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In vitro studies of infectious pancreatic necrosis virus infections in leucocytes isolated from Atlantic salmon (Salmo salar L.)
Aquaculture ELSEVIER
Aquaculture 132 (1995) 91-95
In vitro studies of infectious pancreatic necrosis virus infections in leucocytes isolated from Atlantic salmon (Salmo salar L.) Lill-Heidi Johansen, Ann-Inger Sommer * Norwegian
Institute of Fisheries and Aquaculture Ltd., MB, P.O. Box 251 I, N-9002 Trams), Norway
Abstract Leucocytes were isolated from blood and head kidney of Atlantic salmon carrying infectious pancreatic necrosis virus (IPNV). Results that indicated an IPNV replication were only obtained from adherent head kidney leucocytes (mainly macrophages) . IPNV specific fluorescence was detected in both carrier and in vitro infected macrophages, and the number of fluorescent cells increased during a week in culture. However, the release of infectious IPNV was low. Preliminary results also indicated that in vitro IPNV infections of macrophages seemed to reduce the respiratory burst activity in these cells. Keywords: Infectious pancreatic necrosis virus; Carrier condition; Leucocytes; Sulmo salar
1. Introduction Infectious
pancreatic
necrosis
virus (IPNV)
causes
acute infections
with fatal outcome
in young trout. Several IPN epizootics in Atlantic salmon have recently been observed in Norwegian fish farms, with high losses around smolting (Christie et al., 1988; Krogsrud et al., 1989). Like other salmonid fish, Atlantic salmon surviving an IPNV infection may become asymptomatic carriers of the virus for long periods (Swanson and Gillespie, 1979; Smail and Munro, 1985). An association of IPNV with blood leucocytes from trout (Swanson and Gillespie, 1982; Yu et al., 1982; Ahne and Thomsen, 1986) and with head kidney leucocytes from Atlantic salmon (Knott and Munro, 1986) has previously been reported, but there are still uncertainties regarding the replication of IPNV in these cells (Estepa and Coll, 199 1) . In the present work we have isolated leucocytes from blood and head kidney of Atlantic salmon. The purpose was to study the production of virus in primarily
* Corresponding author. 0044-8486/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIOO44-8486(94)00392-O
92
L.-H. Johansen, A.-I. Sommer/Aquaculture
132 (1995) 91-95
both adherent and non-adherent leucocytes. The effect of IPNV infections on the respiratory burst activity in adherent leucocytes was also investigated.
2. Materials and methods Atlantic salmon, all asymptomatic IPNV carriers, weighing 120-200 g were sacrificed and blood was collected from the caudal vein and diluted in Leibowitz L- 15 medium supplied with 100 mg/ml gentamicin (G) and 100 I.U./ml heparin (H). The head kidney was dissected and teased through a metal mesh into L- 15 + G + H. Discontinuous Percoll density gradients were used for cell separation, based on methods described by Braun-Nesje et al. ( 198 1) . The leucocyte fraction was suspended in L- 15 + G and synthetic serum replacement ( 1: 1000, SSR, Medi-Cult), and 3 X lo6 cells per well were seeded in 24-welled plates with glass slides. All cell culture incubations were done at 17°C supplied with 2.5% CO*. After 1 h the non-adherent cells were removed, counted and transferred to new wells, and the number of adherent cells was estimated to about 4 X 10’ cells per well. According to BraunNesje et al. ( 1981) the isolated adherent head kidney leucocytes are mainly macrophages. Some of the wells were inoculated with IPNV serotype Nl or Sp to a multiplicity of infection (moi) of about 1. After 2 h incubation the cells were washed twice and fresh L- 15 + G + SSR was added. The cells were incubated and cell medium was collected and cells fixed for 15 min in 10% (v/v) formalin in PBS at day 1,3 and 7. The virus yield was tested by duplicated endpoint titration on chinook salmon embryo (CHSE)-214 cells. IPNV or viral products in the fixed cells were detected by an indirect fluorescent antibody technique (IFAT), by using monoclonal antibodies against IPNV protein 3 (VP3). The percentage IPNV specific fluorescent cells was estimated by counting the cells within 8 randomly chosen fields of vision by use of a Nikon UV-microscope. The respiratory burst activity in IPNV carrier and in vitro infected adherent head kidney macrophages was quantified by using reduction of nitroblue tetrazolium (NBT) as a measure of superoxide anion production. Phorbol myristate acetate (PMA) was used to trigger the respiratory burst (Chung and Secombes, 1988). The results from the NBT assays are means of triplicate readings expressed per lo5 cells, and they were analyzed statistically by a two-tailed Student’s t-test at a 5% level of significance.
