Carassius auratus gibelio - Springer Link

2015, Vol.20 No.5, 413-420 Article ID 1007-1202(2015)05-0413-08 DOI 10.1007/s11859-015-1114-9

Histopathology and Ultrastructural Pathology of Cyprinid Herpesvirus II (CyHV-2) Infection in Gibel Carp,

Carassius auratus gibelio

□ JIANG Nan 1,2, XU Jin 1 , MA Jie 1, FAN Yuding 1, ZHOU Yong 1, LIU Wenzhi 1, ZENG Lingbing 1†

0

1. Division of Fish Disease, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, Hubei, China; 2. College of Fisheries, Huazhong Agricultural University, Wuhan 430070, Hubei, China

Cyprinid herpesvirus II (CyHV-2), known as herpesviral haematopoietic necrosis virus (HVHNV) of goldfish or goldfish haematopoietic necrosis virus (GFHNV), is a member of genus Cyprinivirus, which includes cyprinid herpesvirus I (CyHV-1), cyprinid herpesvirus II (CyHV-2) and cyprinid herpesvirus III (CyHV-3) [1-3]. CyHV-2 caused haematopoietic necrosis in goldfish in Japan, USA, Taiwan, Australia, UK and New Zealand, and led to a high mortality in infected goldfish [4-11]. The histopathology of CyHV-2 infection in goldfish includes necrosis of kidney, spleen and intestine, and hyperplasia and fusion of secondary lamellae [8, 11]. Histopathological changes of CyHV-3 are characterized clearly. The changes induced by CyHV-3 include necrosis in gill, kidney and liver, nuclear inclusion bodies in gill, spleen and kidney, degeneration in myocardial cells, congestion of capillaries in brain, and hemorrhaging and fusion of lamella [12-14]. The CyHV-2 infection in gibel carp was first reported in Hungary [15], and then in the cultured gibel carp in China [16]. Gibel carp is one of the most important cultured fish in China [17]. In 2009, a severe hemorrhagic disease in cultured gibel carp occurred in Jiangsu province and spread widely from 2011 to 2012. CyHV-2 was identified as the pathogen of the disease [16]. However, the information about the histopathology and ultrastructural pathology of this disease was limited [18]. In this study, the histopathological and ultrastructural patho-

© Wuhan University and Springer-Verlag Berlin Heidelberg 2015

Abstract: Cyprinid herpesvirus II (CyHV-2) infection is identified in cultured gibel carp, Carassius auratus gibelio, with high mortality in China in recent years. Histological pathology includes acute hepatocellular necrosis, splenic necrosis, kidney necrosis, hyperplasia of the secondary lamellae with focal necrosis. Acute necrotic myocarditis and granulocytes are prominent within the cardiac lumen in infected fish. In addition, necrosis is observed in the submucosa and mucosa epithelium of intestinal tract. Edemas are observed in renal glomerulus, submucosa and mucosa epithelium of intestinal tract, myocardial cells and neurons. Transmission electron microscopy indicates the cytoplasmic inclusions in splenocytes, glomerulus cells and hematopoietic tissue cells of kidney, epithelial cells of gills and brain cells. The histopathology and ultrastructural pathology in CyHV-2 infected gibel carp are characterized with extensive necrosis and cytoplasmic inclusions in spleen, kidney, gill and brain, which suggests that CyHV-2 may mainly infect the spleen, kidney, gill and brain of fish. Key words: Cyprinid herpesvirus II; gibel carp; histopathology; ultrastructural pathology; necrosis CLC number: S941.41+4 Received date: 2015-04-23 Foundation item: Supported by the Earmarked Fund for China Agriculture Research System (CARS-46-11), the Special Scientific Research Funds for Central Non-profit Institute, Chinese Academy of Fishery Sciences (2014A06XY07) and the Wuhan Science and Technology Plan Grant, Hubei Province (2014020202010138). Biography: JIANG Nan, female, Assistant research professor, research direction: fish virus. E-mail: [email protected] † To whom correspondence should be addressed. E-mail: [email protected]

Introduction

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Wuhan University Journal of Natural Sciences 2015, Vol.20 No.5

logical changes of CyHV-2 infection in gibel carp are fully investigated.

