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Pancreas disease in Atlantic salmon: serum neutralisation and passive immunisation
Fish & Shellfish Immunology (1996) 6, 465–472
Pancreas disease in Atlantic salmon: serum neutralisation and passive immunisation G. HOUGHTON
AND
A. E. ELLIS*
SOAEFD Marine Laboratory, PO Box 101, Victoria Road, Aberdeen, Scotland, U.K. (Received 5 March 1996, accepted in revised form 19 May 1996) Antisera raised in fish following experimental infection with pancreas disease either by intraperitoneal injection of infective kidney homogenate or by cohabitation with infected fish was found to give up to 100% neutralisation when incubated with PD infective kidney homogenate and then injected into fish. Complete neutralisation occurred with antisera obtained four, eight and 15 weeks after infection by injection and eight and 15 weeks after infection by cohabitation. When antisera, taken from fish eight weeks following infection by injection, was diluted up to 1:100, 100% neutralisation occurred. Significant levels of neutralisation still occurred at a 1:1000 dilution. Following passive immunisation of Atlantic salmon parr and post-smolts with week 8 antisera either one, two or three days before or at the same time as or one, two or three days after an i.p. injection of pancreas disease infective kidney homogenate, fish were 100% protected against the disease. ? 1996 Academic Press Limited
Key words:
pancreas disease, Atlantic salmon, serum neutralisation, passive immunisation.
I. Introduction Pancreas disease (PD) is a disease of farmed Atlantic salmon (Salmo salar) and leads to a total necrosis of the acinar cells of the exocrine pancreas (Munro et al., 1984; McVicar, 1987). McVicar (1990) demonstrated that the disease was caused by an infectious agent of possible viral aetiology. This was confirmed by Raynard & Houghton (1993) who developed a standardised method for experimental transmission of the disease. In a study on the kinetics of infection of fish experimentally injected with pancreas disease, Houghton (1995) demonstrated that plasma, blood leucocytes and lymphoid tissue were all involved in the dissemination of the PD virus through the fish. Recently, the causative agent of PD has been isolated in tissue culture and identified as a toga-like virus (Nelson et al., 1995). Houghton (1994) provided evidence that Atlantic salmon which had recovered from an experimental infection of pancreas disease developed a very strong resistance to the disease which was maintained for at least nine months post-infection. The exact nature of the response was not known but it was *Author to whom correspondence should be addressed. 465 1050–4648/96/060465+08 $18.00/0
? 1996 Academic Press Limited
466
G. HOUGHTON AND A. E. ELLIS
suggested that antibody may play a role. Neutralising antibodies have already been shown to play an important protective role in other viral diseases of fish such as to viral haemorrhagic septicaemia (VHS) in rainbow trout (Oncorhynchus mykiss) (Olesen & Vestergård-Jørgensen, 1986), infectious haematopoietic necrosis (HN) in rainbow trout (Vestergård-Jørgensen et al., 1991; LaPatra et al., 1993) and infectious salmon anaemia (ISA) in Atlantic salmon (Falk & Dannevig, 1995). In addition, passive immunisation of fish with antisera to fish viruses such as channel catfish virus (CCV) (Hedrick & McDowell, 1987), IHN (LaPatra et al., 1994) and ISA (Falk & Dannevig, 1995) have clearly shown a protective role of antibody to these viruses. The aims of the present work were to investigate whether Atlantic salmon experimentally infected with PD produced serum neutralising antibodies and, whether any antibodies produced could protect against the disease in passive immunisation trials. II. Materials and Methods FISH
