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Dual challenges of infectious pancreatic necrosis virus and Vibrio carchariae in the grouper, Epinephelus sp.
Virus Research 63 (1999) 131 – 134 www.elsevier.com/locate/virusres
Short communication
Dual challenges of infectious pancreatic necrosis virus and Vibrio carchariae in the grouper, Epinephelus sp. K.-K. Lee a,*, T.-I. Yang a, P.-C. Liu a, J.-L. Wu b, Y.-L. Hsu b a
Department of Aquaculture, National Taiwan Ocean Uni6ersity, 2, Pei-Ning Road, Keelung, Taiwan, ROC b Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan, ROC
Abstract The grouper industry in Taiwan faces serious threats from various disease problems. The present study investigated dual challenges with infectious pancreatic necrosis virus (IPNV) and Vibrio carchariae in the grouper (Epinephelus sp.). The fish were infected with IPNV for 2 weeks prior to a secondary infection with the bacteria, or vice versa, by either immersion (103 –104 TCID50 IPNV per ml, 106 –107 colony forming units (CFU) Vibrio per ml) or by intraperitoneal injection (103 –104 TCID50 IPNV per g fish or 107 CFU Vibrio/g fish) challenges. Mass mortalities occurred in fish infected with IPNV for 2 weeks prior to the infection with the bacteria, or vice versa, in either immersion or intraperitoneal injection challenges. The bacterium could only survive in seawater or brackish water similar to that of cultured groupers. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Dual challenge; Grouper; IPNV; Pathogenicity; Vibrio carchariae
Vibrio species are natural habitants of seawater and brackish water and widely distributed throughout the world. However, some species have exhibited clinical significance for aquatic animals and are recognized as potential pathogens. Vibriosis is one of the most serious diseases in cultured fish and shellfish (Ezura et al., 1980; Brisinello et al., 1985; Egidius, 1987; Lightner, 1988; Rasheed, 1989; Kusuda and Salati, 1993; Lee, 1995; Liao et al., 1996). Various Vibrio species have been demonstrated to be the causative * Corresponding author. Tel.: +886-2-24622192; fax: + 886-2-24633150.
agents of disease in cultured marine fish in Japan and Taiwan (Kusuda and Salati, 1993; Liao et al., 1996). Recently, we isolated and characterised Vibrio carchariae as a causative agent of gastroenteritis in groupers (Yii et al., 1997), but which caused mass mortality of the fish only after challenge dosage. Infectious pancreatic necrosis virus (IPNV) is a member of a group of aquatic birnaviruses which have been isolated from a wide range of aquatic animals and which are recognised as important fish pathogens (Mcknight and Roberts, 1976; Chen et al., 1984; Sorimachi and Hara, 1985; Wolf, 1988; Kimura and Yoshimizu, 1991; Hill
0168-1702/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 7 0 2 ( 9 9 ) 0 0 0 6 6 - 0
132
K.-K. Lee et al. / Virus Research 63 (1999) 131–134
and Way, 1995; Johansen and Sommer, 1997). The virus is shed by carriers with their sex products and in their faeces (Mcknight and Roberts, 1976; Wolf, 1988). The virus has been frequently diagnosed in fish in Japan and Taiwan (Chen et al., 1984; Sorimachi and Hara, 1985; Kimura and Yoshimizu, 1991), especially in marine fish such as yellowtail, Japanese flounder and sea bream. As the production and scale of marine fish culture in Taiwan has expanded this decade, the control of diseases caused by viral and bacterial pathogens has become increasingly important. The present study investigates the effect of dual challenges of both IPNV and vibrio in the grouper. Infectious pancreatic necrosis virus strain T42G was a gift from Dr Y.-L. Hsu, Institute of Zoology, Academia Sinica, Taipei, Taiwan. Chinook salmon embryo (CHSE-214) cell lines were grown in minimum essential medium (MEM) in Earle’s salt (Flow Laboratories), supplemented with foetal bovine serum to 10% (MEM-10), penicillin (100 IU/ml), streptomycin (100 mg/ml), gentamicin (25 mg/ml) and fungizone (0.25 mg/ml). The strain T42G was propagated in CHSE-214 cells for 7 days at 18–20°C (Hsu et al., 1993). Vibrio carchariae strain EmI82KL was previously isolated from the transparent yellow fluid of the swollen intestine of diseased grouper, Epinephelus coioides (Yii et al., 1997). Groupers (Epinephelus sp.) were held in tanks (2500 l) supplied with air-lifted 3% salinity seawater at 25–28°C. Fish weighing 0.12 – 0.32 g and
