한국의 양식 넙치에서 발생하는 여윔병의 원인 분석

Abstract

The olive flounder industry, a representative species of domestic aquaculture industry, has shown steady and continuous growth since artificial seedling production technique was developed in National Fisheries Research and Development Institute (NFRDI) early 1980's. As it is a species with aquaculture history longer than 30 years, the study of its diseases has a wide variety compared with that of other species. But in Korea, researches on diseases of cultured fishes have been limited and focused only on several specific diseases (Kim et al., 2006; Cho et al., 2008; Kim et al., 2010), and there are few researches on diseases of which the causes are not known. Recently about 20cm long olive flounders in farms on Jeju Island had been increasingly thinner and perished within 1~3 weeks. Unlike Japan, where the causes of emaciation disease were found, there's no report on its causes in Korea. In this study, to identify emaciation infection in cultured olive flounders in Korea, a primer set useful for aquaculturing olive flounder was developed and a test method was established and applied to the fields. In addition, the features of emaciation were identified using PCR, microscopy, histopathological examinations, molecular phylogenetic systematic, real-time PCR etc. and experiments were performed to determine the causes of emaciation. To analyze the causes of emaciation of olive flounders with emaciation symptoms in Korea, a primer set identical with MM18Sf/MM18Sr primer set was developed. The MM18Sf/MM18Sr primer set had been made from small subunit ribosomal DNA gene (SSU rDNA) of E. leei reported as the cause of emaciated turbot in Japan. In the PCR test, for which DNA was taken from intestinal tissue of emaciated olive flounders in Korea, all of them showed negative reactions. We made the EM-F/EM-R primer set from the base sequences of the Myxidium sp., which is registered in GenBank (NCBI, USA). The primer set was applied to emaciated olive flounders in Korea, and the response was positive. Therefore we came to use it for examining emaciated olive flounders in Korea. To observe the infected area in the emaciated olive flounders, PCR was performed on extracted kidneys, intestines, spleens, brains, livers and gills. Whereas the causes were found mostly in intestines in Japanese report on emaciation, in this study PCR was positive not only in intestines but also in kidneys. In case of significantly emaciated fishes, it was positive in all internal organs except gills. Detection frequency was in order of kidneys, intestines and spleens. To identify the infection situation of emaciation disease, 216 olive flounders out of 24 farms were investigated in the period of 2010 to 2013. Most of the infected fishes showed significant emaciation in abdominal area. The weight of emaciated fish was 30~40% lower than that of uninfected fish and 33 (50%) out of 66 farms were positive in infection test. Yearly infection rates were so high with 8 out of 21 farms (38%) in 2010, 12 out of 25 farms (48 %) in 2011, 2 out of 4 farms (50%) in 2012 and 11out of 16 farms (63.1%) in 2013 that they can cause serious damage to farms. The tissue sections of organs were observed with optical microscope, and many spores of circular or oval shape were detected in kidneys and intestines of emaciated olive flounders. In other organs except for kidneys and intestines, some spores were observed, but it was difficult to examine because the number of detected spores was small compared with that in kidneys and intestines or no specific histological abnormalities were observed. Therefore, histopathologic emaciation examination should be performed mainly in kidneys and intestines. According to gene sequencing analysis of PCR products, the gene homology between pathogens detected in emaciated olive flounders was more than 99%. According to gene comparison analysis using the GenBank database, it showed partial agreement with the gene sequences of Myxosporean sp. and was found to be a new species not registered in GenBank. To investigate the outbreak trend of emaciation infection in wider range, total 900 fish were systematically examined in April, May, Sept., Nov., Dec., 2014 and the infection rate was checked. In 2015, the same examination was conducted in March, May, July and October with the different fish number. As a result, the infection rate was 18.3%~71.6% in 2014 and 16.3%~90.3% in 2015, higher than in 2014. In addition, according to the infection trend analysis depending on the sample size, the infection occurred in any size and the size of 11~30cm showed the highest infection rate. In periodic infection rate, Sept. and Dec. in 2014 and March, July and October in 2015 showed