intestinal mucosa epithelial cells are necrotic, the epithelial cells of the epithelial cells are free, and the whole cell structure is almost completely free; the infiltration of the blood cells in the liver, the atrophy of the liver cells, the rupture, the expansion of the liver nucleus and the reduction of the intracellular organelles, and the dissolution of the outer membrane of the gall bladder, the smooth muscle is denatured into a net structure. in addition to that digestive system, the other major organs also have different degree of pathological change, the dissolution of the inter-filament cells disappear, the epithelial cell proliferation and the micropathology show the dissolution of the membrane of the silk epithelial cell membrane, the nuclear membrane disappears, the nuclear substance is concentrated, the renal tubular cavity of the kidney is closed, the atrophy is almost lost, Glomerular congestion and swelling; loose spleen tissue, accumulation of blood cell necrosis; distortion of cardiac muscle fibers, cell swelling and fracture, etc. At the same time, in order to study the cause of the pathogen and the cause of the black body disease of the grouper, the structure of the intestinal flora of the grouper was investigated, and the method of high-throughput sequencing of Iumina HiSeq PE250 was used to study the health and sub-health. The bacterial diversity and community structure in the intestinal tract and the culture water body and the granular bait were analyzed and the correlation analysis was carried out by the typical black body disease and the oral florfenicol. The results showed that the bacteria in the intestinal tract of the grouper were mainly caused by the strain, the thick wall and the soft wall, and the corresponding OTU accounted for more than 85% of the total OTU. The occurrence of the black body disease can affect the ranking order of the first 20 dominant bacteria species in the intestinal tract of the grouper, in which the relative abundance of the genus Vibrio in the strain is significantly increased, and the black body disease symptoms of the grouper are also gradually increased with the increase of the abundance of the genus Vibrio. The addition of florfenicol to the feed was reduced from 60. 33% to 1.29%, and the bacteria group structure of the intestinal tract was changed to a great extent, and it was confirmed that the florfenicol was effective in preventing and treating the black body. in that meantime, the culture water body and the particle bait have certain influence on the intestinal flora of the grouper, and the effect of the culture water body on the intestinal flora is higher than that of the particle bait. The etiology, histopathology and intestinal flora structure of the black body of the snapper are reported in this study. The results of this study provide a reference and technical support for the healthy breeding and disease prevention and control of the grouper.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S943

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