G. Kalayci*, S. Incoglu and B. Özkan

Transcription

1 Bull. Eur. Ass. Fish Pathol., 26(4) 2006, 157 First isolation of viral haemorrhagic septicaemia (VHS) virus from turbot (Scophthalmus maximus) cultured in the Trabzon coastal area of the Black Sea in Turkey G. Kalayci*, S. Incoglu and B. Özkan Bornova Veterinary Control and Research Institute, Ministry of Agriculture and Rural Affairs, Bornova Izmir, Turkey Abstract Viral haemorrhagic septicaemia virus (VHSV) was isolated from cultured turbot fry and broodstock samples in the Trabzon coastal area of the Black Sea, Turkey in The virus was isolated in EPC and RTG-2 cell lines and identified by immunoperoxidase test, immunofluorescence test and enzyme linked immunosorbent assay. The results of applied immunological identification tests confirmed each other. This is the first report on the isolation of VHSV from cultured turbot in Turkey. Introduction Viral haemorrhagic septicaemia (VHS) is a serious infectious disease caused by a virus belonging to the Novirhabdovirus genus within the family Rhabdoviridae. Viral haemorrhagic septicaemia virus (VHSV) has been isolated from a number of wild marine species and at least 45 different freshwater and marine species have tested positive for the virus (Skall et al., 2005). Stone et.al. (1997) suggested that all marine fish species are potential carriers of VHSV. VHSV has been isolated from freshwater and marine fish species in Europe, North America and East Asia (Dixon et al., 1997, Meyers et al., 1994, Mortensen et al., 1999, Takano et al., 2000). This infection is very important to many European countries because of the economical consequences for farmed salmonids. Many of the VHSV isolates from wild marine species have been shown by experimental bath challenge to infect turbot and cause high mortality (Castric & de Kinkelin, 1984, King et al., 2001). Natural infections with VHSV have also caused significant mortality in turbot in aquaculture (Schlotfeldt et al. 1991, Ross et al. 1994, Skall et al., 2005). Anaemia, massive haemorrhaging and/or congestion and exophthalmia are among the signs of VHS. Necrotic changes are seen in the haematopoietic tissue. Gills, liver and kidney are also pale and contain necrotic areas (Wolf, 1988, Bruno & Poppe, 1996). The diagnosis of VHS is based on the isolation of VHS virus in cell culture followed by identification by neutralisation, immunoperoxidase test (IP), immunofluoresence test (IFT), enzyme linked immunosorbent assay *Corresponding author s gulnurunver@yahoo.com

2 Bull. Eur. Ass. Fish Pathol., 26(4) 2006, 158 (ELISA) or identification by reverse transcription polymerase chain reaction (RT- PCR) (Anonymus 2003, Anonymus 2001). This report describes the first isolation of VHSV from cultured turbot in the Trabzon coastal area of the Black Sea in the North part of Turkey using standard screening methods. Materials and methods Fish samples A total of day old turbot fry (200 from each tank) and organ material from 15 broodstock fish sampled from three different tanks from the Central Fisheries Research Institute (CFRI) in the Trabzon coastal area of the Black Sea were transported on ice (4-8 C) to the Virology Laboratory of Bornova Veterinary Control and Research Institute. All the samples were examined according to standard virological procedures (Anonymus 2001). Two hundred turbot fry from each tank were homogenized together to obtain three samples of turbot fry from three tanks for viral examination. Necropsy was performed in the laboratory using kidney, spleen, heart and brain tissues of five broodstock fish in each sample. Processed samples were incubated with rabbit antisera against both the Sp and Ab serotype of infectious pancreatic necrosis virus (IPNV) for 1 h at 15 C. Both anti-ipnv serum treated and non-treated aliquots of supernatants were used for viral examinations. Virus isolation Epithelioma papulosum cyprini (EPC) and rainbow trout gonad (RTG-2) cell lines were grown on 25cm² tissue culture flasks in cell culture medium (EMEM supplemented with 10% FCS, 1% antibiotic-antimycotic solution and 1% HEPES). 1/10 and 1/100 dilutions of the anti-ipnv serum treated and non-treated aliquots of supernatants of sample homogenates were inoculated into 25cm² tissue culture flasks with 24 h-old cultures of EPC and RTG-2 cell lines. Reference virus strains (VHSV-919, VHSV-961 were obtained from Dr. D. Fichtner and Dr. R. Riebe (FLI, Insel Riems, Germany) in 2004) were also inoculated at the same dilutions with samples following the same procedure. Inoculated cell culture flasks were incubated at 15 C for 1 h for adsorbtion following which cell culture medium was added to each cell culture flask. Cultures were examined for cytopathic effect (CPE) following a 10-day incubation at 15 C. Supernatants were then transferred to new cell cultures of the respective cell lines and incubated at 15 C for one week. Flasks were examined daily for CPE formation. Virus identification Immunoperoxidase test (IP), immunofluorescence test (IFT) and enzyme linked immunosorbent assay (ELISA) were carried out using commercially (BIO-X Diagnostics, Belgium and TEST-LINE Clinical Diagnostics, Czech Republic) available kits for the identification of virus. The supernatants of CPE positive samples and reference virus strain positive controls were inoculated onto 24-well cell culture plates with 24 hour-old cultures of EPC and RTG-2 cell lines and incubated at 15 C for IP and IFT. When the CPE was clearly visible (within h) the media in the wells were discarded and monolayers were fixed using acetone solution.