1.5–2.0 g were challenged by immersion for 2 h in a suspension of IPNV (103 –104 TCID50/ml) or V. carchariae (106 –107 colony forming units (CFU)/ ml) and held for 2 weeks prior to the second immersion with the other pathogen (Table 1). Groupers weighing 4–6 g and 15–20 g were challenged by intraperitoneal (i.p.) injection with IPNV (103 –104 TCID50/g fish) or vibrio (107 CFU/g fish) and held for 2 weeks prior to the second intraperitoneal injection with vibrio or IPNV. Suspensions of Vibrio carchariae strain EmI82KL were held at 25°C in seawater (34 ppt), brackish water (15 and 25 ppt) or freshwater, without supplementing nutrients. At intervals, the concentration of viable bacteria was determined in duplicate by plate count on tryptic soy agar (supplemented with 2% NaCl) for a duration of 14 weeks. Among the small groupers (0.12–0.32 g), all fish survived initial challenge with IPNV (103 TCID50/ml) for 2 weeks, but then died within 2 weeks following a second challenge using 107 CFU/ml V. carchariae (Table 1). Of the larger groupers (1.5–2.0 g) used in the immersion challenge 92% survived initial challenge with IPNV (104 TCID50/ml) for 2 weeks. Of these, 64% survived for 2 weeks following the second challenge using V. carchariae (107 CFU/ml) (Table 1). The results reveal that size of the grouper was inversely related to its degree of susceptibility to V. carchariae. This was further confirmed by chal-
Table 1 Challenges of IPNV strain T42G and V. carchariae strain EmI82KL in grouper fry by immersion Number (size) of groupers used
Agent for first challenge
Surviving fish after 2 weeks
Agent for second challenge
Surviving fish after 2 weeks
50 12 50 20 12
IPNV (103 TCID50/ml) IPNV (104 TCID50/ml) Vibrio (107 CFU/ml) Vibrio (106 CFU/g fish) Vibrio (107 CFU/ml)
50 11 0 12 12
Vibrio (107 CFU/ml) Vibrio (107 CFU/ml)
0 7
IPNV (103 TCID50/ml) IPNV (104 TCID50/ml)
0 0
(0.12–0.32 g) (1.5–2.0 g) (0.12–0.32 g) (0.12–0.32 g) (1.5–2.0 g)
K.-K. Lee et al. / Virus Research 63 (1999) 131–134
133
Table 2 Inoculation of IPNV strain T42G and V. carchariae strain EmI82KL in grouper fry by intraperitoneal injection Number (size) of groupers used
Agent for first challenge
Surviving fish after 2 weeks
Agent for second challenge
Surviving fish after 2 weeks
10 10 10 10
IPNV (103 TCID50/g fish) IPNV (104 TCID50/g fish) Vibrio (107 CFU/g fish Vibrio (107 CFU/g fish)
10 10 10 10
Vibrio (107 CFU/g fish) Vibrio (107 CFU/g fish IPNV (104 TCID50/g fish) IPNV (103 TCID50/g fish)
0 0 0 0
(4–6 g) (4–6 g) (15–20 g) (15–20 g)
lenging 50 smaller groupers with a dose of 107 CFU/ml of V. carchariae which resulted in 100% mortality (Table 1). Of the small groupers (0.12 – 0.32 g) 60% survived for 2 weeks after initial challenge with V. carchariae (106 CFU/ml). All the fish died within 2 weeks after a second challenge using IPNV at a dose of 103 TCID50/ml (Table 1). In a further study using larger groupers, all the fish (1.5 – 2.0 g) survived for 2 weeks after challenge with V. carchariae (107 CFU/ml), while all fish died within 2 weeks following a second challenge using IPNV at a dose of 104 TCID50/ml (Table 1). These results reveal that mass mortalities occur in groups of fish challenged by immersion with both IPNV and V. carchariae in either order, but not by a single immersion challenge with either agent when administered at moderate doses. All the smaller groupers (4 – 6 g) used in the injection challenge survived for 2 weeks after inoculation with IPNV (103 or 104 TCID50/g fish), but then died within 2 weeks following the second inoculation with V. carchariae at 107 CFU/g fish (Table 2). In a further experiment where larger (15 – 20 g) fish were injected in the opposite order, all the groupers survived for 2 weeks after initial inoculation with V. carchariae (107 CFU/g fish), while all the fish died within 2 weeks after the subsequent injection with IPNV at 103 or 104 TCID50/g fish (Table 2). Thus no mortality occurred in the fish challenged by intraperitoneal injection with either IPNV or V. carchariae alone and held for 2 weeks. However, if a second infection with the other agent was given, 100% of all fish died in the subsequent 2-week period. The above results showed that dual infections with IPNV and V. carchariae initiated by either
