| Giant salamander (Andrias davidianus) belongs to Amphibia, Anura, cryptobr--achidae, and it’s the largest remaining precious amphibian species and endemic to China. It has been as Chinese class Ⅱ national key protected wild animals,the World Conservation Union Species Survival Commission (IUCN/SSC) and the Chinese Species Red List of threatened the status quo of the giant salamander listed as critically endangered (CR) grade. In the1970s, China began the domestication and breeding trials of Chinese giant salamander,90years later, with the chartered clear policyof the aquatic wildlife and the successful artificial breeding of giant salamander, adhering to the "protection of the development, and promote the development of protection" concepts, the domestication and breeding flourishof giant salamander, formed "the original ecological protection, bionic cultivation of artificial propagation, intensive farming" model of development. In recent years, it has found in Shaanxi, Sichuan, Gansu, Hunan, Chongqing and Guizhou that other new infectious diseases incidence of giant salamander which mainly as a serious hazard to the giant salamander surface bleeding spots, ulcers, head and swelling of the limbs, it was called the canker or "Bigfoot disease". Once the disease occurred, it often leads to high mortality rates, even up to100%, causing serious economic losses to the farmers, so it is also known as "giant salamander canceramong the majority of farmers. This study, in Leshan, Sichuan a giant salamander farms in the natural incidence of giant salamander as the research object, the infection of Ranavirus by PCR, morphological changes, and on this basis, the EPC cells were used to isolate the Chinese Giant Salamander ranavirus (Chinese giant salamander virus, CGSV-L)and biological characteristics in part, in order to understand the cause of the disease and pathological characteristics of injuries, to provide a theoretical basis of the clinical diagnosis of the disease, prevention and treatment, as well as lay the foundation for the elucidation of the pathogenesis of the disease. ] The detection of ranavirus in giant salamander by PCRTake sick giant salamanders’liver, spleen and kidney tissues to extract virus DNA, primers were designed to amplify the specific Ranavirus major capsid protein (Major capsid protein, MCP)5’end500bp genereference to Mao et al., then the gel electrophoresis showed, extracted DNA of liver, spleen and kidney can be amplified out500bpDNA fragment, i.e. it’s strong positive of Ranavirus infection in giant salamander.2Isolation and physical and chemical characteristics of Chinese giant salamander ranavirusThe Epithelioma Papillosum Cyprini(EPC) cell was used to isolate and proliferate CGSV-L virus, after the virus infected EPC cells showed typical cytopathic effect (CPE) and gained high titer TCID50, up to105.0/0.1ml. Virus cultured in multi-temperature conditions, the results showed that25℃was most suitable for the proliferation of the virus. Diethyl ether, chloroform, acid, alkali, trypsin and heat sensitive, as well as BrdU inhibition study, suggested that the virus mightbe enveloped DNA virus, under the peracids, too alkaline, trypsin treatment and thermal conditions, the virus is easy to inactivation.3Researches of the major functional gene of Chinese giant salamander virusCGSV-L genomic DNA as the template, genes using specific primers to amplify MCP gene, the DNA polymerase gene and NF-H1gene fragment by PCR, and the target fragments were cloned and sequenced, the sequences’analysis were used to construct phylogenetic tree as analysis of its taxonomic status. The results showed that the CGSV-L is a new member of Ranavirus, having high sequence similarity of reported ranavirus genes in GeneBank. Based on MCP genes and DNApolymerase gene, phylogenetic tree revealed CGSV-L virus on the taxonomic status is closer to FV3, Hynobius nebulosus virus, NF-H1gene-based phylogenetic tree:CGSV-L and FV3were in a large classification, but closer to Ranavirus KRV-1, soft-shelled turtle iridovirus and iridovirus Rgrylio.4Pathological studies of Ranavirus natural infectiongiant salamanderUsing the pathological anatomy technology, tissue sections technology, ultra-thin slicing technology to general, tissues and cells lesion of giant salamandernaturally infected with Ranavirus, results showed the giant salamanders’ mainly grossly symptoms including systemic swelling, congestion or hemorrhage in jaw and abdominal, limbs and skin necrosis, muscle ulceration and even amputation phenomena, as well as the liver, spleen and kidney were swelling, necrosis. Pathological manifestations of body tissues and organs revealed generalized edema, hemorrhage, degeneration; the kidney, liver, spleen, skin, muscle and gastrointestinal damages were most severe, and with round or oval eosinophilic cytoplasm of diseased tissue inclusion bodies, some cells could see the round basophilic inclusion bodies. Vacuolar degeneration and necrosis were in renal tubular epithelial cells, with renal interstitial inflammatory cell infiltration; severe vacuolar degeneration of the liver cells with focal cytolytic necrosis, a large number of lymphocyte arranged in interstitial gathered into a group; spleen lymphocyte necrosis, the number reduced, a large number of pink-stained protein-like substance was deposited among the spleen and marrow cells as well ason the reticular fibers; gastrointestinal thehemorrhage-catarrhus inflammation; skin was edema, and epidermal cell was degeneration, necrosis, muscle was waxy necrosis, with inflammatory cell infiltration, toformulcers stove. By transmission electron microscopy, it’s visible that liver’s mitochondria swelling, nuclear chromatin concentrated, the expansion of the endoplasmic reticulum; increase of lysosomes, mitochondrial swelling, partial condensation or the formation of myelin-like structure inrenal tubular epithelial cells; spleen lymphocytes significantly reduced with the proliferation of polymorphous reticular cells and macrophages. Regular hexagon visible inside with diameter of140-180nm for the virus particles scattered or crystal-like arrangement in the cell.5The morphogenesis of Chinese giant salamander ranavirus in the EPC cellsThe EPC cell samples at different times after infected the CGSV-L were collected, then ultrathin sections with uranyl acetate and lead citrate, under the transmission electron microscope the morphology and assembly processof CGSV-L in the EPC cell, showed the progeny virions was visible after infected16h,72h peaked. The virus particles had unique morphological characteristics, the size of the virus was approximately160nm, a hexagonal shape, in line with the morphological characteristics that belong to the Ranavirus. The completion of thereplication of the viral genome, amplification, synthesis, assembly and nucleocapsidin the nucleus of the host cell; assembly areas with low electron density was formed in the cytoplasm with the mature virus particles and empty capsids gather into a crystal-like arrangement in the cytoplasm, or scattered in the cytoplasm, or the formation of inclusion bodies; final maturation of the virus particle was released from membrane cells by budding. |
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