Susceptibility of Oblong Rockfish Sebastes oblongus to Viral Hemorrhagic Septicemia Virus

Transcription

1 Aquaculture Sci Susceptibility of Oblong Rockfish Sebastes oblongus to Viral Hemorrhagic Septicemia Virus Tadashi ISSHIKI 1,, Giannakou ELENI 1, Yutaka UETA 2,3 and Taizou NAGANO 2 Abstract: Susceptibilities of the oblong rockfish Sebastes oblongus to viral hemorrhagic septicemia virus (VHSV) were examined by experimental infections via an intramuscular route with an isolate KRRV-9822 from Japanese flounder Paralichthys olivaceus. Mortalities for these fish injected with doses of , and TCID 50 /fish occurred rapidly, and took 10, 5 and 3 days to reach 90, 100 and 100%, respectively, compared with 10 days to reach 100% even for Japanese flounder injected with TCID 50 /fish. The cumulative mortalities for oblong rockfish maintained at 15 and 20 for 16 days following a viral injection of TCID 50 /fish were significantly different, being 70 and 13%, respectively. When oblong rockfish, which had survived a VHSV natural outbreak, received a viral injection of TCID 50 /fish, the mortality for 16 days at 15 was 17%. In comparison, other oblong rockfish, which were previously naïve to a VHSV infection, had a 70% mortality following the same viral infection. These results revealed that the viral susceptibility of oblong rockfish is much higher than that of Japanese flounder and it depends on lower temperature. In addition, it is likely that oblong rockfish develops a protective immunity against VHSV infection. Key words: Sebastes oblongus; Viral hemorrhagic septicemia virus; Susceptibility; Experimental infection Viral hemorrhagic septicemia virus (VHSV), the etiological agent of viral hemorrhagic septicemia (VHS), is an enveloped negativesense ss-rna virus belonging to the genus Novirhabdovirus within the family Rhabdoviridae (Tordo et al. 2005). VHS was a serious viral disease of farmed rainbow trout Oncorhynchus mykiss in Europe, and VHSV was recognized as a virus that only affected freshwater fish species (Wolf 1988; Smail 1999). In the late 1980s, however, VHSV was reported from marine fish species in Europe (Vestergard-Jorgensen and Olesen 1987; Schlotfeldt et al. 1991; Ross et al. 1994; Mortensen et al. 1999; Smail 2000), from returning adult chinook salmon O. tshawytscha and coho salmon O. kisutch (Brunson et al. 1989; Hopper 1989), and from marine fishes in North America (Meyers 1992, 1994, 1999; Meyers and Winton 1995; Amos et al. 1998). VHSV has been isolated from an increasing number of free-living marine fish species. So far, it has been isolated from over 40 species of wild and farmed marine fish and from some freshwater species throughout the northern hemisphere including the USA, Canada, Japan, Korea and Europe (Skall et al. 2005), indicating host and geographic range extensions of VHSV in the marine environment. In Japan, the first isolation of VHSV was from farmed Japanese flounder Paralichthys olivaceus during a natural outbreak in 1996 (Isshiki et al. 2001). VHSV has caused serious mortalities in flounder farms over a wide area in western Japan. So far, outbreaks associated with VHSV Received 16 June 2010; Accepted 17 August Graduate School of Bioresources, Mie University, Tsu, Mie , Japan. 2 Kagawa Prefectural Fisheries Experimental Station, Takamatsu, Kagawa , Japan. 3 Present address: Fisheries Section of Kagawa Prefectural Office, Takamatsu, Kagawa , Japan. Corresponding author: Tel: (+81) ; FAX: (+81) ; isshiki@bio.mie-u.ac.jp (T. Isshiki).