immersion or intraperitoneal injection cause mass mortality in the groupers. In the immersion study, a higher dosage of V. carchariae EmI82KL exhibited similar pathogenicity in the groupers as that described previously (Yii et al., 1997). Recently, Johansen and Sommer (1997) demonstrated that challenge with Vibrio salmonicida (intraperitoneal injection) 3 weeks after IPNV infection (bath challenge) gave a cumulative mortality of 39.7% in the group of Atlantic salmon post-smolts infected with both IPNV and the bacterium while significantly fewer fish (15%) died in a control group infected with only the bacterium. It seems that challenge with both IPNV and V. salmonicida were necessary to cause higher cumulative mortality in these post-smolts. Similarly, in our present study, we obtained mass mortality in both immersion and intraperitoneal injection challenges only in dual infections if the pathogens were delivered
Fig. 1. Survival of Vibrio carchariae strain EmI82KL in seawater (34 ppt) and brackish water (25 and 15 ppt) at 25°C without supplementing nutrients. Each data point represents the mean of two similar determinations.
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K.-K. Lee et al. / Virus Research 63 (1999) 131–134
at moderate doses. Our results confirmed the synergistic effects of these fish pathogens and showed that a second challenge with either IPNV or V. carchariae caused mass mortality. Vibrio carchariae strain EmI82KL did not survive in freshwater, while it remained viable in seawater or brackish water without supplementing nutrients for at least 14 weeks (Fig. 1). An apparent increase of viable counts, with highest counts in 15 ppt brackish water, was determined at week 1, indicating that numbers of the bacteria increased as a response to nutrient deprivation especially in lower salinity water in the early phase. This is in agreement with findings by Morita (1982) who suggested that a response to starvation by marine bacteria might be rapid division. The present results suggest that this Vibrio species might survive in marine or brackish systems much longer because of the presence of high amounts of organic matter in systems where marine fish are reared. While IPNV is generally recognised as a primary pathogen and frequently diagnosed in cultured marine fish, our results indicate the control of secondary invaders such as Vibrio species may be crucial to successful fish health management.
Acknowledgements The work was supported by a grant NSC-862311-B-019-002-B23 from the National Science Council, ROC. We thank W.-M. Su and C.-C. Tu for their technical assistance.
.
References Brisinello, W., Doimi, M., Giorgetti, G., Sarti, M., 1985. Vaccination trials against vibriosis in sea bass (Dicentrarchus labrax) fry. Bull. Eur. Assoc. Fish Pathol. 5, 55– 56. Chen, S.-N., Chi, S.-C., Guu, J.-J., Chen, J.-C., Kou, G.-H., 1984. Pathogenicity of birnavirus isolated from loach (Mis-
