양식 뱀장어의 Nocardia seriolae 감염에 대한 병리학적 연구

Abstract

In 2018, a high cumulative mortality rate was observed suddenly in eel farms. We investigated and identified the causative agent as Nocardia seriolae. Nocardia sp. is an aerobic, filamentous, Gram-positive and acid-fast bacteria. To date, it has been shown to be susceptible to various species except Japanese eel, Anguilla japonica. In the eel farm of Incheon, 2,000 fish per day perished for several months, with a cumulative mortality of 20 %. Eight Japanese with skin lesions, disseminated red spots or ulcers, were sampled. Mixture of gill epithelium and radial bacterial clusters, ranging from 30 to 40㎛ in diameter, were observed in whole primary lamella by gill biopsy. Bacteria was isolated from spleen and head kidney, and was performed for amplifying 16S rRNA gene then obtaining nucleotide sequence by Sanger method(accession number in GenBank: MH196537), and was most closely related to N. seriolae AF254418 and N. seriolae LC144544. Histopathologically, massive hemorrhage, disseminated leucocyte infiltration and aggregation, and pyknotic nuclei of parenchymal cells or stroma cells were observed in various organs, and Giemsa staining revealed numerous filamentous bacteria scattered in the inflammatory activated zone. In liver, numerous hepatic artery and central veins were filled with a mixture of erythrocytes and leucocytes, including mostly macrophages or monocytes, and formation of emboli. Ten Nocardia seriolae strains isolated from diseased fish were compared by genetic analysis. N. seriolae could be clustered with 2 groups; 1 group include N. seriolae strains isolated from Anguilliformes and Anabantiformes, and other group include N. seriolae strains isolated from Perciformes. Difference between 1 group and 2 group was observed to numbers of gene in terpenoid biosynthesis. In N. seriolae isolated from eel, presence of genes for an internalin-like protein is seen to be beneficial in the invasion of bacteria. For pathological analysis against N. seriolae infection in eel, artificial infection was conducted with visceral injection method. N. seriolae dose (1.52X106 CFU/fish) similar to LD50 (1.04X106 CFU/fish) was treated, and observed the changes in various organs with light microscope and transmission electron microscope. As a result, the regressive changes(necrosis, atrophy, changes associated with functional impairment) and inflammatory changes were evident in eel treated with N. seriolae compared with control. At 7d post-infection especially, necrosis and inflammatory lesions caused by invasions of N. seriolae were shown in parenchymal tissue in various organs. At 7d post-infection, the production of protein in rough endoplasmic reticulum of liver and activated leukocytes increased cell volume and euchromatin was observed with transmission electron microscope, and Differentially expressed genes(DEG) revealed that many genes associated with endoplasmic reticulum, mitochondria, cytoplasmic vesicle, golgi apparatus, cytosol, vacuole were increased.