養(yǎng)殖斑石鯛黑身病的病原病理學(xué)及藥物干預(yù)下腸道菌群結(jié)構(gòu)分析
發(fā)布時間：2018-11-26 20:11
【摘要】：近年來,斑石鯛(Oplegnathus punctatus)在養(yǎng)殖過程中發(fā)生一種黑身病,患病魚體色變黑、活力差、攝食減少、身體消瘦,解剖發(fā)現(xiàn)腹腔內(nèi)有大量無色或淡黃色積水,腸道透明萎縮,肝臟充血易碎,鰓絲出血黏連,累計死亡率達50~90%,給斑石鯛養(yǎng)殖業(yè)帶來重大的經(jīng)濟損失,嚴(yán)重威脅了斑石鯛養(yǎng)殖產(chǎn)業(yè)的健康發(fā)展。本研究針對斑石鯛黑身病,從病原學(xué)、病理學(xué)和腸道菌群結(jié)構(gòu)變化等方面進行了系統(tǒng)研究,以明確該病的發(fā)病原因,并為臨床防控提供理論依據(jù)。利用傳統(tǒng)細(xì)菌分離方法,從自然發(fā)病斑石鯛的腸、肝、鰓、腎等器官中分離得到一株優(yōu)勢細(xì)菌OD1,經(jīng)人工感染試驗表明該菌為斑石鯛黑身病的致病菌。對致病菌進行形態(tài)學(xué)分析發(fā)現(xiàn)該菌是革蘭氏陰性桿菌,電鏡下發(fā)現(xiàn)該菌的具有一條端生鞭毛,大小為1.8~2×0.8~1μm(長×寬)。生理生化特征及16S rDNA基因序列鑒定顯示該病原菌與哈維氏弧菌(Vibrio harveyi)相似性高達99%。藥敏試驗結(jié)果表明,該菌對四環(huán)素、新生霉素、強力霉素和氟苯尼考等藥物高度敏感,對頭孢拉定、克拉霉素等藥物中度敏感,對青霉素、紅霉素等藥物不敏感。對自然發(fā)病的斑石鯛進行病理學(xué)觀察,結(jié)果發(fā)現(xiàn),患病斑石鯛大部分組織器官都發(fā)生了不同程度的病理變化。其中,消化系統(tǒng)的病變最為嚴(yán)重,腸道黏膜上皮細(xì)胞壞死、脫落,上皮細(xì)胞核游離,幾乎無完整細(xì)胞結(jié)構(gòu);肝臟內(nèi)血細(xì)胞浸潤,肝細(xì)胞萎縮、破裂,肝細(xì)胞核膨脹,胞內(nèi)細(xì)胞器減少;膽囊外膜溶解消失,平滑肌變性成網(wǎng)狀結(jié)構(gòu)。除消化系統(tǒng)外,其他主要器官也發(fā)生了不同程度的病變,鰓絲間細(xì)胞溶解消失,上皮細(xì)胞增生相融,超微病理顯示鰓絲上皮細(xì)胞膜溶解,核膜消失,核質(zhì)濃縮;腎臟腎小管腔閉合、萎縮幾近消失,腎小球充血腫脹;脾臟組織疏松,血細(xì)胞壞死堆積;心肌纖維扭曲,細(xì)胞腫脹斷裂等。同時為了深入研究斑石鯛黑身病的病原與發(fā)病原因,探究患黑身病及藥物治療后斑石鯛腸道菌群的結(jié)構(gòu)變化,本研究采用Ilumina HiSeq PE250高通量測序的方法對健康、亞健康、典型黑身病和口服氟苯尼考條件下的斑石鯛腸道以及養(yǎng)殖水體和顆粒餌料中的細(xì)菌多樣性及群落結(jié)構(gòu)進行了解析和相關(guān)性分析。結(jié)果顯示,不同健康程度及氟苯尼考干預(yù)下斑石鯛腸道中細(xì)菌均以變形菌門、厚壁菌門和軟壁菌門為主,且對應(yīng)的OTU占樣品全部OTU的比例均達到85%以上。黑身病的發(fā)生可影響斑石鯛腸道中豐度最高的前20種優(yōu)勢細(xì)菌種類的排名次序,其中變形菌門中的弧菌屬的相對豐度顯著增加,且隨著弧菌屬豐度的增加,斑石鯛的黑身病癥狀也逐漸加重。餌料中添加氟苯尼考投喂斑石鯛能使患病魚腸道弧菌屬的豐度從60.33%下降到1.29%,較大程度的改變了腸道的菌群結(jié)構(gòu),并證實氟苯尼考能有效防治黑身病。同時,養(yǎng)殖水體和顆粒餌料對斑石鯛腸道菌群也有一定影響,且養(yǎng)殖水體對其的影響高于顆粒餌料。本研究分析報道了斑石鯛黑身病的病原學(xué)、組織病理學(xué)和腸道菌群結(jié)構(gòu),其研究結(jié)果為今后斑石鯛的健康養(yǎng)殖和疾病防控提供了參考依據(jù)和技術(shù)支撐。
[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.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S943
本文編號：2359508
[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.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S943
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