gurnus anguillicaubulatus). Fish Dis. Res. 6, 6 – 11. Egidius, E., 1987. Vibriosis: pathogenicity and pathology. A review. Aquaculture 67, 15 – 28. Ezura, Y., Tajima, K., Yoshimizu, M., Kimura, T., 1980. Studies on the taxonomy and serology of causative organisms of fish vibriosis. Fish Pathol. 14, 167 – 179. Hill, B.J., Way, K., 1995. Serological classification of infectious pancreatic necrosis (IPN) virus and other aquatic birnaviruses. Annu. Rev. Fish Dis. 5, 55 – 77. Hsu, Y.-L., Chen, B.-S., Wu, J.-L., 1993. Demonstration of infectious pancreatic necrosis virus strain VR-299 in Japanese eel, Anguilla japonica Temminck & Schlegel. J. Fish Dis. 16, 123 – 129. Johansen, L.-H., Sommer, A.-I., 1997. The interference of an IPN virus infection with other viral and bacterial infections in Atlantic salmon post-smolts. In: VIII EAFP International Conference, Edinburgh, 14 – 19 September 1997, abstract P-083. Kimura, T., Yoshimizu, M., 1991. Viral diseases of fish in Japan. Annu. Rev. Fish Dis. 1, 67 – 82. Kusuda, R., Salati, F., 1993. Major bacterial diseases affecting mariculture in Japan. Annu. Rev. Fish Dis. 3, 69 – 85. Lee, K.-K., 1995. Pathogenesis studies on Vibrio alginolyticus in the grouper, Epinephelus malabaricus, Bloch et Schneider. Microb. Pathog. 19, 39 – 48. Liao, I.-C., Lee, K.-K., Chien, Y.-H., 1996. Practical approaches to marine fish health problems in Taiwan. In: Main, K.L., Rosenfeld, C. (Eds.), Aquaculture Health Management Strategies for Marine Fish in Asia and the United States. The Oceanic Institute, Honolulu, pp. 57 – 67. Lightner, D.V., 1988. Vibrio disease of penaeid shrimp. In: Sindermann, C.J., Lightner, D.V. (Eds.), Disease Diagnosis and Control in North American Marine Aquaculture. Elsevier, Amsterdam, pp. 42 – 47. Mcknight, I.J., Roberts, R.J., 1976. The pathology of IPNV. I. The sequential histopathology of naturally occurring conditions. Br. Vet. J. 132, 76 – 86. Morita, R.Y., 1982. Starvation-survival of heterotrophs in the marine environment. Adv. Microb. Ecol. 6, 171 – 198. Rasheed, V.M., 1989. Diseases of cultured brown-spotted grouper Epinephelus tau6ina and silvery black porgy Acanthopagrus cu6ieri in Kuwait. J. Aquat. Anim. Health 1, 102 – 107. Sorimachi, M., Hara, T., 1985. Characteristics and pathogenicity of a virus isolated from yellowtail fingerlings showing ascites. Fish Pathol. 19, 231 – 238. Wolf, K., 1988. Fish Viruses and Fish Viral Diseases. Cornell University Press, Ithaca, NY. Yii, K.-C., Yang, T.-I., Lee, K.-K., 1997. Isolation and characterization of Vibrio carchariae, a causative agent of gastroenteritis in the groupers, Epinephelus coioides. Curr. Microbiol. 35, 109 – 115.
Short communication
Dual challenges of infectious pancreatic necrosis virus and Vibrio carchariae in the grouper, Epinephelus sp. K.-K. Lee a,*, T.-I. Yang a, P.-C. Liu a, J.-L. Wu b, Y.-L. Hsu b a
Department of Aquaculture, National Taiwan Ocean Uni6ersity, 2, Pei-Ning Road, Keelung, Taiwan, ROC b Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan, ROC
Abstract The grouper industry in Taiwan faces serious threats from various disease problems. The present study investigated dual challenges with infectious pancreatic necrosis virus (IPNV) and Vibrio carchariae in the grouper (Epinephelus sp.). The fish were infected with IPNV for 2 weeks prior to a secondary infection with the bacteria, or vice versa, by either immersion (103 –104 TCID50 IPNV per ml, 106 –107 colony forming units (CFU) Vibrio per ml) or by intraperitoneal injection (103 –104 TCID50 IPNV per g fish or 107 CFU Vibrio/g fish) challenges. Mass mortalities occurred in fish infected with IPNV for 2 weeks prior to the infection with the bacteria, or vice versa, in either immersion or intraperitoneal injection challenges. The bacterium could only survive in seawater or brackish water similar to that of cultured groupers. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Dual challenge; Grouper; IPNV; Pathogenicity; Vibrio carchariae
Vibrio species are natural habitants of seawater and brackish water and widely distributed throughout the world. However, some species have exhibited clinical significance for aquatic animals and are recognized as potential pathogens. Vibriosis is one of the most serious diseases in cultured fish and shellfish (Ezura et al., 1980; Brisinello et al., 1985; Egidius, 1987; Lightner, 1988; Rasheed, 1989; Kusuda and Salati, 1993; Lee, 1995; Liao et al., 1996). Various Vibrio species have been demonstrated to be the causative * Corresponding author. Tel.: +886-2-24622192; fax: + 886-2-24633150.
