![]() Five randomly selected fish were confirmed to be free from pathogens (the absence of parasites under microscopic observation, absence of bacteria on BHIA and no amplified CyHV-2 DNA polymerase gene product by PCR). The resulted sequence was used to search for similarity using BLASTN of NCBI.įor experimental infections, a total number of 60 healthy goldfish (10.4 ± 1.1 cm, 16.9 ± 0.5 g) were obtained from local fish farm in Suncheon, Jeollanam-do, Korea. The PCR products were resolved from the agarose gel, and purified using AccuPrep ® Gel purification kit (Bioneer) and cloned into pGEM ®-T easy vector for sequencing (Bioneer) according to manufacturer’s protocol. The amplified PCR products of virus were analyzed in 1.5% agarose gels containing ethidium bromide and visualized under UV light. PCR amplification conditions were as follows: 94☌ for 1 min, followed by 30 cycles of 94☌ for 30 s, 45☌ for 30 s and 72☌ for 3 min ( Goodwin et al., 2006). The PCR reactions were performed using AccuPower ® PCR PreMix (Bioneer) in a final reaction volume of 20 μL, containing 1 μL of each primer (10 μM), 13 μL of DEPC-treated water and 5 μL of the template (genomic DNA) by MyGenie 32 Thermal Block (Bioneer). Extracted genomic DNA was dissolved in 100 μL of diethyl pyrocarbonate (DEPC)-treated water and stored at –20☌ until use. The genomic DNA was isolated from the kidney and spleen from goldfish using an AccuPrep ® Genomic DNA extraction kit (Bioneer, Daejeon, Korea) according to the manufacturer’s instructions. To confirm the CyHV-2 infection, PCR was conducted by amplifying DNA polymerase gene of CyHV-2 using a primer set (F 5’ CGGAATTCTAGAYTTYGCNWSNYTNTAYCC 3’ Y = C, T N = ACGT W = AT S = CG R = AG and R 5’ CCCGAATTCAGATCTCNGTRTCNCCRTA 3’) ( Goodwin et al., 2006). Virus identification by PCR and sequence analysis Additionally, artificial infection experiment of CyHV-2 was carried out to confirm that the cause of goldfish mortality was CyHV-2. This study describes two cases of CyHV-2 detection in Korea. The tendency of CyHV-2 spreading globally is significant. Moreover, CyHV-2 has been detected in prussian carp ( Carassius giberio B.) and crucian carp ( Carassius carassius L.) from Czech, China, Hungary, and Italy, causing massive mortality in recent years ( Daněk et al., 2012 Doszpoly et al., 2011 Fichi et al., 2013 Luo et al., 2013 Wang et al., 2012). Since then, CyHV-2 outbreaks in goldfish have been reported in many countries, including Australia, India, France, Taiwan, UK, and USA ( Boitard et al., 2016 Chang et al., 1999 Goodwin et al., 2006 Groff et al., 1998 Jeffery et al., 2007 Sahoo et al., 2016 Stephens et al., 2004). This disease first occurred in Japan (19), causing severe mortality (up to 100%) of goldfish ( Jung & Miyazaki, 1995). CyHV-2 causes hematopoietic necrosis, resulting in herpesviral hematopoietic necrosis. Members of the cyprinid herpesviridae family are divided into carp pox herpesvirus (Cyprinid herpesvirus 1, CyHV-1), goldfish hematopoietic necrosis herpesvirus (Cyprinid herpesvirus 2, CyHV-2) and koi herpesvirus (Cyprinid herpesvirus 3, CyHV-3) ( Davison et al., 2013 Waltzek et al., 2005). However, several diseases can cause mortality of goldfish ( Groff et al., 1998 Iqbal et al., 1999 Jung & Miyazaki, 1995). Goldfish ( Carassius auratus L.) is a freshwater fish belonging to family Cyprinidae of order Cypriniformes. In the present study, CyHV-2 was detected and identified as the causative pathogen of the epizootic in goldfish in Korea. Artificial infection trials using affected tissues filtrates gave cumulative mortalities of 30% for virus injected goldfish. The hematopoietic necrosis herpes-virus (Cyprinid herpesvirus 2, CyHV-2) DNA polymerase gene successfully detected in DNA extracted from kidney and spleen of affected fish using PCR assay and showed 100% identity with already deposited CyHV-2 DNA polymerase gene in NCBI. The histological examination led to hypothesize the implication of a virus in the mortality. In addition, nucleus exhibiting peripherally displaced chromatin were particularly abundant in the kidney of affected fish. The predominant histopathological changes were severe necrosis of hematopoietic tissue. ![]() Moreover, the histopathological characteristics observed mainly in hematopoietic tissues of kidney, gill lamellae and intestine. The principal signs included pale gills, severely enlarged and softened spleen and kidney and red liver. In April and June 2014, mortalities of goldfish occurred at Korea.
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