Atlantic salmon were reared and maintained at the Fish Cultivation Unit of the Marine Laboratory, Aultbea, Wester Ross, Scotland. The unit has a history of being free of PD and IPN. Fish which were used to raise antisera were post-smolts maintained in sea water at 12) C in 2 m2 tanks at a flow rate of 20 l min "1. For the serum neutralisation experiments, salmon parr were used which were maintained in fresh water at 14) C in 1 m diameter tanks at a flow rate of 5 l min "1. For the passive immunisation experiments, both parr and post-smolts were used. Parr were maintained in fresh water and post-smolts were maintained in sea water as described above. Fish were fed a standard commercial diet by automatic feeders to satiation (Mainstream diets, BP nutrition). Fish were acclimated in tanks 2–4 weeks prior to experimentation. Prior to injection, freshwater fish were anaesthetised in ethyl-4-aminobenzoate (benzocaine, Sigma, UK) dissolved in ethanol, and sea water fish were anaesthetised in 3-aminobenzoic acid ethyl ester (MS222, Sigma, U.K.). HISTOLOGY
For histological examination of the pancreas, pyloric caeca with pancreas were fixed in 20% bu#ered formol saline, embedded in para$n wax and 5 ìm sections were cut and stained with haematoxylin and eosin. Assessment of PD pathology was carried out according to Raynard & Houghton (1993). PREPARATION OF PD INFECTIVE AND CONTROL KIDNEY HOMOGENATES
Pools of PD-infective and control non-infective kidney homogenates for use in experiments were prepared as described by Raynard & Houghton (1993). The infective and non-infective pools were homogenised, diluted in phosphate bu#ered saline (PBS, pH 7·2, w/o Ca2, Mg2 Gibco, U.K.), filtered (120 ìm and then 22 ìm, Millipore) and stored in liquid nitrogen before use.
PANCREAS DISEASE IN ATLANTIC SALMON
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The protein concentration was determined following procedure 690-A (Sigma, U.K.).
PRODUCTION OF SALMON ANTISERA TO PANCREAS DISEASE FOLLOWING INFECTION BY I.P. INFECTION OR COHABITATION
Sibling Atlantic salmon post-smolts (mean wt 189·7&33·5 g), held at 12) C, were infected by i.p. injection of PD infective kidney homogenate (N=180) with controls receiving non-infective kidney homogenate (N=60), at a dose of 10 ìg protein g "1 body wt. At the same time, non-injected fish were dye marked using a panjet (Wright Dental Health Group, U.K.) and added to the same tanks as the i.p. infected fish at a ratio of injected to non-injected of 3:1. These fish therefore became infected by cohabitation. Non-infected controls were kept in a separate tank. Samples (N=10) of pyloric caeca and pancreas were taken at weeks 1, 2, 3 and 4 following injection or cohabitation to assess pathology. Fish (N=10) were bled at weeks 4, 8 and 15, and sera from each sample time were pooled and stored at "80) C until used.
SERUM NEUTRALISATION
Undiluted antiserum, or antiserum diluted 1:10, 1:50, 1:100 and 1:1000 in PBS, and non-immune control sera were mixed with an equal volume of PD infective or non-infective control kidney homogenate and incubated at room temperature (20) C) for 1·5 h. This was then injected i.p. into salmon parr (mean 21·5 g, N=50) at a dose of 6 ìg kidney protein g "1 body wt which was twice the minimum infective dose (Raynard & Houghton, 1993). Fish were kept at 14) C to test for infectivity. Controls consisted of PD infective homogenate incubated with non-immune control serum or PBS and non-infective homogenate incubated with antiserum, non-immune control serum or PBS. Samples of pyloric caeca and pancreas were taken from 10 fish within each group at weekly intervals for up to five weeks.
PASSIVE IMMUNISATION
Fish were passively immunised with antiserum taken from fish eight weeks following an i.p. injection of PD-infective kidney homogenate. Salmon parr (N=40 for each group, mean weight 25 g) and post-smolts (N=40 for each group, mean weight 39 g) were injected with week 8 antiserum on days 3, 2 and 1 before, or at the same time as, or one, two or three days after an i.p. injection of 0·1 ml of PD infective kidney homogenate at a dose of 6 ìg protein g "1 body wt. The volume of serum (undiluted) injected into the parr was 0·1 ml and 0·3 ml for the post-smolts. Controls (N=40 parr and 40 post-smolts for each group at each time) consisted of fish injected with PBS or non-immune control serum at the same intervals as for the antiserum. Fish (N=10 for each group) were sampled for pyloric caeca and pancreas at weekly intervals to assess pancreas pathology.