2 T. ISSHIKI, G. ELENI, Y. UETA and T. NAGANO have also occurred in cultured black rockfish Sebastes inermis (Isshiki et al. 2003), Japanese sand lance Ammodytes personatus (Isshiki et al. 2003), and red sea bream Pagrus major (Nakaoka N, pers. comm. 2006). Occurrences of VHSV have been found in wild populations of Japanese flounder and Japanese sand lance that were caught in several coastal areas of Japan (Takano et al. 2000, 2001; Watanabe et al. 2002). Several species of marine fish have been shown to be susceptible to Japanese isolates of VHSV (KRRV-9601 or JF00Ehi1) under experimental conditions. These include black sea bream Acanthopagrus schlegeli, red spotted grouper Epinephelus akaara, Schlegel s black rockfish Sebastes schlegeli and yellowtail Seriola quinqueradiata (Isshiki et al. 2003; Ito et al. 2004). During April to May of 2004, VHSV infection occurred in juveniles of oblong rockfish Sebastes oblongus (mean body weight 5 g) that were produced and reared in the Kagawa Prefectural Fisheries Experimental Station, Japan, where the cumulative mortality reached about 64%. Moreover, it also prevailed in 2-year-old fish (mean body weight 91 g) leading to a cumulative mortality of about 10%. Thus, oblong rockfish was noted as a susceptible host of VHSV. The oblong rockfish is commonly distributed from southern Hokkaido to Kyushu, Japan, and in southern Korean (Amaoka et al. 1984). Recently, this species has been regarded as having aquaculture potential because of its scarcity in coastal waters, and marketability in western Japan including Kagawa Prefecture. Since 2002 its seed production has been successfully conducted in the Kagawa Prefectural Fisheries Experimental Station. Therefore, occurrences of VHSV in oblong rockfish resulted in a significant impact on seed production. Control measures need to be developed against the viral disease, such as giving protective immunity by vaccination. In this paper, we describe experimental infection to determine the susceptibility of oblong rockfish to VHSV by comparing results with those from Japanese flounder, as it is of fundamental importance in determining the efficacy of vaccination by experimental challenge with VHSV. Additional infectivity experiments were also attempted to estimate the effect of temperature on fish susceptibility, and to examine whether oblong rockfish surviving a disease outbreak possess protection against re-infection of VHSV. This is the first report of susceptibility of oblong rockfish to fish viruses including VHSV. Materials and Methods Virus The VHSV isolate KRRV-9822, which was obtained from Japanese flounder in Japan in 1998 (Nishizawa et al. 2002), was used in this study. This strain was propagated in the fathead minnow (FHM) cell line at 20 in Eagle s minimum essential medium (Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum. Fish Hatchery-reared oblong rockfish (mean body weight 13 g) were obtained from Kagawa Prefectural Fisheries Experimental Station. They included two different case history groups. One group (naïve fish) was previously naïve to a VHSV infection. Another group (survivors) had experienced a VHSV natural outbreak causing 91% mortality over an approximately 2 mo period and survived the outbreak one year prior to this study. The neutralizing antibody against VHSV was detected in the sera of 10 fish of the survivors ranging from 1 : 16 to 1 : 48 ND 50. Japanese flounder (mean body weight 6 g) from Kagawa Prefectural Sea Farming Center with no history of a VHSV infection was also used for comparison. Prior to experimental infection, fish of each species were randomly selected and processed for virus isolation using FHM cells to confirm the absence of VHSV. All the fish were acclimatized to experimental conditions for at least 7 days. Experimental infection Experiment 1 was carried out to compare the susceptibilities of two species, oblong rockfish and Japanese flounder, to VHSV. Three test