3. Results IPNV was not detected in cultured blood leucocytes nor in non-adherent head kidney leucocytes from IPNV carrier fish. Both inoculation of collected medium onto CHSE-214 cells and IFAT gave negative results. In vitro infections of these negative cells with IPNV serotype Nl or Sp yielded very low, stable titers of lo’-10’ I.U./ml throughout a week in culture. IPNV specific fluorescence was detected in adherent head kidney leucocytes (mainly macrophages) from carrier fish. Increasing amounts of IPNV positive cells were observed during 1 week in culture, and pinpoint fluorescence was detected in about 50% of the macrophages on day 7. The initial number of fluorescent cells on day 1 correlated to the
L.-H. Johansen, A.4
Sommer/Aquaculture
132 (1995) 91-95
Table 1 IPNV carrier titers obtained by standard isolation procedures and percent immunofluorescent kidney macrophages detected day 1,3 and 7 after isolation of the cells Fish no.
Titre (I.U./g
tissues)
5x103 lo6 5x105 5x104
93
IPNV positive head
IPNV positive cells (%) Day 1
Day 3
Day 7
5 10 15 7
25 25 20 20
40 45 40 50
titers obtained by the standard isolation procedure (Table 1) . IPNV specific fluorescence was also detected in about 50% of the in vitro IPNV Nl or Sp infected macrophages on day 7 (data not shown). The virus yield obtained from macrophages carrying IPNV was very low and did not increase during the first 3 days in culture (Table 2). However, the only carrier cells tested for virus production on day 7 (fish no. 9) demonstrated a 1000 times increase in titer from day 1 to day 7. A 100 to 1000 times increase in virus yield was detected even by day 3 in some of the in vitro IPNV serotype Nl infected macrophage cultures (Table 2).
0.16 0.16
I
Day 1
Fish no.
Carrier
0 In vitro infected
Fig. 1. NBT reduction after PMA stimulation of head kidney macrophages from 5 fish at day 1 and day 3 after isolation and in vitro infection. The individual results are corrected for the NBT reduction by macrophages without PMA added (background activity).
94
L.-H. Johansen, A.-I. Sommer/Aquaculture
132 (1995) 91-95
Table 2 IPNV released from carrier aad in vitro IPNV serotype Nl infected head kidney macrophages period in culture. The IPNV yield is expressed as infectious units (I.U. ) /ml Fish no.
5 6 7 8 9
throughout
a 7-day
In vitro infected
Carrier Day 1
Day 3
10’ IO’ 10’ 10’ 0
10’ IO’ 10’ 10’ 0
Day 7
Day 1
Day 3
Day 7
103
103 IO3 lo* lo* lo*
103 IO4 lo5 lo4 5x102
104
Note: the five fish are the same as used for the NBT assay.
Except for fish number 6, in vitro IPNV serotype Nl infected macrophages demonstrated a statistically significant reduction of the respiratory burst activity on day 1, compared to carrier macrophages (P < 0.05). The same tendency was observed by day 3, but the reduction was only significant for fish numbers 8 and 9 (Fig. 1).
