Epidemiology, Pathology, And Pathogen Of The "Chrysanthemum Season Disease" Of Meretrix Meretrix Linnaeus Cultured In Jiangsu Province

The hard clam, Meretrix meretrix Linnaeus is one of the economically important bivalve molluscs, a maior marine culture species in the coastal areas of Jiangsu province (Rudong and Qidong prefecture). One disease we named"Chrysanthemum season disease"occurred recent years has influenced the sustainable development of the marine aquaculture industry as a serious disease which caused a mass mortality. Epidemiology, histopathology, cytopathology of the disease, separation and purification of a suspicious virus, artificial reinfection and the identification of the virus have been studied in this thesis. It showed that a globose virus identified as aquticbirnavirus was associated with the"Chrysanthemum season disease"of the hard clam. Many measures of prevention and controlment of the disease were discussed about improving the mudflat environment and biological factors of the clam.Epidemiology investigations were progressed from 2003 to 2005 in several culture area such as the natural larvae culture area, the natural clam culture area, the purse net culture area and the culture in sandbank. The diseased clam were too weak to burrow into the sand protrude out of the substrate. It showed that"Chrysanthemum season disease"of Meretrix meretrix Linnaeus outbreaked in Rudong and Qidong prefecture. It was paroxysmal and high-mortality rate. The mortality increased markedly from low tide level to high tide level, from the low density culture area to high density culture area, and from the mudflat near the river to mudflat away from river. It occurred in May to July and September to October and was often continuous in two months. The temperature of the water was 25 to 30℃when the disease occurred. It was influenced by the tide. "Chrysanthemum season disease"Only associated with Meretrix meretrix Linnaeus, not with the other clam on the mudflat. No any bacteria and parasites were found in the diseased and moribund Meretrix meretrix Linnaeus.The histopathological changes of Meretrix meretrix Linnaeus suffering"Chrysanthemum season disease"in Nantong coast were studied, by means of routine parafin-section technique, with normal individuals as control. The results showed that the different tissues of the Meretrix meretrix Linnaeus suffered explosive epidemic disease was showing pathological change. The pathological changes mainly appeared, the mantle swell, its epithelium detached, and connective tissue proliferated or dissolved, hemocytes proliferated, muscle fiber is breaked off; the gill filament were disordered, its epithelium degenerated, the number of basophilic cell is arised, sometimes it became necrocytosis if serious; The gland alveolus of digestive diverticul is damaged, its epithelium were disordered, swell and necrosis, the duct dissolved; the epithelium of digestive tract swell, its connective tissue proliferated; the structure of the epithelium and muscle fiber of foot became loose and disordered, the mucus cell of the connective tissue proliferated. These serious pathological changes in these tissues might affect their physiological functions, which could be the major cause of the disease and the death of Meretrix meretrix Linnaeus.Electron microscopic examinations of ultrathin sections revealed the presence of spherical virus particles in the intercellular substance, cytoplasm of the gill, digestive diverticula, mantle, and digestive tract of the moribund hard clam Meretrix meretrix Linnaeus in 2005. And the virions are approximately 45 to 55 nm in diameter, having no envelope, no ambit between the core and nucleocapsid. In the infected cells with the virus, the cellular organelles presented obvious pathological changes. For example, the endoplasmic reticulum was swelled and mostly turned into vesicles, the nuclear chromatin condensed, the mitochondira swelled and dissolved, the muscle fiber was disarraied, the numbers of lysosome arised, and so on.The method purifying the virus from liquid of organism homogenization by differential centrifugation, potassium tartrate cushion ultracentrifugation and CsCl density gradient centrifugation. We also compared the sucrose and potassium tartrate-glycerol density gradient ultracentrifugation. With the negative stained, the virions are isometric symmetry, and approximately 60 to 75 nm in diameter. And there were many obvious capsomers on the surface of the virions. The virions have buoyant density of 1.554 to 1.582g/ml in the isopycnic CsCl gradient by the metage mothod. In this paper, we also discussed the systematic status of the virus by the morphological characters.Two step reverse transcription PCR were used to ensure the virus systematic status in the study. The first step was a reverse transcription using the M-MLV Rtase cDNA Kit from TaKaRa company. The second step was PCR using two Primer PrA and PrB which reported by Blake for detection of aquatic Birnavirus. The results showed that the fisrt primer PrA did not identify the 1180-bp fragment, while the other primer PrB identified the 524-bp fragment. The sequence of PrB is PrB 1:5’-GCCGACATCGTCAACTCCAC-3’,PrB2:5’-GACAGGATCATCTTGGCATA-3’. So we suggested the spherical-shaped virus could be the aquticbirnavirus.The eaperimental infections was studied by means of intrapalleal cavity injection or water boren method at 25℃. The reinfetious virions, which were isolated from the moribund hard clam Meretrix meretrix Linnaeus in the outbreak area were used to reinfect the healthy cultured hard clam Meretrix meretrix Linnaeus. The artificial infection experimental results indicated that the cumulant mortality of the hard clam infected by injection reached 100% in the 17th day while the median lethal time was 14-16d after the infection by virus injection. The cumulant mortality of the hard clam immersed to virus solutions reached 100% in the 15th day. The cumulant mortality of the hard clam in the control was only 10%. Vinous could be reisolated from the dead clam and the moribund clam while no virus was reisolated from the survivors. Our preliminary suggestion was that the aquticbirnavirus could be the pathogen of the "Chrysanthemum season disease"of Meretrix meretrix Linnaeus.The results of this study showed that the synergy of the virus and environment were the cause of the "Chrysanthemum season disease" of Meretrix meretrix Linnaeu which occurred recent years in the coastal areas of Jiangsu province. The aquticbirnavirus which were encysted in the clam were the direct cause, while the contaminated environment, unreasonable culture method such as high density culture and purse net culture, degradation of the germ plasm of Meretrix meretrix Linnaeus, and the biology of the clam were the indirect derivational reason of the "Chrysanthemum season disease". When the development of factor became limited, Meretrix meretrix Linnaeus will couldn’t resist the infection from any phogens. So the disease will occurred.Strengthen the protection and seed selection of the germ plasm of Meretrix meretrix Linnaeus, strengthen the protection of environment, arrange rationale clam density, sterilize the mudflat, and further research will be the the effective measures of prevention and controlment of the "Chrysanthemum season disease"of Meretrix meretrix Linnaeus.