3 Susceptibility of Sebastes oblongus to VHSV groups (10 3.3, and test groups) of 10 naïve fish for each species were intramuscularly injected with 0.1 ml of cultured VHSV at three doses of , and TCID 50 /fish, respectively. An additional 10 fish of each species were injected with cell culture supernatant of non-infected FHM cells as the control groups. After injection, each group was reared in a 30-l aquarium at 15 for 14 days without feeding. Dead fish were removed daily and subjected to virus recovery test. Experiment 2 was performed to evaluate the effect of temperature on the susceptibility of oblong rockfish to VHSV. We subjected two test groups maintained at 15 and 20, respectively, and a corresponding two control groups. Each test group of 30 naïve fish was intramuscularly injected with 0.1 ml of cultured VHSV at TCID 50 /fish. The control groups of 30 fish were injected with cell culture supernatant of noninfected FHM cells. After injection, each group was reared in a 30-l aquarium at the respective temperature for 16 days without feeding. Dead fish were treated as described above. Experiment 3 was accomplished using naïve fish and survivors to demonstrate resistance development to VHSV infection in oblong rockfish following exposure to VHSV. Thirty survivors were intramuscularly injected with 0.1 ml of cultured VHSV at TCID 50 /fish. As a comparison, 30 naïve fish received VHSV in the same manner as survivors. The controls for survivors or naïve fish were injected with cell culture supernatant of non-infected FHM cells. After injection, each group was reared in a 30-l aquarium at 15 for 16 days without feeding. Dead fish were treated as described above. Virus recovery and measurement of the virus infectivity VHSV-associated mortalities in the experiments were confirmed by virus isolation from the heart of dead fish. Virological examinations were performed in 24-well plates (Falcon, Franklin Lakes, NJ, USA) with FHM cells and using an antiserum against KRRV-9822 according to the procedure previously described (Isshiki et al. 2003). The virus infectivity titers of the heart of dead fish were also measured as TCID 50 by the Behrens-Kaber s method. Statistical analysis Significant differences between experimental groups in levels of cumulative mortality were detected by the chi-square test. Results Experiment 1 Cumulative mortalities of oblong rockfish and Japanese flounder injected with VHSV at different doses are shown in Fig. 1. For oblong rockfish, mortality in the test groups commenced 5 days after injection (DAI), increasing to 90% mortality 10 DAI, subsequently lasting Fig. 1. Cumulative mortalities of oblong rockfish (A) and Japanese flounder (B) injected intramuscularly with a Japanese isolate KRRV-9822 of VHSV at doses of ( ), ( ) and TCID 50 /fish ( ) and maintained at 15. No mortalities occurred in the controls injected with non-infected cell culture supernatant (not shown).

4 T. ISSHIKI, G. ELENI, Y. UETA and T. NAGANO up to the end of the experimental period. In the and test groups, mortalities commenced 4 and 2 DAI and reached 100% 5 and 3 DAI, respectively. By contrast, mortalities slowly occurred for Japanese flounder and took 10 days to reach 100% even in the test groups, while the mortality rate in the or test groups was lower than that in the test group, and reached 40 or 70% at the end of the experimental period, respectively. No moralities occurred in any control groups. The moribund oblong rockfish usually exhibited the following clinical signs: dark body coloration, exophthalmia, hemorrhage of ventral fins and expanded abdomen due to ascites, and internally, pale coloration of the gills, ascitic fluid in the peritoneal cavity, congested liver, splenomegaly and swollen kidneys (Fig. 2). These signs were very similar to those observed in naturally diseased fish. Experiment 2 Cumulative mortalities of oblong rockfish maintained at 15 and 20 following VHSV injection are shown in Fig. 3. In the 15 test group, mortality commenced 7 DAI and then increased up to 15 DAI, resulting in a cumulative mortality of 70%. In the 20 test group, mortality occurred only 4-6 DAI and the cumulative mortality was 13%. Statistical analysis showed a significant difference of the cumulative mortalities between Fig. 3. Cumulative mortalities of oblong rockfish injected intramuscularly with a Japanese isolate KRRV-9822 of VHSV at a dose of TCID 50 /fish and maintained at 15 ( ) and 20 ( ). No mortalities occurred in the controls injected with non-infected cell culture supernatant and maintained at the corresponding temperatures (not shown). Fig. 2. External and internal signs of moribund oblong rockfish in the infectivity experiments. (A) A fish externally shows an expanded abdomen due to ascites and exophthalmia (scale bar = 20 mm). (B) Internally, the fish shows pale coloration of the gills, ascitic fluid in the peritoneal cavity, congested liver, splenomegaly and swollen kidney (scale bar = 10 mm). Fig. 4. Cumulative mortalities of oblong rockfish with two different case histories, naïve fish ( ) and survivors ( ), injected intramuscularly with a Japanese isolate KRRV-9822 of VHSV at a dose of TCID 50 /fish and maintained at 15. No mortalities occurred in the controls for naïve fish and survivors injected with non-infected cell culture supernatant (not shown).