4. Discussion The present work investigated the ability of Atlantic salmon leucocytes to support an IPNV replication. Results that indicated IPNV multiplication were only obtained from adherent head kidney leucocytes, mainly macrophages according to Braun-Nesje et al. (1981). Others (Yu et al., 1982; Saint-Jean et al., 1991) have also reported both naturally and in vitro IPNV infected blood leucocytes (from trout) to be negative by use of IFAT, while Ahne and Thomsen (1986) did detect IPNV positive leucocyes from blood. This may be a matter of detection level for the IFAT, as demonstrated by Saint-Jean et al. ( 1991) applying the more sensitive flow cytometry. Yu et al. ( 1982) found that only a minor proportion of adherent blood leucocytes from trout were capable of replicating IPNV with relatively high yields. In the present work no significant virus yield was detected from carrier macrophages from Atlantic salmon during the first 3 days in culture. However, IPNV carrier cells from the only fish examined on day 7 demonstrated a 1000 times increase in titer. As these cells had been thoroughly washed after isolation and were not inoculated with virus, the increase both in titer and number of IFAT positive cells during the 7-day period in culture indicated production of new IPNV particles. The virus release was delayed compared to in vitro infected macrophages and may reflect a low number of initially infected carrier cells. The possibility that the titers obtained, especially from in vitro infected cells, may be due to excretion or release of still intact phagocytized or adherent virus particles should be taken into account. Further studies of the intracellular virus content are in progress and will help to elucidate this question. It has previously been concluded that trout leucocytes harbouring IPNV may not contribute significantly to the high titers found in kidneys by standard isolation methods (Yu
L.-H. Johansen, A.-I. Sommer/Aquaculture
132 (1995) 91-95
95
et al., 1982)) and that these cells are not lytic target cells for IPNV (Estepa and Coll, 199 1). However, low virus yield from non-lytic IPNV infected cells may not imply that the intracellular virus content is low. In Atlantic salmon this was supported by the IFAT results, and could explain the higher titers obtained from head kidneys by a procedure where the cells are disrupted. Knott and Munro ( 1986) reported that Atlantic salmon leucocytes from IPNV carriers demonstrated suppressed response to phytohemagglutinin stimulation. The respiratory burst is an important defence mechanism in macrophages activated by, for instance, invading microorganisms. The present preliminary results indicated that an IPNV infection may inhibit rather than stimulate the macrophages, but the in vivo importance of these findings is uncertain.
Acknowledgements We thank Dr. K.E. Christie for the kind gift of IPNV serotype Nl and monoclonal antibodies and Dr. J. Krogsrud for the gift of IPNV serotype Sp.
References Ahne, W. and Thomsen, I., 1986. Infectious pancreatic necrosis: detection of virus and antibodies in rainbow trout IPNV-carrier (Salvo gairdneri). J. Vet. Med. B., 33: 552-554. Braun-Nesje, R., Bertheussen, K., Kaplan, K. and Seljelid, R., 1981. Salmonid macrophages: separation, in vitro culture and characterization. J. Fish Dis., 4: 141-151. Christie, K.E., Havarstein, L.S., Djupvik, H.O., Ness, S. and Endresen, C., 1988. Characterization of a new serotype of infectious pancreatic necrosis virus isolated from Atlantic salmon. Arch. Virol., 103: 167-177. Chung, S. and Secombes, C.J., 1988. Analysis ofevents occurring within teleost macrophages during the respiratory burst. Comp. Biochem. Physiol., 89B, 3: 539-544. Estepa, A. and Coll, J.M., 1991. Infection of mitogen-stimulated trout leucocytes with salmonid viruses. J. Fish Dis., 14: 555-562. Knott, R.M. and Munro, A.L.S., 1986. The persistence of infectious pancreatic necrosis virus in Atlantic salmon. Vet. Immunol. Immunopathol., 12: 359-364. Krogsrud, J., H&stein, T. and Ronningen, K., 1989. Infectious pancreatic necrosis virus in Norwegian fish farms. In: W. Ahne and E. Kurstak (Editors), Viruses of Lower Vertebrates. Springer-Verlag. Berlin, pp. 284291. Saint-Jean, R., Minondo, P.V., Palacios, A. and Prieto, P., 1991. Detection of infectious pancreatic necrosis in a carrier population of rainbow trout, Oncorhynchus mykiss (Richardson), by flow cytometry. J. Fish Dis., 14: 545-553. Smail, D.A. and Munro, A.L.S., 1985. Infectious pancreatic necrosis virus persistence in farmed Atlantic salmon (Salmo salar). In: A.E. Ellis (Editor), Fish and Shellfish Pathology. Academic Press Inc., London, pp. 277288. Swanson, R.N. and Gillespie, J.H., 1979. Pathogenesis of infectious pancreatic necrosis in Atlantic salmon (Salmo salar). J. Fish. Res. Board Can., 36: 587-591. Swanson, R.N. and Gillespie, J.H., 1982. Isolation of infectious pancreatic necrosis virus from the blood and blood components of experimentally infected trout. Can. J. Fish. Aquat. Sci., 39: 225-228. Yu, K. K-Y., Macdonald, R.D. and Moore, A.R., 1982. Replication of infectious pancreatic necrosis virus in trout leucocytes and detection of the carrier state. J. Fish Dis., 5: 401410.
