Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/12962
標題: 中華鱉之解剖、血液相觀察及首次分離到類痘病毒之研究
The Studies of Anatomy, Hematology, and the First Isolated Poxvirus-like Virus in the Soft Shell Turtle, Pelodiscus sinensis
作者: 劉邦成
Liu, Pan-Chen
關鍵字: anatomy
中華鱉
hematology
poxvirus
soft shell turtle
Pelodiscus sinensis
解剖
血液相
類痘病毒
出版社: 獸醫學系暨研究所
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摘要: 本論文為世界第一個發現養殖鱉皮膚類痘病毒感染症,本研究的目的是觀察和研究中華鱉之解剖和血液相並建立胚胎初代細胞培養以進行鱉類痘病毒複製及其特性之研究。鱉由尾側靜脈抽血,傷害性最小,較容易抽到血。血液抹片檢查可見紅血球有細胞核,單核球細胞質有空泡,細胞核形為多形性,嗜酸性球核有分葉,異嗜球細胞質呈長條桿狀。雄鱉副睪與腎臟相連,精液經陰莖溝流入雌鱉泄殖腔內。1.5 歲齡成鱉肝臟為黃棕色,隨著年齡增加,黑色素吞噬細胞增多,顏色愈暗沉,7 歲齡成鱉肝臟呈黑色。肝臟分左右兩大葉,腹側面可見右葉比左葉大,肝臟右葉可分為外葉、中葉和內葉,中葉又可分成中外葉和中內葉,左葉可分成分離葉和內葉;7 歲齡成鱉肝臟葉數跟 1.5 歲齡成鱉相同,但左葉的分離葉會變細長。2 月齡鱉肝臟組織無明顯肝小葉結構,門脈三角區不明顯,少量黑色素吞噬細胞呈散發狀。鱉肺臟無肺泡結構,但有非常多的末端氣體交換單元,有氣管軟骨與蛇相似,肺間質有少量黑色素吞噬細胞。心臟為二心房一心室,有一對大動脈和一對大靜脈,心室和心房間有瓣膜,心室腔中有一軟骨結構之不完全間隔。鱉腎臟為分葉型腎,呈黃棕色或暗紅色,有絲球體和腎小管,無明顯皮質跟髓質區分。組織學檢查雌鱉泄殖腔,有消化道開口、泌尿道開口和輸卵管開口黏膜層,輸卵管黏膜層有形成蛋殼的上皮結構,輸卵管和泄殖腔交界處黏膜層有纖毛,但無形成蛋殼的上皮結構。某一飼養 20,500 隻 50 日齡中華鱉的養殖場,因感染鱉類痘病毒,於一個月內鱉大量死亡,死亡率約 44 %,導致農民嚴重的經濟損失。臨床症狀可見病鱉厭食、昏睡、頸部腫大,在鱉甲及腿部皮膚有許多水疱及大疱。組織病理學檢查可見典型的急性病變,包括氣球樣變性及表皮角質細胞急性細胞腫脹,在空泡可見小的嗜伊紅性質內包涵體;壞死表皮脫落及真皮層有炎症反應細胞和大量的異噬球浸入,皮膚潰瘍並有深層的炎症反應及細菌二次感染。以 6 隻鱉進行人工接種類痘病毒液,於接種 14 天內實驗鱉陸續死亡,臨床症狀呈現厭食和昏睡,皮膚病灶與養殖場病鱉類似,在頭、鱉甲及腿部皮膚有中至大型水疱,白色環形薄層膿疱及壞死剝落的表皮。鱉胚胎於氣室接種類痘病毒液,3 天後在絨毛尿囊膜有許多白色的痘斑產生。以穿透式電子顯微鏡檢查養殖場病鱉和實驗接種鱉病變處皮膚,可見大量呈卵圓形、有封套,平均直徑為 239 nm,長度為 483 nm 的病毒顆粒。依型態及大小作比較,此病毒與痘病毒十分相似。為進行鱉類痘病毒複製及特性之研究,因此建立鱉胚胎初代細胞體外培養,至目前為止,在 25 T 細胞培養瓶內,鱉胚胎初代細胞已培養至第 25 代,依型態學判斷,大多為纖維母細胞。細胞培養基之研究結果顯示,L15 培養基最適合鱉胚胎初代細胞培養;RPMI 培養基雖然可用於鱉胚胎初代細胞培養,但細胞生長效果較 L15 培養基差;然而 MEM培養基並不適合鱉胚胎初代細胞培養。鱉胚胎初代細胞,在 37 °C 的環境下生長速度最快。加入 200 μL 病鱉皮膚類痘病毒乳劑,於 24 孔長滿鱉胚胎初代細胞的培養盤內,在第 2 天可見細胞病變;然而僅加入 20 μL 病鱉皮膚類痘病毒乳劑,在第 5 天才可見細胞病變。以穿透式電子顯微鏡檢查產生細胞病變的初代細胞,可見大量呈卵圓形、有封套的類痘病毒顆粒。類痘病毒毒力顯著的被 5-iodo-2’-deoxyuridine (IUDR)、氯仿和放線菌素D (actinomycin D) 抑制,然而病毒力價則無顯著性被5-bromo-2’-deoxyuridine (BUDR) 抑制;受類痘病毒感染的初代細胞,以acridine orange染色結果顯示細胞呈黃綠色。綜合以上結果顯示,鱉類痘病毒為雙股核酸有封套的 DNA病毒。本研究發展鱉胚胎初代細胞培養,已成功地複製鱉類痘病毒,實驗結果可提供學者對養殖鱉類痘病毒感染症有更深刻的了解。
The aims of this study are to investigate the anatomy and hematology of the soft shell turtle, Pelodiscus sinensis and to establish a primary cell culture for our isolated poxvirus-like virus replication and studies. Blood collection from caudal veins is the least harmful for the soft shell turtle;meanwhile, it is more easily to get blood from the turtles. The results of the soft shell turtle hematology revealed that their erythrocytes were with nucleus, monocytes were with pleomorphic nuclear shape and some vacuoles in the cytoplasm, eosinophils were with lobed nucleus, and heterophils were with rod-shaped cytoplasm. The results of the soft shell turtle anatomy showed that the epididymides of soft shell turtles were connected with the kidney. Their semen passed through the dorsal groove of penis into female soft shell turtle's cloaca. Interestingly, the color of livers of 1.5-year-old and 7-year-old turtles were yellow-brown color and black color, respectively. In ventral, the right lobes of the liver were larger than the left lobes. The right lobes of the liver were divided into outer lobe, middle lobes, and inner lobe. The middle lobes were divided into outer lobe and inner lobe. The left lobes were divided into separation lobe and inner lobe. The separation lobe of 7-year-old turtle was slender than the lobe of 1.5-year-old turtle. No remarkable hepatic lobule structure and portal tract was seen in the liver of 2-month-old turtles. The tracheal cartilages and numerous terminal gas exchange units were seen in soft shell turtle's lung. However, there was no alveolus was seen in the lung. A soft shell turtle's heart had a pair of large arteries, a pair of large veins, one semi-circular ventricle and two atriums, a cartilage, and atrioventricular valves. Their kidneys had no remarkably cortex and medulla differention but there were many glomeruli and tubules. The lobulated kidneys showed yellow-brown or dark red color. Cloaca of female turtle revealed the mucosa layer of digestive tract opening, the mucosa layer of urinary tract opening, and the mucosa layer of oviduct