1

Materials and Methods

1) Animals Moribund gibel carp (15-28 cm in length) were collected from gibel carp culture ponds in Sheyang county of Jiangsu province from May to June in 2012. The diseased fish were kept in oxygenated bags and then transferred to the laboratory for experiments [16]. Healthy gibel carp (18-20 cm in length) were obtained from the breeding station at Yangtze River Fisheries Research Institute, which has no record of CyHV-2 infection. 2) Histopathology examination Samples of liver, spleen, kidney, intestinal tract, gill, heart and brain from the spontaneously diseased fish and the healthy fish were fixed in 4% paraformaldehyde (PFA ) for 24 hours at 4 ℃, and then washed with Dulbecco’s phosphate-buffered saline (DPBS, Sigma, USA), dehydrated with 30% sucrose/DPBS, and embedded in optimum cutting temperature compound (OCT). Samples were sectioned (8 µm in thickness) at -20 ℃ with cryostat (CM1950, Leica, Germany), stained with haematoxylin and eosin (HE) and examined with light microscopy with CCD picture system (DM2500, Leica, Germany). 3) Electron microscopy The liver, spleen, kidney and gill of the spontaneously diseased fish were fixed in 2.5% glutaraldehyde, postfixed in 1% osmium tetraoxide, dehydrated with gradient acetone and embedded in Epon 812. The ultra-thin sections (50-60 nm in thickness) were prepared with mictome machine (Leica UC7, Germany), stained with 2% uranyl acetate and lead citrate, and then viewed by transmission electron microscopy (Hitachi-7650, Japan) at 80 kV.

2 2.1

Results and Discussion

Histopathology Changes The histopathological lesions in the liver, spleen, kidney, intestinal tract, gill, heart and brain of the diseased fish consist of necrosis and focal necrosis throughout the entire organ. Cytoplasmic inclusion bodies, staining in basophilic to amphophilic, are observed in the necrotic splenocytes, hematopoietic tissue cells of kidney, and submucosa cells of intestine and the adjacent

areas. 1) Liver Multifocal acute hepatocellular necrosis is randomly distributed throughout the liver (Fig. 1(A)). Infiltration of melanomacrophages and granulocytes exist in hepatic sinusoids, and the endothelial cells of hepatic sinusoids hypertrophied are found (Fig. 1(B)). Large lesions show necrosis with margination or fragmentation of nuclei (Fig. 1(B)). The widespread vascular necrosis of hepatocytes are found in both the affected liver (Fig. 1(B)) and the healthy liver (Fig. 1(C)). The narrow hepatic sinusoids of the healthy liver are lined by endothelial cells, and hepatocytes are stained evenly. The macrophages and granulocytes are not found in the healthy sinusoids (Fig. 1(C)). 2) Spleen Severely diffused necrosis are found in the entire spleen. The melanomacrophage centers are adjacent to the areas of necrosis, and the scattered melanin granules of melanomacrophage centers are prominent within necrotic areas (Fig. 1(D)). The affected splenocytes have enlarged, pale stained and marginated nuclei. Splenocytes with amphophilic inclusions and nuclear debris are scattered throughout the necrotic tissue (Fig. 1(E)). There is no obvious melanomacrophage center in the healthy spleen, and the splenocytes are stained evenly with regular cell shape (Fig. 1(F)). 3) Kidney The kidney of diseased gibel carp shows extensive necrosis of the entire organ. The serious edema of the renal glomerulus and the increased number of melanomacrophage centers are found in the affected kidney (Fig. 2(A)). There is necrosis with basophilic cytoplasmic inclusions and a large number of nuclear debris in glomerulus cells and hematopoietic tissue cells (Fig. 2(B) and (C)). The necrotic cells are seen within the tubular epithelium, but cytoplasmic inclusions are not present (Fig. 2(C)). The nuclear changes of the affected cells are similar to that of hepatocytes previously described. Hyperplasia of glomerulus cells and proliferation of mesangial cells are also present in the affected kidney (Fig. 2(B) and (C)). Typical scattering of melanomacrophage centers are found in the healthy kidney. And the hematopoietic tissue cells, glomerulus cells and tubular epithelia are stained evenly without necrosis (Fig. 2(D)). 4) Intestinal tract Edema and necrosis appear in both submucosa and mucosa epithelium. Hemorrhaging is found in intestinal tract submucosa (Fig. 3(A)). The nuclear changes of the