Aquaculture 132 (1995) 91-95
In vitro studies of infectious pancreatic necrosis virus infections in leucocytes isolated from Atlantic salmon (Salmo salar L.) Lill-Heidi Johansen, Ann-Inger Sommer * Norwegian
Institute of Fisheries and Aquaculture Ltd., MB, P.O. Box 251 I, N-9002 Trams), Norway
Abstract Leucocytes were isolated from blood and head kidney of Atlantic salmon carrying infectious pancreatic necrosis virus (IPNV). Results that indicated an IPNV replication were only obtained from adherent head kidney leucocytes (mainly macrophages) . IPNV specific fluorescence was detected in both carrier and in vitro infected macrophages, and the number of fluorescent cells increased during a week in culture. However, the release of infectious IPNV was low. Preliminary results also indicated that in vitro IPNV infections of macrophages seemed to reduce the respiratory burst activity in these cells. Keywords: Infectious pancreatic necrosis virus; Carrier condition; Leucocytes; Sulmo salar
1. Introduction Infectious
pancreatic
necrosis
virus (IPNV)
causes
acute infections
with fatal outcome
in young trout. Several IPN epizootics in Atlantic salmon have recently been observed in Norwegian fish farms, with high losses around smolting (Christie et al., 1988; Krogsrud et al., 1989). Like other salmonid fish, Atlantic salmon surviving an IPNV infection may become asymptomatic carriers of the virus for long periods (Swanson and Gillespie, 1979; Smail and Munro, 1985). An association of IPNV with blood leucocytes from trout (Swanson and Gillespie, 1982; Yu et al., 1982; Ahne and Thomsen, 1986) and with head kidney leucocytes from Atlantic salmon (Knott and Munro, 1986) has previously been reported, but there are still uncertainties regarding the replication of IPNV in these cells (Estepa and Coll, 199 1) . In the present work we have isolated leucocytes from blood and head kidney of Atlantic salmon. The purpose was to study the production of virus in primarily
* Corresponding author. 0044-8486/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIOO44-8486(94)00392-O
92
L.-H. Johansen, A.-I. Sommer/Aquaculture
132 (1995) 91-95
both adherent and non-adherent leucocytes. The effect of IPNV infections on the respiratory burst activity in adherent leucocytes was also investigated.
2. Materials and methods Atlantic salmon, all asymptomatic IPNV carriers, weighing 120-200 g were sacrificed and blood was collected from the caudal vein and diluted in Leibowitz L- 15 medium supplied with 100 mg/ml gentamicin (G) and 100 I.U./ml heparin (H). The head kidney was dissected and teased through a metal mesh into L- 15 + G + H. Discontinuous Percoll density gradients were used for cell separation, based on methods described by Braun-Nesje et al. ( 198 1) . The leucocyte fraction was suspended in L- 15 + G and synthetic serum replacement ( 1: 1000, SSR, Medi-Cult), and 3 X lo6 cells per well were seeded in 24-welled plates with glass slides. All cell culture incubations were done at 17°C supplied with 2.5% CO*. After 1 h the non-adherent cells were removed, counted and transferred to new wells, and the number of adherent cells was estimated to about 4 X 10’ cells per well. According to BraunNesje et al. ( 1981) the isolated adherent head kidney leucocytes are mainly macrophages. Some of the wells were inoculated with IPNV serotype Nl or Sp to a multiplicity of infection (moi) of about 1. After 2 h incubation the cells were washed twice and fresh L- 15 + G + SSR was added. The cells were incubated and cell medium was collected and cells fixed for 15 min in 10% (v/v) formalin in PBS at day 1,3 and 7. The virus yield was tested by duplicated endpoint titration on chinook salmon embryo (CHSE)-214 cells. IPNV or viral products in the fixed cells were detected by an indirect fluorescent antibody technique (IFAT), by using monoclonal antibodies against IPNV protein 3 (VP3). The percentage IPNV specific fluorescent cells was estimated by counting the cells within 8 randomly chosen fields of vision by use of a Nikon UV-microscope. The respiratory burst activity in IPNV carrier and in vitro infected adherent head kidney macrophages was quantified by using reduction of nitroblue tetrazolium (NBT) as a measure of superoxide anion production. Phorbol myristate acetate (PMA) was used to trigger the respiratory burst (Chung and Secombes, 1988). The results from the NBT assays are means of triplicate readings expressed per lo5 cells, and they were analyzed statistically by a two-tailed Student’s t-test at a 5% level of significance.