3 Bull. Eur. Ass. Fish Pathol., 26(4) 2006, 159 Results Macroscopic observations Exophthalmia displayed in both eyes, darkening of the body, pale coloration of gills, haemorrhaging of the head and the fin bases, pale and off-colour areas in liver and haemorrhages in the intestinal tract were observed macroscopically during the necropsy of broodstock samples (Figure 1). Virus isolation After ten days of incubation, CPE was clearly visible in both EPC and RTG-2 cultures Figure 1. External (a) and internal (b) signs of VHSV in infected turbot broodstock. Immunoperoxidase test (IP) and Immunofluorescence test (IFT) The tests were carried out according to manufacturers instructions of the kit ( BIO- PEROX VHS, BIO-X Diagnostics, Belgium and BIO-fluo VHS-BioK006, BIO-X Diagnostics Belgium). Enzyme linked immunosorbent assay (ELISA) The supernatants of CPE positive samples were tested by ELISA using the VHSV Ag ELISA kit of TEST- LINE Clinical Diagnostics Ltd., Czech Republic, according to their recommendations. Figure 2. Cytopathic effect (CPE) on EPC cells infected with viral haemorrhagic septicaemia virus (VHSV) from turbot a) cell control, normal EPC cells b) EPC cells with VHSV CPE.

4 Bull. Eur. Ass. Fish Pathol., 26(4) 2006, 160 were observed in the infected cell layer with field isolates and also with reference virus strains (positive controls, data not shown). There was no red plaque formation in the negative control or cell control wells. Granular positive staining was observed around the peripheral part of the cytoplasm of infected cells by IFT. Staining was never detected in any negative controls (Figure 3). In ELISA, cell culture isolates of both turbot fry and broodstock were tested againist both a positive and negative control antigen. The absorbance values of tested samples were compared with the mean absorbance values of positive and negative antigen wells. As a results of repeated tests the mean absorbance values of tested samples were calculated to be at least three or four times greater than the mean absorbance of negative controls. According to the kit instructions, these results were interpreted to be VHSV positive. Figure 3. Immunofluorescence staining of a) VHSV infected EPC cells b) VHSV infected RTG-2 cells. inoculated with samples of turbot fry and broodstock. The supernatants of cell cultures were passaged to new cultures of respective cell lines. In second and third passages CPE was clearly visible within seven days. CPE was observed both in cultures inoculated with anti-ipnv treated and non-treated supernatants of sample homogenate (Figure 2). Virus identification For virus identification in cell culture three different test methods were used. VHS virus was detected in samples from both turbot fry and broodstock and identified using IP. As a result of the IP test, red plaque formations Discussion This study represents the first isolation of VHSV from cultured turbots in Turkey. The standard methods for virus isolation in cell culture and subsequent immunological identification using tests recommended by the Office International des Epizooties (OIE) and European Commission Directive (Anonymus 2003, Anonymus 2001) were used for the isolation and identification of VHSV from three pooled samples of turbot fry and three pools of turbot broodstock from different tanks. Virus isolates obtained from samples considered to be VHSV positive in both cell lines were identified by IPT, IFA and ELISA. The results of the tests confirmed the findings in all cases. Turbot fry and broodstock