Jiang Nan et al : Histopathology and Ultrastructural Pathology of Cyprinid Herpesvirus …

Fig. 1

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Histological changes of liver and spleen in CyHV-2 infected gibel carp

(A) Extensive necrosis of liver parenchyma. HE. Scale bars=100 µm; (B) Marginated nuclei (arrow), and melanomacrophages (asterisk) of hepatocytes. HE. Scale bars=20 µm; (C) Healthy liver parenchyma. HE. Scale bars=100 µm; (D) Extensive necrosis of splenic parenchyma and melanomacrophage centers (asterisk). HE. Scale bars=100 µm; (E) Cytoplasmic inclusions (arrowhead) and marginated nuclei (arrow) of splenocytes. HE. Scale bars=20 µm; (F) Healthy spleen parenchyma. HE. Scale bars=100 µm

Fig. 2

Histological changes of kidney in CyHV-2 infected gibel carp

(A) Extensive necrosis of kidney parenchyma and melanomacrophage centers (asterisk). HE. Scale bars=100 µm; (B) Cytoplasmic inclusions (arrowhead) and marginated nuclei (arrow) of glomerulus cells and hematopoietic tissue cells. HE. Scale bars=20 µm ; (C) Cytoplasmic inclusions (arrowhead) and marginated nuclei (arrow) of glomerulus cells, hematopoietic tissue cells and tubular epitheliums. HE. Scale bars=20 µm; (D) Melanomacrophage centers (asterisk) and glomerulus (arrow) of healthy kidney. HE. Scale bars=100 µm

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affected submucosa and mucosa cells are similar to that of the affected hepatocytes (Fig. 3(B)). There is no hemorrhaging in the submucosa of the healthy intestinal tract, and the epithelium and submucosa cells are stained evenly with regular cell shape (Fig. 3(C)). 5) Gill The gills of the diseased gibel carp are infiltrated by numbers of granulocytes. Multifocal to diffuse hypertrophy and hyperplasia of the branchial secondary lamellae epithelium that result in extensive fusion of the adjacent lamellae (Fig. 3(D)). Meanwhile, the degeneration, necrosis, and sloughing of the affected branchial epithelial cells occurr. The affected epithelial cells also present in marginated nuclei. Lamellae are congested with a large number of macrophages, and melanomacrophage centers are also found in the primary lamellae (Fig. 3(E)). The healthy branchial secondary lamellae with one layer epithelium are stained evenly, and the melanomacrophage centers are not seen in primary lamellae (Fig. 3(F)).

Fig. 3

6) Heart Cardiac muscle cells are hypertrophied and associated with serious and wide spread infiltrates of granulocytes (Fig. 4(A)). Diffuse necrosis and edema of the muscle fibers lead to disorder and disruption of muscular trabeculae. The affected cells have condensed and marginated nuclear chromatin (Fig. 4(B)). The healthy myocardial cells are regular in arrangement and stained evenly, and there is no granulocyte in the cardiac chamber (Fig. 4(C)). 7) Brain Ventriculomegaly and extensive necrosis of neurons are detected in the brain of diseased gibel carp (Fig. 4(D)). The neurons are hypertrophied, swollen, disordered and loose, and the condensation and margination of the nuclear chromatin in neurons are similar to that of the affected hepatocytes (Fig. 4(E)). An enlarged vessel is detected in brain, and associated with hypertrophied and necrotic vessel endothelial cells and inflammation of the vessel wall (Fig. 4(E)). The healthy brain vessel is

Histological changes of intestinal tract and gill in CyHV-2 infected gibel carp

(A) Extensive necrosis of intestinal tract. HE. Scale bars=100 µm; (B) Marginated nuclei (arrow) of submucosa cells. HE. Scale bars=20 µm; (C) Healthy intestinal tract. HE. Scale bars=50 µm; (D) Extensive necrosis of gill. HE. Scale bars=100 µm; (E) Marginated nuclei (arrow) of the branchial secondary lamellae epithelium and melanomacrophage centres (asterisk) of the primary lamellae. HE. Scale bars=20 µm; (F) Healthy gill. HE. Scale bars=20 µm

narrow and fill with blood cells, and the healthy neurons are typical in arrangement and stain evenly (Fig. 4(F)). The most consistent changes in all tissues (liver, spleen, kidney, intestinal tract, gill, heart, brain) are the presence of margination of nuclei. The pathologies from CyHV-2 infection in gibel carp are similar to the symptoms of HVHNV infection in Japan, USA, Taiwan, Australia, UK and New Zealand and CyHV-3 infection in