agents of disease in cultured marine fish in Japan and Taiwan (Kusuda and Salati, 1993; Liao et al., 1996). Recently, we isolated and characterised Vibrio carchariae as a causative agent of gastroenteritis in groupers (Yii et al., 1997), but which caused mass mortality of the fish only after challenge dosage. Infectious pancreatic necrosis virus (IPNV) is a member of a group of aquatic birnaviruses which have been isolated from a wide range of aquatic animals and which are recognised as important fish pathogens (Mcknight and Roberts, 1976; Chen et al., 1984; Sorimachi and Hara, 1985; Wolf, 1988; Kimura and Yoshimizu, 1991; Hill
0168-1702/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 7 0 2 ( 9 9 ) 0 0 0 6 6 - 0
132
K.-K. Lee et al. / Virus Research 63 (1999) 131–134
and Way, 1995; Johansen and Sommer, 1997). The virus is shed by carriers with their sex products and in their faeces (Mcknight and Roberts, 1976; Wolf, 1988). The virus has been frequently diagnosed in fish in Japan and Taiwan (Chen et al., 1984; Sorimachi and Hara, 1985; Kimura and Yoshimizu, 1991), especially in marine fish such as yellowtail, Japanese flounder and sea bream. As the production and scale of marine fish culture in Taiwan has expanded this decade, the control of diseases caused by viral and bacterial pathogens has become increasingly important. The present study investigates the effect of dual challenges of both IPNV and vibrio in the grouper. Infectious pancreatic necrosis virus strain T42G was a gift from Dr Y.-L. Hsu, Institute of Zoology, Academia Sinica, Taipei, Taiwan. Chinook salmon embryo (CHSE-214) cell lines were grown in minimum essential medium (MEM) in Earle’s salt (Flow Laboratories), supplemented with foetal bovine serum to 10% (MEM-10), penicillin (100 IU/ml), streptomycin (100 mg/ml), gentamicin (25 mg/ml) and fungizone (0.25 mg/ml). The strain T42G was propagated in CHSE-214 cells for 7 days at 18–20°C (Hsu et al., 1993). Vibrio carchariae strain EmI82KL was previously isolated from the transparent yellow fluid of the swollen intestine of diseased grouper, Epinephelus coioides (Yii et al., 1997). Groupers (Epinephelus sp.) were held in tanks (2500 l) supplied with air-lifted 3% salinity seawater at 25–28°C. Fish weighing 0.12 – 0.32 g and
1.5–2.0 g were challenged by immersion for 2 h in a suspension of IPNV (103 –104 TCID50/ml) or V. carchariae (106 –107 colony forming units (CFU)/ ml) and held for 2 weeks prior to the second immersion with the other pathogen (Table 1). Groupers weighing 4–6 g and 15–20 g were challenged by intraperitoneal (i.p.) injection with IPNV (103 –104 TCID50/g fish) or vibrio (107 CFU/g fish) and held for 2 weeks prior to the second intraperitoneal injection with vibrio or IPNV. Suspensions of Vibrio carchariae strain EmI82KL were held at 25°C in seawater (34 ppt), brackish water (15 and 25 ppt) or freshwater, without supplementing nutrients. At intervals, the concentration of viable bacteria was determined in duplicate by plate count on tryptic soy agar (supplemented with 2% NaCl) for a duration of 14 weeks. Among the small groupers (0.12–0.32 g), all fish survived initial challenge with IPNV (103 TCID50/ml) for 2 weeks, but then died within 2 weeks following a second challenge using 107 CFU/ml V. carchariae (Table 1). Of the larger groupers (1.5–2.0 g) used in the immersion challenge 92% survived initial challenge with IPNV (104 TCID50/ml) for 2 weeks. Of these, 64% survived for 2 weeks following the second challenge using V. carchariae (107 CFU/ml) (Table 1). The results reveal that size of the grouper was inversely related to its degree of susceptibility to V. carchariae. This was further confirmed by chal-
Table 1 Challenges of IPNV strain T42G and V. carchariae strain EmI82KL in grouper fry by immersion Number (size) of groupers used
Agent for first challenge
Surviving fish after 2 weeks
Agent for second challenge
Surviving fish after 2 weeks
50 12 50 20 12
IPNV (103 TCID50/ml) IPNV (104 TCID50/ml) Vibrio (107 CFU/ml) Vibrio (106 CFU/g fish) Vibrio (107 CFU/ml)
50 11 0 12 12
Vibrio (107 CFU/ml) Vibrio (107 CFU/ml)
0 7
IPNV (103 TCID50/ml) IPNV (104 TCID50/ml)
0 0
(0.12–0.32 g) (1.5–2.0 g) (0.12–0.32 g) (0.12–0.32 g) (1.5–2.0 g)
K.-K. Lee et al. / Virus Research 63 (1999) 131–134
133
Table 2 Inoculation of IPNV strain T42G and V. carchariae strain EmI82KL in grouper fry by intraperitoneal injection Number (size) of groupers used
Agent for first challenge