5 Susceptibility of Sebastes oblongus to VHSV the 15 and 20 test groups (P 0.01). No mortalities occurred in any control groups. Experiment 3 Cumulative mortalities of oblong rockfish with two different case histories, naïve fish and survivors, injected with VHSV are shown in Fig. 4. For naïve fish, the changes in mortality showed a similar trend to those in Experiment 2, as the mortality increased gradually from 7 DAI reaching a maximum level of 70% 15 DAI. Conversely, for survivors, moderate mortality occurred 8-12 DAI, reaching a maximum level of 17%. The cumulative mortality for naïve fish was significantly higher than that for survivors (P 0.01). No mortalities occurred in any control groups. Virus recovery and measurement of the virus infectivity VHSV was recovered from all of the dead fish with the infectivity titers ranging from to TCID 50 /g, indicating that mortalities in all the Experiments were associated with VHSV. Discussion Virulence of flounder isolates of VHSV against Japanese flounder has been recognized in infectivity experiments (Mori et al. 2002). Our data on cumulative mortalities of Japanese flounder in Experiment 1 are consistent with this previous finding. In all the test groups injected with VHSV at different doses, occurrences of the mortality for oblong rockfish were more acute than those for Japanese flounder. In addition, the virus injection at a low dose of TCID 50 /fish to oblong rockfish allowed the efficient propagation of the virus in the organs leading to the high mortality. These reveal that the viral susceptibility of oblong rockfish is much higher than that of Japanese flounder. Therefore, VHS appears to be a threat to oblong rockfish rather than to Japanese flounder. The present study showed that the clinical signs of VHS in oblong rockfish generally resemble those in Japanese flounder. The characteristic signs of VHS have been reported in other susceptible marine fish species including black sea bream, red spotted grouper, and Shlegel s black rockfish, although the severity of hemorrhaging varied among the fish species (Isshiki et al. 2003). In this report, the authors suggested that some differences in the severity of hemorrhaging among fish species might depend on the susceptibility of capillary endothelial cells to the VHSV isolate. No histopathological examinations in the experimental fish were conducted in the present study. Thus, in order to determine a cause for the high susceptibility of oblong rockfish observed in the present study, further investigations are needed taking into account the histopathological changes of oblong rockfish and the tissuetropism of VHSV isolate. The results of Experiment 2 revealed that the viral susceptibility of oblong rockfish depends on a lower temperature, as was the case with Japanese flounder (Isshiki et al. 2002). In fact, increasing numbers of dead fish during a natural outbreak of VHS in oblong rockfish in Kagawa Prefecture, Japan were observed when the water temperature was about 12 (data not shown). Because the seed production for oblong rockfish commonly starts from winter and extends until spring (Tochino 2004), the reproductive seasons can be closely related to the epidemic of VHS. This may be the reason for a significant impact of VHS on seed production. Although the VHSV isolates from Japanese flounder have been reported to grow well in vitro in fish cell lines such as RSBK-2 and FHM at 20 (Isshiki et al. 2001; Mori et al. 2002), little mortality of oblong rockfish was induced by virus injection at 20 in this study. For Japanese flounder infected with VHSV, no mortality occurred at 20, whereas mortality was recorded at 15. (Isshiki et al. 2002). Castric and de Kinkekin (1984) have also reported similar findings that two marine species, sea bass Dicentrarchus labrax and turbot Scophthalmus maximus, were not susceptible to VHSV at 20, and they suggested that the production of interferon probably explains the lack of susceptibility at 20. In general, the water temperature affects the immune response of fish that are