Japan and Israel [4-8,10-14]. Jung et al [4] reported that there was very slight necrosis of renal glomeruli, liver, heart and brain in HVHNV infected goldfish in Japan. While in CyHV-2 infected gibel carp, there are serious lesions of glomerulus, liver, heart and brain, including necrosis of glomerulus cells, hepatocytes, myocardial cells and neurons. Moreover, cytoplasmic inclusions, nuclear debris and margination of the nuclear chromatin are found


in glomerulus cells in our study. Melanomacrophage centers were not found in CyHV-2 infected spleen and kidney of goldfish in New Zealand [10]. However, more diffused melanomacrophage centers are detected in affected spleen and kidney of gibel carp (Fig. 1(D) and Fig. 2(A)). The necrosis, fusion and hyperplasia of the secondary lamellae epithelium are associated with impaired blood supply, and the lesions of gills are similar to that of those described in HVHNV infection of goldfish and

Fig. 4

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CyHV-3 infection of koi carp [5, 7, 11, 14]. Melanomacrophage centers are first detected in primary lamellae in this report, which are not found in HVHNV and CyHV-3 infection (Fig. 2(E)). Infiltration of granulocytes in affected heart and necrotic vessel in affected brain are also similar to HVHNV infection in goldfish and CyHV-3 infection in common carp [8, 13]. The different histopathology changes between HVHNV and CyHV-2 infection are summarized in Table 1.

Histological changes of heart and brain in CyHV-2 infected gibel carp

(A) Extensive necrosis of heart. HE. Scale bars=100 µm; (B) Marginated nuclei (arrow) of myocardial cells. HE. Scale bars=20 µm; (C) Healthy heart. HE. Scale bars=100 µm; (D) Extensive necrosis of brain. HE. Scale bars=100 µm; (E) The necrotic vessel endothelial cells (arrowhead) and marginated nuclei (arrow) of neurons. HE. Scale bars=20 µm; (F) Healthy brain. HE. Scale bars=50 µm

Table 1 Tissue

Histopathology of HVHNV infection

Histopathology of CyHV-2 infection

Liver

Slight necrosis

Melanomacrophages and granulocytes in hypertrophied hepatic sinusoids

Spleen

Slight necrosis, no melanomacrophage centers

Cytoplasmic inclusions and nuclear debris in splenocytes, more diffused melanomacrophage centers

Kidney

Slight necrosis

Serious necrosis of glomerulus cells (cytoplasmic inclusions, nuclear debris and margination of the nuclear chromatin), more diffused melanomacrophage centers

Necrosis, fusion and hyperplasia of the secondary lamellae

Melanomacrophage centers

Gill

2.2

The different histopathology changes between HVHNV and CyHV-2 infection

Ultrastructural Pathology In the ultrastructural examination it illustrates virus particles and clear pathological changes in the tissues of diseased gibel carp. The hematopoietic cells of kidney show marginated nuclear chromatin, swollen mitochondria and vacuolated cytoplasm (Fig. 5(A)). The intranu-

clear virus particles are protruding from the inner nuclear membrane, and some virus particles are found in the perinuclear space between the inner and outer nuclear membranes (Fig. 5(A)). The virus particles in the nucleus and cytoplasm could be classified into three types. The fist type is the incomplete particles which have empty to

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partially full cores inside the capsid. The second type has full electron-dense cores which are perfect particles. The third type appears in the cytoplasm, and the virus particles that have empty to full electron-dense cores are double enveloped. Meanwhile, the third type virus particles usually are surrounded by membrane structure in the cytoplasm (Fig. 5(B)). The nucleocapsid of the first and the second type is 90-120 nm in diameter, while the third type is 170-200 nm (Fig. 5(A) and (B)). A number of cytoplasm virus particles aggregate together to form cytoplasmic inclusions (Fig. 5). The serious infected cells show nuclear pyknosis and fragmentation (Fig. 5(C)), and finally the apoptotic bodies arise (Fig. 5(D)). In addition, the virus particles are embedded in the apoptotic bodies (Fig. 5(D)). The hepatocytes display nuclear margination, mitochondria swelling and cytoplasm vacuola-