3. Results IPNV was not detected in cultured blood leucocytes nor in non-adherent head kidney leucocytes from IPNV carrier fish. Both inoculation of collected medium onto CHSE-214 cells and IFAT gave negative results. In vitro infections of these negative cells with IPNV serotype Nl or Sp yielded very low, stable titers of lo’-10’ I.U./ml throughout a week in culture. IPNV specific fluorescence was detected in adherent head kidney leucocytes (mainly macrophages) from carrier fish. Increasing amounts of IPNV positive cells were observed during 1 week in culture, and pinpoint fluorescence was detected in about 50% of the macrophages on day 7. The initial number of fluorescent cells on day 1 correlated to the
L.-H. Johansen, A.4
Sommer/Aquaculture
132 (1995) 91-95
Table 1 IPNV carrier titers obtained by standard isolation procedures and percent immunofluorescent kidney macrophages detected day 1,3 and 7 after isolation of the cells Fish no.
Titre (I.U./g
tissues)
5x103 lo6 5x105 5x104
93
IPNV positive head
IPNV positive cells (%) Day 1
Day 3
Day 7
5 10 15 7
25 25 20 20
40 45 40 50
titers obtained by the standard isolation procedure (Table 1) . IPNV specific fluorescence was also detected in about 50% of the in vitro IPNV Nl or Sp infected macrophages on day 7 (data not shown). The virus yield obtained from macrophages carrying IPNV was very low and did not increase during the first 3 days in culture (Table 2). However, the only carrier cells tested for virus production on day 7 (fish no. 9) demonstrated a 1000 times increase in titer from day 1 to day 7. A 100 to 1000 times increase in virus yield was detected even by day 3 in some of the in vitro IPNV serotype Nl infected macrophage cultures (Table 2).
0.16 0.16
I
Day 1
Fish no.
Carrier
0 In vitro infected
Fig. 1. NBT reduction after PMA stimulation of head kidney macrophages from 5 fish at day 1 and day 3 after isolation and in vitro infection. The individual results are corrected for the NBT reduction by macrophages without PMA added (background activity).
94
L.-H. Johansen, A.-I. Sommer/Aquaculture
132 (1995) 91-95
Table 2 IPNV released from carrier aad in vitro IPNV serotype Nl infected head kidney macrophages period in culture. The IPNV yield is expressed as infectious units (I.U. ) /ml Fish no.
5 6 7 8 9
throughout
a 7-day
In vitro infected
Carrier Day 1
Day 3
10’ IO’ 10’ 10’ 0
10’ IO’ 10’ 10’ 0
Day 7
Day 1
Day 3
Day 7
103
103 IO3 lo* lo* lo*
103 IO4 lo5 lo4 5x102
104
Note: the five fish are the same as used for the NBT assay.
Except for fish number 6, in vitro IPNV serotype Nl infected macrophages demonstrated a statistically significant reduction of the respiratory burst activity on day 1, compared to carrier macrophages (P < 0.05). The same tendency was observed by day 3, but the reduction was only significant for fish numbers 8 and 9 (Fig. 1).