6 T. ISSHIKI, G. ELENI, Y. UETA and T. NAGANO poikilotherms (Ellis 2001; Magnadottir 2006). The optimum temperatures are different among these four fish species: around 10 for oblong rockfish, sea bass and turbot, which are coldwater fish; but around 20 for Japanese flounder, which is a warm-water fish. Nevertheless, the susceptibilities of all the four fish species to VHSV decreased at 20. This suggests that the range of temperature where an optimal immune response can be established might not be in clearly agreement with the optimum temperature for some fish species. On the other hand, it is well known that the pathogenicity of fish viruses is influenced by the host fish size as well as the rearing temperature (Wolf 1988). Isshiki et al. (2001) have reported that pathogenicity of VHSV against Japanese flounder decreases with increasing body weight of the host fish; however, mortality still occurs even in large fish weighing about 1000 g. As mentioned above, the natural outbreak in oblong rockfish occurred in 2-year-old fish (mean body weight 91 g) with mortalities. Further studies will be needed to determine whether adult brood stocks of oblong rockfish are susceptible to VHSV. The results of Experiment 3 revealed that the survivors had higher protection than the naïve fish, suggesting induction of a protective immunity in the survivors through a primary viral infection as shown in previous studies on rainbow trout (de Kinkelin 1988). From this observation, some vaccinations against VHS possibly induce a similar protective immunity in oblong rockfish, and thus studies on vaccination against VHS could be conducted not only in rainbow trout but also in marine fish. In conclusion, the viral susceptibility of oblong rockfish is much higher than that of Japanese flounder and depends on a lower temperature. In addition, it is likely that oblong rockfish develops a protective immunity against VHSV infection. Development of vaccines against VHS is required for oblong rockfish as well as for Japanese flounder. Acknowledgments This work was partly supported by a Grant-in-Aid for Scientific Research from JSPS, Japan ( ). References Amaoka, K. (1984) Family Scorpaenidae. In The Fishes of the Japanese Archipelago (ed. by H. Masuda, K. Amaoka, C. Araga, T. Ueno and T. Toshino), Tokai Univ. Press, Tokyo, pp Amos, K., J. Thomas and K. Hopper (1998) A case history of adaptive management strategies for viral hemorrhagic septicemia virus (VHSV) in Washington state. J. Aquat. Anim. Health., 10, Brunson, R., K. True and J. Yancey (1989) VHS virus isolated at Makah National Fish Hatchery. Fish Health. Sect. Am. Fish Soc. Newsl., 17, 3-4. de Kinkeline, P. (1988) Vaccination against viral haemorrhagic septicaemia. In Fish Vaccination (ed. by A. E. Ellis), Academic Press, London, pp Ellis, A. E. (2001) Innate host defense mechanisms of fish against viruses and bacteria. Dev. Comp. Immunol., 25, Hopper, K. (1989) The isolation of VHSV from Chinook salmon at Glenwood Springs, Orcas Island, Washington. Fish Health Sect. Am. Fish Soc. Newsl., 17, 1. Isshiki, T., T. Nishizawa, T. Kobayashi, T. Nagano and T. Miyazaki (2001) An outbreak of VHSV (viral hemorrhagic septicemia virus) infection in farmed Japanese flounder Paralichthys olivaceus in Japan. Dis. Aquat. Org., 47, Isshiki, T., T. Nagano and T. Miyazaki (2002) Effect of water temperature on pathological states of Japanese flounder experimentally infected with viral hemorrhagic septicemia virus, a flounder isolate KRRV Fish Pathol., 37, Isshiki, T., T. Nagano and T. Miyazaki (2003) Susceptibility of various marine fish species to viral hemorrhagic septicemia virus isolated from Japanese flounder. Fish Pathol., 38, Ito, T., K.-I. Mori, M. Arimoto and K. Nakajima (2004) Virulence of viral hemorrhagic septicemia virus (VHSV) isolated from Japanese flounder Paralichthys olivaceus in rainbow trout and several species of marine fish. Fish Pathol., 39, Magnadottir, B. (2006) Innate immunity of fish (overview). Fish Shellfish Immunol., 20, Meyers, T. R., J. Sullivan, E. Emmenegger, J. Follett, S. Short, W. N. Batts and J. R. Winton (1992) Identification of viral hemorrhagic septicemia virus isolated from Pacific cod Gadus macrocephalus in Prince William Sound, Alaska, USA. Dis. Aquat. Org., 12, Meyers, T. R., S. Shorts, K. Lipson, W. N. Batts, J. R. Winton, J. Wilcock and E. Brown (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. Dis. Aquat. Org., 19, Meyers, T. R. and J. R. Winton (1995) Viral hemorrhagic

