Establishment Of Channel Catfish Kidney Cell Line And Pathology Study Of Channel Catfish Hemorrhagic Disease

Since channel catfish(Ictalurus punctatus) was introduced into China in1984, it has become an important export species farmed in nearly20provinces in China. According to production estimates, the annual output of farmed channel catfish in China was about220,000metric tons. With the increasing production there has been more epizootic diseases detected in farmed channel catfish in China because of high density stocking and genetic depression. Recently, a hemorrhagic disease in cultured channel catfish fingerlings broke out and spread in the most part of China. It caused large economic losses to channel catfish industry. We attempted to establish a cell line from channel catfish tissues for the isolation of viral pathogen from the hemorrhagic diseased fish. Fortunately, a channel catfish kidney cell line was obtained and it was apllied in the pathology and vaccine research of channel catfish hemorrhagic disease. The results were present as below:1. Establishment of channel catfish kidney cell lineA channel catfish kidney cell line (CCK), was established from the kidney of channel catfish, Ictalurus punctatus, Rafinnesque by using explant techqiques. CCK cell line had been continuously subcultured over70times and been fully characterized in the aspects of morphology observation, optimal growth kinetics, plating efficiency, karyotyping, cryopreservation and28S ribosome RNA genotyping, etc. The CCK cell monolayer was consisted of fibroblast-like cells and the optimal growth condition for CCK cells was:medium, M199; temperature,28-32℃; FBS,10%. The plating efficiency of CCK cells was about74%. Following cryopreservation in liquid nitrogen, thawed cells exhibited a viability of86.69±1.04%after6months storage period. Chromosome typing of CCK cell line revealed that at9th pasage the modal diploid chromosome numeber was2n=58, and at33rd passage the modal number was60. Polymerase chain reaction amplification of partial28S ribosome RNA and sequence analysis indicated94.0%identity to the sequence found in Genbank from channel catfish source, confirming that the cell line was of Channel catfish origin.2. Pathology of channel catfish hemorrhagic disease.A reovirus, designated as CCRV-730, was isolated from channel catfish, Ictalurus punctatus, fingerlings suffering a severe hemorrhage in Hubei province in China. Experimental infection confirmed the pathogenicity of the virus and proved it to be the causative pathogen. Signs of the diseased channel catfish included abdominal distention, eyes bulging and hemorrhages of operculum, lower jaw, skin and fin bases. The CCK cell line was used for viral pathogen isolation. The electron microscopy observation of the virus infected cells revealed that there were a large number of reovirus-like particles measuring60～70nm in diameter in cytoplasm arrayed in crystalline. The viral genomic RNA was extracted and analyzed by SDS-PAGE and the complete S-class (S7-S11) segments of genome RNA were cloned and sequenced. The sequence alignment analysis of the S-class segments of CCRV-730with corresponding sequences of other Aquareovirus members in the genetic sequence database of NCBI indicated that the virus had high similarity to grass carp reovirus, especially shared99%-100%nucleotide sequence identity with the grass carp reovirus873strain (GCRV-873). The results implied that the genetic variation of GCRV-873potentially arose in natural environment and resulted in the viral host conversion or expansion and made it pathogenic to channel catfish.3. Inactivated vaccine of channel catfish hemorrhagic diseaseCell culture inactivated vaccine of channel catfish hemorrhagic disease was produced by using (3propiolactone as the inactivator. The safety and immune effect of the vaccine were assessed.The results showed that CCRV-730virus could be completely inactivated within a final concentration of0.025%(V/V) β-propiolactone at4℃for24h. The vaccine was test by infecting cells and fish to confirm the virus had been completely inactivated. Bacterial test showed no bacterial contamination of the vaccine. These results confirmed the safty of the produced vaccine. Immune protective effect test results show that, under laboratory conditions, the8～10cm channel catfish fingerlings intraperitoneal injection0.2mL105TCID5o/mL vaccine could obtain up to89.1%protective rate. Immune protective effect test in the fish cultured in ponds showed that4～6cm small size fingerlings, which were immunized by immersion, could obtain72.6%protective rate. These results indicate that channel catfish fingerlings immunized with P-propiolactone inactivated channel catfish hemorrhagic disease vaccine by injection or immersion could obtain good protective force. This vaccine can be widely used in the production in order to prevent the channel catfish hemorrhagic disease.