| Summary: | 博士 === 國立中興大學 === 獸醫學系暨研究所 === 100 === 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.
|
|---|