Fig. 5

tion. And the virus particles distribute in the cytoplasm (Fig. 6(A) and (B)). The infected splenocytes show marginated or condensed chromatin and sparse cytoplasm. Some cytoplasm virus particles aggregate together to form cytoplasmic inclusions (Fig. 6(C) and (D)). The vacuolated epithelial cells in diseased gills show condensed nuclear chromatin, and a large number of viral particles also form cytoplasmic inclusions (Fig. 7(A)). The infected brain cells reveal disintegrative nucleus, condensed nuclear chromatin, swollen mitochondria and sparse cytoplasm. A large number of virus particles distribute in the nuclear and cytoplasm, and the virus particles include three types of particles (Fig. 7(B)). No virus particle is found in the intestinal tract and heart with transmission electron microscopy.

Electron micrograph of kidney in CyHV-2 infected gibel carp

(A) Marginated nuclei (N), swollen mitochondria (M), vacuolated cytoplasm (asterisk) and cytoplasmic inclusions (black arrow) of hematopoietic cells. Some virus separate in the perinuclear space between the inner and outer nuclear membranes (white arrow). The virus particles in nucleus and cytoplasm can be classified into three types (arrowheads with numbers) based on their morphology: 1, capsids containing empty to partially full cores; 2, capsids containing full cores; 3, double enveloped capsids. Scale bars=400 nm; (B) The third type virus particles (arrowheads with 3) aggregate together to form cytoplasmic inclusions (black arrow). Scale bars=200 nm; (C) Pyknosis and fragmented nuclei (N) of hematopoietic cells. The virus particles form cytoplasmic inclusions (black arrow). Scale bars=200 nm; (D) Apoptotic cell in the kidney, and apoptotic bodies (apb) present. Some virus in apoptotic bodies (black arrow). Scale bars=200 nm


Fig. 6

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Electron micrograph of liver and spleen in CyHV-2 infected gibel carp

(A) Marginated nuclei (N), lipochondria (L) and vacuolated cytoplasm (asterisk) of hepatocytes. Scale bars=400 nm; (B) The virus particles (black arrow) and swollen mitochondria (M) in the cytoplasm of hepatocytes. Scale bars=200 nm; (C) Marginated nuclei (N), swollen mitochondria (M) and cytoplasmic inclusions (black arrow) of splenocytes. Scale bars=400 nm; (D) Pyknosis (N) and cytoplasmic inclusions (black arrow) of splenocytes. Scale bars=400 nm

Fig. 7

Electron micrograph of gill and brain in CyHV-2 infected gibel carp

(A) Condensed nuclei (N) and cytoplasmic inclusions (black arrow) of vacuolated epithelial cells in gill. Scale bars=400 nm; (B) Disintegrative and condensed nuclei (N), swollen mitochondria (M) and three types virus particles (arrowheads with numbers) of brain cell. Scale bars=200 nm

Similar morphological variation of virus has also been shown for CyHV-3 [19], which suggests that the process of CyHV-2 maturation be similar to CyHV-3. It is the first report that virus particles are presented in the hepatocytes and brain cells, which indicates that liver and brain are the target organs of CyHV-2. The virus

inclusion bodies were often found in the nucleus of HVHNV infected spleen, kidney and lamellae [5, 7, 8]. In this study, cytoplasmic inclusions are detected in spleen, kidney, gill and brain of CyHV-2 infected gibel carp with transmission electron microscopy, but cytoplasmic inclusion is not found, though detected in HVHNV infected

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goldfish [5]. Hence, spleen, kidney, gill and brain all play an important role in the infection, proliferation and transmission of CyHV-2.

3

Conclusion

In conclusion, we have characterized the histopathology and ultrastructural pathology of CyHV-2 infected gibel carp. There are extensive necrosis and cytoplasic inclusions in the spleen, kidney, gill and brain, which suggests that CyHV-2 may mainly infect the spleen, kidney, gill and brain of the fish. CyHV-2 was difficult to be cultured in cell lines [1, 20], our results will contribute to establishing a sensitive cell line for this virus. Furthermore, these results will be helpful to investigation of the pathogenesis of CyHV-2 and in control of the disease in gibel carp.

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