Surviving fish after 2 weeks
Agent for second challenge
Surviving fish after 2 weeks
10 10 10 10
IPNV (103 TCID50/g fish) IPNV (104 TCID50/g fish) Vibrio (107 CFU/g fish Vibrio (107 CFU/g fish)
10 10 10 10
Vibrio (107 CFU/g fish) Vibrio (107 CFU/g fish IPNV (104 TCID50/g fish) IPNV (103 TCID50/g fish)
0 0 0 0
(4–6 g) (4–6 g) (15–20 g) (15–20 g)
lenging 50 smaller groupers with a dose of 107 CFU/ml of V. carchariae which resulted in 100% mortality (Table 1). Of the small groupers (0.12 – 0.32 g) 60% survived for 2 weeks after initial challenge with V. carchariae (106 CFU/ml). All the fish died within 2 weeks after a second challenge using IPNV at a dose of 103 TCID50/ml (Table 1). In a further study using larger groupers, all the fish (1.5 – 2.0 g) survived for 2 weeks after challenge with V. carchariae (107 CFU/ml), while all fish died within 2 weeks following a second challenge using IPNV at a dose of 104 TCID50/ml (Table 1). These results reveal that mass mortalities occur in groups of fish challenged by immersion with both IPNV and V. carchariae in either order, but not by a single immersion challenge with either agent when administered at moderate doses. All the smaller groupers (4 – 6 g) used in the injection challenge survived for 2 weeks after inoculation with IPNV (103 or 104 TCID50/g fish), but then died within 2 weeks following the second inoculation with V. carchariae at 107 CFU/g fish (Table 2). In a further experiment where larger (15 – 20 g) fish were injected in the opposite order, all the groupers survived for 2 weeks after initial inoculation with V. carchariae (107 CFU/g fish), while all the fish died within 2 weeks after the subsequent injection with IPNV at 103 or 104 TCID50/g fish (Table 2). Thus no mortality occurred in the fish challenged by intraperitoneal injection with either IPNV or V. carchariae alone and held for 2 weeks. However, if a second infection with the other agent was given, 100% of all fish died in the subsequent 2-week period. The above results showed that dual infections with IPNV and V. carchariae initiated by either
immersion or intraperitoneal injection cause mass mortality in the groupers. In the immersion study, a higher dosage of V. carchariae EmI82KL exhibited similar pathogenicity in the groupers as that described previously (Yii et al., 1997). Recently, Johansen and Sommer (1997) demonstrated that challenge with Vibrio salmonicida (intraperitoneal injection) 3 weeks after IPNV infection (bath challenge) gave a cumulative mortality of 39.7% in the group of Atlantic salmon post-smolts infected with both IPNV and the bacterium while significantly fewer fish (15%) died in a control group infected with only the bacterium. It seems that challenge with both IPNV and V. salmonicida were necessary to cause higher cumulative mortality in these post-smolts. Similarly, in our present study, we obtained mass mortality in both immersion and intraperitoneal injection challenges only in dual infections if the pathogens were delivered
Fig. 1. Survival of Vibrio carchariae strain EmI82KL in seawater (34 ppt) and brackish water (25 and 15 ppt) at 25°C without supplementing nutrients. Each data point represents the mean of two similar determinations.
134
K.-K. Lee et al. / Virus Research 63 (1999) 131–134
at moderate doses. Our results confirmed the synergistic effects of these fish pathogens and showed that a second challenge with either IPNV or V. carchariae caused mass mortality. Vibrio carchariae strain EmI82KL did not survive in freshwater, while it remained viable in seawater or brackish water without supplementing nutrients for at least 14 weeks (Fig. 1). An apparent increase of viable counts, with highest counts in 15 ppt brackish water, was determined at week 1, indicating that numbers of the bacteria increased as a response to nutrient deprivation especially in lower salinity water in the early phase. This is in agreement with findings by Morita (1982) who suggested that a response to starvation by marine bacteria might be rapid division. The present results suggest that this Vibrio species might survive in marine or brackish systems much longer because of the presence of high amounts of organic matter in systems where marine fish are reared. While IPNV is generally recognised as a primary pathogen and frequently diagnosed in cultured marine fish, our results indicate the control of secondary invaders such as Vibrio species may be crucial to successful fish health management.
