Study On The Pathogen Of Ulcer Disease In Catfish-like Loach (Triplophysa Siluorides)

A communicable diseases of cultured catfish-like loach (Triplophysa siluorides) was outbreak in Leshan area of Sichuan Province from Febbruary to March at 2014, which caused mass mortalities of catfish-like loach (approximately 30%). Necropsy and histopathological examination were observed. External clinical signs included the depigmentation of body, ulcerations on the skin and muscle, hemorrhage of belly, and swelling, hyperaemia of gill filament. Based on the pathological examination, we developed research on the aetiology. The anatomy of moribund fish showed bloody ascites, necrosis of the liver, the yellow mucoid fluid in the intestine, hyperemia and hemorrhages in meninges, and swelling of spleen and kidney. On the pathological histology, skeletal muscle was degeneration, necrosis and dissolution with widened muscle space. Hyperemia and hemorrhages were seen in myocardium, and the epicardium was severely hyperplasia and edema. Gill epithelium was swelling and exfoliative, deeply hyperplasia together as one. There was lymphocytopenia in spleen, amounts of hemosiderin. Kidney hematopoietic tissue necrosis, hemorrhage, severe renal tubular epithelial cells vacuoles degeneration, necrosis and disintegration were shown in kidney, while granular and vacuolar degeneration of liver cell, disorder hepatic cords, and some lymphocytes infiltration around blood vessel were observed. Cerebral pia mater was edema, and blood capillary of brain was extravasated blood. Focal necrosis of brain, hyperplasia of miscroglial cells and degeneration of neuron were showed.After examining gill, surface mucus and ulcer lesions via microscopes, there were not parasite, fungus, and gram-negative. In sterile conditions, liver, spleen and kidney of moribund fish were streaked directly onto LB and BHI plate, and inoculated at 28℃ for 48h with no bacteria growning. Then, the organization filtering aseptic liquid of liver, spleen and kidney was inoculated into epithelioma papulossum of carp (EPC) cells. After three passages, the monolayer cells desquamated, forming a network-like monolayer, which formed significant plaque-leision characteristics. After collection and centrifugation, the supernate of cells was inoculated into fresh monolayer cells. So far, we gained a new isolated virus. In order to test Koch’s postulates, the EPC-grown virus was used to inoculate 20 healthy catfish-like loach by intramuscular (IM) and intraperitoneal (IP) injection respectively. The clinical sign and post-mortem lesions of infected fish were similar to those described above, while uninfected control fish remained normal. These observations indicated that the isolated virus was the aetiological agent of this disease. In addition, electron microscopy analysis revealed iridovirus-like particles within the inclusions. All virions were regular hexagon, icosahedral with an outer diameter of 103±7 nm from vertex to vertex. Virions were regularly crystalline-arranged forming a inclusionbody. What’s more, the EPC cells were used to isolate and proliferate virus. Inoculating EPC cells, the virus showed high titer TCID50 upto 1054/0.1ml. After BrdU,, aether, acid, alkali, trypsin and heat treatments, each TCID50 of virus sharply declined, that was to say, the isolated virus might be enveloped DNA virus. With overheating treatment, the isolated virus tended to inactivation. Genomic DNA which was extracted from internal organs was used as a template and amplified by PCR using a pair primers (5’-GAC TTG GCC ACT TAT GAC-3’,5’-GTC TCT GGA GAA GAA GAA-3’) ofranavirus’ maior capsid protein (MCP) gene. The internal organs of moribund fish and the EPC-grown virus were both successfully amplified 500bp target fragments of ranavirus. The PCR products of MCP gene was purified and sequenced. A GenBank NCBI-Blast search on the sequence revealed above 97% identities to ranavirus.To determine the taxonomy of the isolated virus,4 pairs primers of MCP, DNApol, RNR-a and RNR-β genes of ranavirus were syntheticed. Then the DNA of isolated virus was regarded as templatetoamplify, T-clone and sequence.The analysis sequences were used to construct phylogenetic trees as basis of its taxonomic status.The phylogenetic tree which constructed on the multiple alignments of MCP, DNApol, RNR-a and RNR-β genes indicated that the isolated virus was clustered with Chinese giant salamander virus (CGSV), Zuerich pelophylax collection ranavirus (ZPCR), Pike-perch iridovirus (PPIV) and Frog virus 3 (FV3). Among them, CGSV was the most approachto the isolated virus. Combinedwith the 4 genes sequences, the phylogeny tree showed the isolated virus belonged to the amphibian-likeranavirus. This is the first report on a natural ranavirus infection and mortality caused by the pathogen in cultured catfish-like loach.