opening. The mucosa layer of oviduct suggested the shell forming epithelial structure. The mucosa layer of oviduct cloaca junction had cilia but it lacked the shell forming epithelial structure. An outbreak of a poxvirus-like infection in a population of 20,500 at 50-day-old soft shell turtles in a cultured farm. The mortality rate was approximately 44 %. The clinical findings of the diseased turtles included anorexia, lethargy, swollen necks and several unusual vesicles, and bullas were present on the legs and shell skin. Histopathological examination results revealed typical acute lesions, such as ballooning degeneration and acute cell swelling of keratinocytes of the epidermis. Furthermore, tiny eosinophilic cytoplasmic inclusion bodies were observed in the vacuoles. Sloughed necrotic epidermis, inflammatory cells, and a predominance of heterophils were found to have infiltrated the area of the exposed underlying dermis. In addition, patches of skin ulceration and deep layer inflammation were present, and secondary bacterial infection was suspected. Six soft shell turtles were challenged with a 0.45 μm filtered viral suspension. They showed anorexia and lethargy. The observed cutaneous lesions of the 14 days post-challenge were similar to the reported clinical findings including medium to large size vesicles, white and circular thin layered pustules and sloughed necrotic epidermis over the legs, head and shell skin of the infected turtles in the cultured farm. In addition, numerous white pock lesions on the chorioallantoic membranes (CAM) of embryonated soft shell turtle eggs inoculated with the viral suspension and observed 3 days post-challenge. The result of a transmission electron microscopy (TEM) study conducted from both farmed and experimentally infected turtles revealed enveloped, ovoid-shaped viral particles, measuring approximately 239 nm in diameter and 483 nm in length. According to its morphology and size, the virus was very similar to a poxvirus. In order to establish a primary embryo cell culture from a soft shell turtle for the first isolated soft shell turtle poxvirus-like replication and characteristics studies. Until today, the primary culture cells have been subcltured more than 25 passages on the 25 T cell culture flask. Morphology of these cells most are fibroblast cells. From our study results, the L15 medium is the optimal culture medium for those cells; meanwhile, the MEM is not suited for soft shell turtle primary cells growing. Interestingly, those soft shell turtle primary cells grow more fast at 37 °C than below 30 °C. We found that the lysis cellular pathogenic effects (CPE) presented on the mono-layer cell culture inoculationed with 200 μL of our isolated poxvirus-like viral suspension on the 2nd postinoculation day. However, the CPEs showed among the mono-layer cultured cells inoculationed with 20 μL of above viral suspension on the 5th postinoculation day. From the TEM results, the harvested virus from the infected primary cells revealed that the same our inoculated poxvirus-like virus with enveloped and ovoid shaped viral particles. The virulence of poxvirus-like virus was significantly inhibited by 5-iodo-2'-deoxyuridine (IUDR), chloroform and actinomycin D but not significantly affected by 5-bromo-2'-deoxyuridine (BUDR). According the acridine orange staining results revealed the viral infected primary cells shown yellow-green color, the poxvirus-like virus is identified as a double stranded enveloped DNA virus. Our set primary culture cell is able to be very successfully infected with the soft shell turtle poxvirus-like virus. This may be the first study about the primary culture from soft shell turtle embryo for poxvirus-like virus replication. The findings detailed in this study may allow researchers to gain a better understanding of the infectious diseases present in cultured soft shell turtle farms.
URI: http://hdl.handle.net/11455/12962
其他識別: U0005-2012201114292600
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2012201114292600
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