relatively higher infection rate. The reason for the high infection rate was not identified yet, but it is assumed that it might be related with marine environment of Jeju Island region. It might also be related with the fact that olive flounder seedlings are brought in from other regions to Jeju Island. To identify whether emaciation disease could be transmitted within a same fishes or among the other fishes (red sea bream, black sea bream), two groups were built after PCR infection test a donor group showing positive reactions and a recipient group showing negative reactions. The test result showed that infection occurred within olive flounders, the same fishes, but was not observed in red sea bream and black sea bream, the other species. This result, in relation to species specificity, is estimated that emaciated disease causes infection only to olive flounder of the same fishes not to those of different fishes because factors such as host environment and living conditions do not fit for fish of different fishes. However, up to now, more investigation is required and in the future, a more detailed study is required to find out infection occurs to fish of different species. Moreover, according to the result of the histological examination of the renal tissue after cohabitation, the same spores as those where emaciation is generally observed were observed for the same group (recipient group) detected in PCR and for red sea bream and black sea bream of different fishes, the PCR result and spores were not observed. To establish diagnosis index of emaciation disease, hematologic analysis was performed and non-specific immune response to emaciated and non-emaciated fish was observed. The result of the hematological analysis shows that hematocrit and GPT (farm-B) displayed higher experimental values than control values, and that GPT, GLU, total glucose, total cholesterol, and total protein exhibited lower experimental values than control values. As a result of measuring Lysozyme, NBT and MPO which are non-specific immune responses, lower experimental values were observed in three experiments. It is assumed that the stress by emaciation cause and the damage of internal organs destroy the primary defense system. According to phylogenetic systematics analysis, all of 4 isolates belong to Parvicapsula petuniae and gene homology with the 4 isolates identified in Korea was 99.7%~99.76 %. In addition, according to the result of the gene comparative analysis using GenBank database, P. petuniae and high homology of 92% were observed for 18S rRNA and emaciation occurring domestically has been identified as Parvicapsula sp. For morphological identification, according to the result of the microscopic inspection of the fish species suffering from emaciation, the spores were classified into two forms. One from form was where two spores gathered in the same or different direction and spores with lengths of 16~18㎛ And widths of 4~5㎛ Were observed. Also, spores with two polar capsules gathered on one side were observed. The other form was a form of small spores with lengths of 5~8㎛ and widths of 7~9㎛ And a form with one polar capsule and lacking part of spore was observed. In this result, spores morphologically similar to P. anisocaudata and P. pseudobranchicola n. sp. occurring in a olive flounder farm has been observed and spores typically observed only in Parvicapsula sp. have been confirmed. Therefore, emaciation occurring domestically has been identified as Parvicapsula sp. in the same way shown by phylogenetic analysis result. Real-time PCR we analyzed the number of pathogens in tissues of Parvicapsula sp. and the order of infection. Through Histopathologic examination we checked its correlation with the results of real-time PCR. Infection concentration of Parvicapsula sp. in tissues was investigated. The highest infection concentration was observed in kidney with 2.6 × 103 ~ 1.7 × 107copies/mg, followed by intestines, spleens, brains and livers. With the result we could identify more infected cases that were not detected on PCR. It is assumed that minimum infection concentration for PCR examination of Parvicapsula sp. is 103copy/mg. To check the result of real-time PCR of Parvicapsula sp. we performed correlation analysis through Histopathologic examination. A number of spores were observed in all of the organs, and the presence or absence of infecting spores corresponds with the results of real-time PCR. As Histopathologic examination is accurate but unfavorable in immediacy, it is difficult to respond to infection outbreaking immediately. Real-time PCR is assumed to be more useful for the Parvicapsula sp.examination, with which it is easy to diagnose latency and initial stage of infection.