The Research On Isolation And Identification Of Pathogeny From The Hermorrage Disease Of Prussian Carp(Carassius Gibelio) In Jiangsu China And Its Detection Method

Prussian carp (Carassius gibelio) are closely related to goldfish (Carassius auratus L.) and have been cultured on a large scale in the past decade in China. Despite the wide culture and distribution of Prussian carp, few viral diseases have been reported and characterized from this fish. Cyprinid herpesvirus2(CyHV-2), also known as herpesviral hematopoietic necrosis virus (HVHNV) and goldfish hematopoietic necrosis virus (GFHNV), is a very important viral pathogen in cultured goldfish. It was first reported from ornamental goldfish in Japan, where it caused severe haematopoietic necrosis and high mortality (100%) in the spring of1992and1993. Subsequently, CyHV-2was reported occurring in private ponds and aquaria or in small scale breeding facilities in the USA, Taiwan, Australia, southern England and New Zealand. These reports confirm that CyHV-2is widespread in world goldfish populations.From2011to2012, an acute haematopoietic necrosis disease outbreak in farmed Prussian carp in central China caused serious loss, with mortality levels of100%in some cases. This paper reports the investigation of this outbreak and is the first report of CyHV-2causing large-scale mortalities in cultured Prussian carp.Signs of diseased fish included lethargy and inappetence, gill hemorrhages, haemorrhagic spots on body surface. Internal gross pathology included hyperaemia, hepatic hypertrophy and splenomegaly. Large numbers of viral particles, approximately90-170nm in diameter with a spherical shape, were observed by electron microscopy. PCR assays with CyHV-2specific primers were used to detect the virus in DNA extracted from the infected cell supernatant and tissue homogenates of diseased fish brain, liver, spleen and kidney. Sequencing of the PCR product showed more than98%nucleotide identity with the published sequence of CyHV-2. Experimental infection with healthy Prussian carp using filtrates of tissues by intramuscular injection caused100%mortality and presented similar clinical signs to those seen in wild infected fish, in which the presence of the virus was regarded as the cause of the high mortalities. The GFHNV derived from disease Prussian carp, was identified that caused HVNV when challenging healthy test fish by intraperitoneal inoculation, the virus infected healthy goldfish, one population of hybrid Prussian carp under15-25℃and the optimum temperature for infection was20-25℃. Our trial demonstrated no age-related was resistance to GFHNV among Prussian carp, but the juveniles were more susceptible to the virus.The histopathology research conducted with tissue technology, hematoxylin an eosin staining. The most pronounced histopathological changes were observed in the spleen and kidney. The haematopoietic cells of kidneys displayed both karyopyknosis and karyorrhexis and renal tubule epithelia occasionally exhibited cloudy degeneration. In the spleen, necrosis with intranuclear inclusions were seen in some cells. Necrotic splenocytes showing nuclear hypertrophy, margination of chromatin, or pyknosis was observed. Infected renal hematopoietic cells showed swelling of the cytoplasm and similar nuclear pathology.Following the development of molecular biotechnology, some new technologies,such as Loop-mediated isothermal amplification method(LAMP), are developed in the recent years and supply new skills for the early and rapid examination of GFHNV. The gene of GFHNV was downloaded from GenBank, the outer, inner and loop peimers of LAMP were the designed in their conservative region using software Primer Explorer V3. The primers used in this method were shown to be specific for GFHNV and did not amplify Koi Herpes Virus, Viral Nervous Necrosis Virus, Viral Hemorrhagic Septicemia Virus and等Therefore, the rapid and simple assay is potential useful technique for GFHNV detection in field conditons.