7 Susceptibility of Sebastes oblongus to VHSV septicemia virus in North America. Annu. Rev. Fish Dis., 5, Meyers, T. R., S. Shorts and K. Lipson (1999) Isolation of the North American strain of viral hemorrhagic septicemia virus (VHSV) associated with epizootic mortality in two new host species of Alaska marine fish. Dis. Aquat. Org., 38, Mori, K.-I., H. Iida, T. Nishizawa, M. Arimoto, Z. Nakajima and K. Muroga (2002) Properties of viral hemorrhagic septicemia virus (VHSV) isolated from Japanese flounder Paralichthys olivaceus. Fish Pathol., 37, Mortensen, H. F., O. E. Heueer, N. Lorenzen, L. Otte and N. J. Olesen (1999) Isolation of viral haemorrhagic septicaemia virus (VHSV) from wild marine fish species in the Baltic Sea, Kattegat, Skagerrak and the North Sea. Virus Res., 63, Nishizawa, T., H. Iida, R. Takano, T. Isshiki, K. Nakajima and K. Muroga (2002) Genetic relatedness among Japanese, American and European isolates of viral hemorrhagic septicemia virus (VHSV) based on partial G and P genes. Dis. Aquat. Org., 48, Ross, K., U. McCarthy, P. J. Huntly, B. P. Wood, D. Stuart, E. I. Rough, D. A. Smail and D. W. Eruno (1994) An outbreak of viral haemorrhagic septicaemia (VHS) in turbot (Scophthalmus maximus) in Scotland. Bull. Eur. Assoc. Fish Pathol., 14, Schlotfeldt, H. J., W. Ahne, P. E. Vestergard-Jorgensen and W. Glende (1991) Occurrence of viral haemorrhagic septicaemia in turbot (Scophthalmus maximus) - a natural outbreak. Bull. Eur. Assoc. Fish Pathol., 11, Smail, D. A. (1999) Viral haemorrhagic septicaemia. In Fish Diseases and Disorders, Vol 3. Viral, Bacterial and Fungal Infections (ed. by P. T. K. Wood and D. W. Bruno), CABI Publishing, NY, pp Smail, D. A. (2000) Isolation and identification of viral haemorrhagic septicaemia (VHS) viruses from cod Gadus mothua with the ulcus syndrome and from haddock Melanogrammus aeglefinus having skin haemorrhages in the North Sea. Dis. Aquat. Org., 41, Takano, R., T. Nishizawa, M. Arimoto and K. Muroga (2000) Isolation of viral hemorrhagic septicemia virus (VHSV) from wild Japanese flounder, Paralichthys olivaceus. Bull. Eur. Assoc. Fish Pathol., 20, Takano, R., K.-I. Mori, T. Nishizawa, M. Arimoto and K. Muroga (2001) Isolation of viruses from wild Japanese flounder, Paralichthys olivaceus. Fish Pathol., 36, Tochino, M. (2004) Possibility of culturing oblong rockfish. Aquaculture magazine, 41(7), (in Japanese). Tordo, N., A. Benmansour, C. Calisher, R. G. Dietzgen, R. X. Fang, A. O. Jackson, G. Kurath, S. Nadin-Davies, R. B. Tesh and P. J. Walker (2005) Family Rhabdoviridae. In Virus Taxonomy. Eight Report of the International Committee on Taxonomy of Viruses (ed. by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger and L. A. Ball), Academic Press, San Diego, CA, pp Vestergard-Jorgensen, P. E. and N. J. Olesen (1987) Cod ulcus syndrome rhabdovirus is indistinguishable from the Egtved (VHS) virus. Bull. Eur. Assoc. Fish Pathol., 7, Skall, H. F., N. J. Olesen and S. Mellergaad (2005) Viral haemorrhagic septicaemia virus in marine fish and its implications for fish farming - a review. J. Fish Dis., 28, Watanabe, L., R. Pakingking Jr, H. Iida, T. Nishizawa, Y. Iida, M. Arimoto and K. Muroga (2002) Isolation of aquabirnavirus and viral hemorrhagic septicemia virus (VHSV) from wild marine fishes. Fish Pathol., 37, Wolf, K. (1988) Viral hemorrhagic septicemia. In Fish Viruses and Fish Viral Diseases (ed. by K. Wolf), Cornell University Press, Ithaca, NY, pp Giannakou ELENI VHSV TCID 50 / VHSV TCID 50 / VHSV VHSV TCID 50 / 15 17