Proceedings of the Third World Fisheries Congress: Feeding the World with Fish in the Next Millenium—The Balance between Production and Environment
Development of Polymer Chain Reaction and Nucleic Acid Probe Diagnostic Methods for the Detection of Infectious Spleen and Kidney Necrosis Virus from Mandarin Fish
Min Deng, Jian-guo He, Shao-ping Weng, Lin Lu, Yan-qi Wang, Qing-xin Long, Siu-ming Chan
The predatory freshwater mandarin fish Siniperca chuatsi is an economically important species cultured in southern China. The spread of a disease caused by infectious spleen and kidney necrosis virus (ISKNV) has resulted in significant economic loss in many Chinese fishponds. Outbreaks of ISKNV were reported between 1994 and 1999 (Wu et al. 1997; He et al. 1998).
We previously reported the virus outbreak in mandarin fish. The histopathology of the infection is characterized by cell hypertrophy of the spleen, kidney, cranial connective tissue, and endocardium. In enlarged cells, many icosahedral viral particles (150 nm) were present in the cytoplasm (Weng et al. 1998). The virus was later identified as ISKNV of the family Iridoviridae on the basis of morphological evidence and partial nucleotide sequences (He et al. 1998; Deng et al. 2000). Iridoviruses are icosahedral cytoplasm DNA viruses that have been isolated from insect invertebrate and vertebrate host species. Two genera in the Iridoviridae family infect vertebrates including fish, amphibians, and reptiles: Lymphocystivirus and Ranavirus (Regenmortel et al. 1999).
Because of the demand for cultured mandarin fish for food consumption and the negative economic impact of ISKNV infections on the fish farms, sensitive and rapid diagnostic methods is urgently needed. Thus far, ISKN disease has been diagnosed with a histopathological method. Infected fish show cell hypertrophy in spleen and kidney (Weng et al. 1998). Unfortunately, this method cannot distinguish tissues from infected and uninfected fish at early stages of infection. Because the isolation of purified virus using fish cell lines has not been successful, the cell culture techniques cannot be used to diagnose the disease.
In this paper, we report the results of our studies on the isolation of ISKNV from cultured mandarin fish and describe our attempt to obtain genomic information about ISKNV. We also discuss the development of a rapid, sensitive, and specific diagnostic method based on polymer chain reaction (PCR) to detect early and latent stages of infection. In addition, dot-blot and in situ hybridization were performed using the ISKNV fragments as gene probes to localize the virus in different tissues.
Moribund mandarin fish with symptoms of ISKNV infection were collected from the fish farm in Nanhai, Guangdong. They were kept at –80°C before use. The fish were examined by gross anatomy, light microscopy, and electron microscopy to confirm the presence of virus by using the methods described in Weng et al. (1998).
Spleen and kidney were removed from mandarin fish and pulverized in liquid nitrogen. The powdered tissue was added slowly to 10 volumes of phosphate-buffered saline (PBS; pH 7.4) and centrifuged at 5,000 gravity (g) for 20 min at 4°C. The supernatant was centrifuged at 35,000 g for 30 min at 4°C, and the pellet was suspended in PBS. Additional purification was achieved by overlayering the resuspended pellet on a 20–50% (weight/ weight) sucrose gradient and centrifuging for 2 h at 23,000 revolutions/min with a SW40 rotor (Beckman). The visible viral band was removed and diluted threefold with PBS and pelleted by centrifugation for 30 min at 50,000 g at 4°C. The pellet was resuspended with PBS.