5 Bull. Eur. Ass. Fish Pathol., 26(4) 2006, 161 samples were sent to Virology Laboratory of Bornova Veterinary Control and Research Institue (NRL) because of observed levels of high cumulative mortality at the hatchery of CFRI. As shown in Figure 1, most of the external and internal clinical signs of VHS disease were observed in the broodstock samples during necropsy. The VHS outbreak occurred in spring 2004 in Turkey when water temperatures were fluctuating and the mortality rates reached 99% in day-old turbot fry. The broodstock turbot were collected by CFRI from the Black Sea. There is a filtration system at the marine water inlet to prevent bacterial contamination originating from sea water but this system was not able to exclude viruses. This factor may be considered as a possible source of infection. Acknowledgements The authors would like to thank Dr.D. Fichtner and Dr. R. Riebe (FLI, Insel Riems, Germany) for providing EPC, RTG-2 cell lines and VHSV reference virus strain used in this study. References Anonymus (2001). Commission Decision 2001/183/EC of 22 February 2001 laying down the sampling plans and diagnostic methods for the detection and confirmation of certain fish disease and repealing Decision 92/532/ EEC. Anonymus (2003). Viral haemorrhagic septicaemia. Manual of Diagnostic Tests for Aquatic Animals. Copyright 2003 OIE. World Organisation for Animal Health / A_0005.htm.updated: Bruno DW & Poppe TT (1996). A colour atlas of salmonid diseases. Academic Press. Castric J & Kinkelin de P (1984). Experimental study of the susceptibility of two marine fish species, sea bass (Dicentrarchus labrax) and turbot (Scophthalmus maximus), to viral haemorrhagic septicaemia. Aquaculture 41, Dixon PF, Feist S, Kehoe E, Parry L, Stone DM & Way K (1997). Isolation of viral haemorrhagic septicaemia virus (VHSV) from Atlantic herring Clupea harengus from the English Channel. Diseases of Aquatic Organisms 30, King JA, Snow M, Skall HF & Raynard RS (2001). Experimental susceptibility of Atlantic salmon Salmo salar and turbot Scophthalmus maximus to European freshwater and marine isolates of viral hemorrhagic septicaemia virus. Diseases of Aquatic Organisms 47, Meyers TR, Short S, Lipson K, Batts WN, Winton JR, Wilcock J & Brown E (1994). Association of viral hemorrhagic septicemia virus with epizootic hemorrhages of the skin in Pacific herring Clupea harengus pallasi from Prince William Sound and Kodiak Island, Alaska, USA. Diseases of Aquatic Organisms 19, Mortensen HF, Heuer OE, Lorenzen N, Otte L & Olesen NJ (1999). Isolation of viral haemorrhagic septicaemia virus (VHSV) from wild marine fish species in the Baltic Sea, Kattegat, Skagerrak and the North Sea. Virus Research 63, Ross K, McCarthy U, Huntly PJ, Wood BP, Stuart D, Rough El, Smail DA & Bruno DW (1994.) An outbreak of viral hemorrhagic septicemia (VHS) in turbot (Scophthalmus maximus) in Scotland. Bulletin of the European Association of Fish Pathologists 14,

6 Bull. Eur. Ass. Fish Pathol., 26(4) 2006, 162 Schlotfeldt HJ, Ahne W, Jørgensen PEV & Glende W (1991). Occurence of viral hemorrhagic septicemia in turbot (Scophthalmus maximus)- a natural outbreak. Bulletin of the European Association of Fish Pathologists 11, Skall HF, Olesen NJ & Mellergaard S (2005). Viral haemorrhagic septicaemia virus in marine fish and its implication for fish farming - a review. Journal of Fish Diseases 28, Takano R, Nishizawa T, Arimoto M & Muroga K (2000). Isolation of viral haemorrhagic septicaemia virus (VHSV) from wild Japanese flounder, Paralichthys olivaceus. Bulletin of the European Association of Fish Pathologists 20, Wolf K (1988). Fish viruses and fish viral disease. Cornell University Press. Ithaca NY. Stone DM, Way K & Dixon PF (1997). Nucleotide sequence of the glycoprotein gene of viral haemorrhagic septicaemia (VHS) viruses from different geographical areas: a link between VHS in farmed fish species and viruses isolated from North sea cod (Gadus morhua L.). Journal of General Virology 78,