Acknowledgements The work was supported by a grant NSC-862311-B-019-002-B23 from the National Science Council, ROC. We thank W.-M. Su and C.-C. Tu for their technical assistance.
.
References Brisinello, W., Doimi, M., Giorgetti, G., Sarti, M., 1985. Vaccination trials against vibriosis in sea bass (Dicentrarchus labrax) fry. Bull. Eur. Assoc. Fish Pathol. 5, 55– 56. Chen, S.-N., Chi, S.-C., Guu, J.-J., Chen, J.-C., Kou, G.-H., 1984. Pathogenicity of birnavirus isolated from loach (Mis-
gurnus anguillicaubulatus). Fish Dis. Res. 6, 6 – 11. Egidius, E., 1987. Vibriosis: pathogenicity and pathology. A review. Aquaculture 67, 15 – 28. Ezura, Y., Tajima, K., Yoshimizu, M., Kimura, T., 1980. Studies on the taxonomy and serology of causative organisms of fish vibriosis. Fish Pathol. 14, 167 – 179. Hill, B.J., Way, K., 1995. Serological classification of infectious pancreatic necrosis (IPN) virus and other aquatic birnaviruses. Annu. Rev. Fish Dis. 5, 55 – 77. Hsu, Y.-L., Chen, B.-S., Wu, J.-L., 1993. Demonstration of infectious pancreatic necrosis virus strain VR-299 in Japanese eel, Anguilla japonica Temminck & Schlegel. J. Fish Dis. 16, 123 – 129. Johansen, L.-H., Sommer, A.-I., 1997. The interference of an IPN virus infection with other viral and bacterial infections in Atlantic salmon post-smolts. In: VIII EAFP International Conference, Edinburgh, 14 – 19 September 1997, abstract P-083. Kimura, T., Yoshimizu, M., 1991. Viral diseases of fish in Japan. Annu. Rev. Fish Dis. 1, 67 – 82. Kusuda, R., Salati, F., 1993. Major bacterial diseases affecting mariculture in Japan. Annu. Rev. Fish Dis. 3, 69 – 85. Lee, K.-K., 1995. Pathogenesis studies on Vibrio alginolyticus in the grouper, Epinephelus malabaricus, Bloch et Schneider. Microb. Pathog. 19, 39 – 48. Liao, I.-C., Lee, K.-K., Chien, Y.-H., 1996. Practical approaches to marine fish health problems in Taiwan. In: Main, K.L., Rosenfeld, C. (Eds.), Aquaculture Health Management Strategies for Marine Fish in Asia and the United States. The Oceanic Institute, Honolulu, pp. 57 – 67. Lightner, D.V., 1988. Vibrio disease of penaeid shrimp. In: Sindermann, C.J., Lightner, D.V. (Eds.), Disease Diagnosis and Control in North American Marine Aquaculture. Elsevier, Amsterdam, pp. 42 – 47. Mcknight, I.J., Roberts, R.J., 1976. The pathology of IPNV. I. The sequential histopathology of naturally occurring conditions. Br. Vet. J. 132, 76 – 86. Morita, R.Y., 1982. Starvation-survival of heterotrophs in the marine environment. Adv. Microb. Ecol. 6, 171 – 198. Rasheed, V.M., 1989. Diseases of cultured brown-spotted grouper Epinephelus tau6ina and silvery black porgy Acanthopagrus cu6ieri in Kuwait. J. Aquat. Anim. Health 1, 102 – 107. Sorimachi, M., Hara, T., 1985. Characteristics and pathogenicity of a virus isolated from yellowtail fingerlings showing ascites. Fish Pathol. 19, 231 – 238. Wolf, K., 1988. Fish Viruses and Fish Viral Diseases. Cornell University Press, Ithaca, NY. Yii, K.-C., Yang, T.-I., Lee, K.-K., 1997. Isolation and characterization of Vibrio carchariae, a causative agent of gastroenteritis in the groupers, Epinephelus coioides. Curr. Microbiol. 35, 109 – 115.