4. Discussion The present work investigated the ability of Atlantic salmon leucocytes to support an IPNV replication. Results that indicated IPNV multiplication were only obtained from adherent head kidney leucocytes, mainly macrophages according to Braun-Nesje et al. (1981). Others (Yu et al., 1982; Saint-Jean et al., 1991) have also reported both naturally and in vitro IPNV infected blood leucocytes (from trout) to be negative by use of IFAT, while Ahne and Thomsen (1986) did detect IPNV positive leucocyes from blood. This may be a matter of detection level for the IFAT, as demonstrated by Saint-Jean et al. ( 1991) applying the more sensitive flow cytometry. Yu et al. ( 1982) found that only a minor proportion of adherent blood leucocytes from trout were capable of replicating IPNV with relatively high yields. In the present work no significant virus yield was detected from carrier macrophages from Atlantic salmon during the first 3 days in culture. However, IPNV carrier cells from the only fish examined on day 7 demonstrated a 1000 times increase in titer. As these cells had been thoroughly washed after isolation and were not inoculated with virus, the increase both in titer and number of IFAT positive cells during the 7-day period in culture indicated production of new IPNV particles. The virus release was delayed compared to in vitro infected macrophages and may reflect a low number of initially infected carrier cells. The possibility that the titers obtained, especially from in vitro infected cells, may be due to excretion or release of still intact phagocytized or adherent virus particles should be taken into account. Further studies of the intracellular virus content are in progress and will help to elucidate this question. It has previously been concluded that trout leucocytes harbouring IPNV may not contribute significantly to the high titers found in kidneys by standard isolation methods (Yu
L.-H. Johansen, A.-I. Sommer/Aquaculture
132 (1995) 91-95
95
et al., 1982)) and that these cells are not lytic target cells for IPNV (Estepa and Coll, 199 1). However, low virus yield from non-lytic IPNV infected cells may not imply that the intracellular virus content is low. In Atlantic salmon this was supported by the IFAT results, and could explain the higher titers obtained from head kidneys by a procedure where the cells are disrupted. Knott and Munro ( 1986) reported that Atlantic salmon leucocytes from IPNV carriers demonstrated suppressed response to phytohemagglutinin stimulation. The respiratory burst is an important defence mechanism in macrophages activated by, for instance, invading microorganisms. The present preliminary results indicated that an IPNV infection may inhibit rather than stimulate the macrophages, but the in vivo importance of these findings is uncertain.
Acknowledgements We thank Dr. K.E. Christie for the kind gift of IPNV serotype Nl and monoclonal antibodies and Dr. J. Krogsrud for the gift of IPNV serotype Sp.
References Ahne, W. and Thomsen, I., 1986. Infectious pancreatic necrosis: detection of virus and antibodies in rainbow trout IPNV-carrier (Salvo gairdneri). J. Vet. Med. B., 33: 552-554. Braun-Nesje, R., Bertheussen, K., Kaplan, K. and Seljelid, R., 1981. Salmonid macrophages: separation, in vitro culture and characterization. J. Fish Dis., 4: 141-151. Christie, K.E., Havarstein, L.S., Djupvik, H.O., Ness, S. and Endresen, C., 1988. Characterization of a new serotype of infectious pancreatic necrosis virus isolated from Atlantic salmon. Arch. Virol., 103: 167-177. Chung, S. and Secombes, C.J., 1988. Analysis ofevents occurring within teleost macrophages during the respiratory burst. Comp. Biochem. Physiol., 89B, 3: 539-544. Estepa, A. and Coll, J.M., 1991. Infection of mitogen-stimulated trout leucocytes with salmonid viruses. J. Fish Dis., 14: 555-562. Knott, R.M. and Munro, A.L.S., 1986. The persistence of infectious pancreatic necrosis virus in Atlantic salmon. Vet. Immunol. Immunopathol., 12: 359-364. Krogsrud, J., H&stein, T. and Ronningen, K., 1989. Infectious pancreatic necrosis virus in Norwegian fish farms. In: W. Ahne and E. Kurstak (Editors), Viruses of Lower Vertebrates. Springer-Verlag. Berlin, pp. 284291. Saint-Jean, R., Minondo, P.V., Palacios, A. and Prieto, P., 1991. Detection of infectious pancreatic necrosis in a carrier population of rainbow trout, Oncorhynchus mykiss (Richardson), by flow cytometry. J. Fish Dis., 14: 545-553. Smail, D.A. and Munro, A.L.S., 1985. Infectious pancreatic necrosis virus persistence in farmed Atlantic salmon (Salmo salar). In: A.E. Ellis (Editor), Fish and Shellfish Pathology. Academic Press Inc., London, pp. 277288. Swanson, R.N. and Gillespie, J.H., 1979. Pathogenesis of infectious pancreatic necrosis in Atlantic salmon (Salmo salar). J. Fish. Res. Board Can., 36: 587-591. Swanson, R.N. and Gillespie, J.H., 1982. Isolation of infectious pancreatic necrosis virus from the blood and blood components of experimentally infected trout. Can. J. Fish. Aquat. Sci., 39: 225-228. Yu, K. K-Y., Macdonald, R.D. and Moore, A.R., 1982. Replication of infectious pancreatic necrosis virus in trout leucocytes and detection of the carrier state. J. Fish Dis., 5: 401410.