G. HOUGHTON AND A. E. ELLIS
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Table 1. Percentage of Atlantic salmon post-smolts showing pancreas disease pathology following experimental infection by i.p. injection of infective kidney homogenate or following infection by cohabitation (cohab). Ten fish were sampled per group per week. Temperature 12)C Percentage PD, weeks post-infection Group
Control i.p. Control cohab PD i.p. PD cohab
1
2
3
4
0 0 90E 0
0 0 80 50E
0 0 80 50
0 0 80 90
E=early PD.
III. Results PD PATHOLOGY OF FISH USED FOR RAISING ANTISERA
The incidence of PD in the fish infected by i.p. and cohabitation is shown in Table 1. I.p. infected fish showed acute PD by week 2 following infection while the cohabiting fish showed pathology one week later. Infection levels of 80–90% were recorded in both groups. SERUM NEUTRALISATION
Serum neutralisation with sera from fish infected by injection and cohabitation Undiluted sera obtained from fish four, eight and 15 weeks after injection of PD-infective kidney homogenate gave a 100% reduction in PD pathology (100% neutralisation) when incubated with PD infective kidney homogenate and tested by i.p. injection into salmon (Table 2). The week 4 sera obtained from fish infected by cohabitation produced no neutralisation, with PD pathology levels of 70–100% occurring, similar to those obtained in the PBS-controls and control serum. However, sera obtained from fish eight and 15 weeks following cohabitation infection gave a reduction in PD pathology of 100% (100% neutralisation) (Table 2). No PD pathology occurred in fish injected with non-infective control kidney homogenate incubated with PBS, control serum or antisera. Serum neutralisation following dilution of antisera A reduction of PD pathology of 100%, representing a neutralisation of 100%, occurred when week 8 antisera (from fish infected by i.p. injection), which was either undiluted or diluted in PBS to 1:10, 1:50, 1:100, was incubated with PD-infective kidney homogenate and tested by i.p. injection of salmon parr (Table 3). At a dilution of 1:1000, a reduction in pathology occurred although levels of neutralisation were less than 100%. At week 2 postinjection, where peak levels of PD pathology of 100% occurred in the PBS and control serum groups, only 20% PD pathology (neutralisation of 80%) was
PANCREAS DISEASE IN ATLANTIC SALMON
469
Table 2. Percentage infection of Atlantic salmon parr with pancreas disease following injection of PD-infective kidney homogenate which had been incubated with antisera taken at weeks 4, 8 and 15 from fish following their infection by injection or cohabitation. Ten fish sampled per group per week. Temperature 12)C Percentage PD weeks post-injection
Fish injected with PD-infective kidney homogenate incubated with:
1
2
3
4
5
Antisera from fish i.p. infected with PD taken after: wk 4 wk 8 wk 15
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
Antisera from fish infected with PD by cohabitation taken after: wk 4 wk 8 wk 15 PBS Control sera
0 0 0 0 0
70E 0 0 40E 70E
80 0 0 90 90
100 0 0 80 90
100 0 0 80 90
E=early PD.
Table 3. Percentage infection of Atlantic salmon parr with pancreas disease following incubation of PD-infective kidney homogenate with week 8 anti-serum. Ten fish sampled per group per week. Temperature 14)C. Percentage PD weeks post-injection Group 1 PD-infective kidney incubated with: Immune serum neat Immune serum 1:10 Immune serum 1:50 Immune serum 1:100 Immune serum 1:1000 Control serum neat PBS
0 0 0 0 0 10E 70E
2
3
4
0 0 0 0 20 100 100
0 0 0 0 40 90 90
0 0 0 0 70 90 90
E=early PD.
seen in the 1:1000 dilution group. By week 4 post-injection, levels of PD pathology had increased but this may just represent multiplication of non-neutralised virus in the fish. PASSIVE IMMUNISATION
Atlantic salmon parr and post-smolts that were injected with week 8 antisera at daily intervals ranging from three days before to three days following injection of PD-infective kidney homogenate were 100% protected
470
G. HOUGHTON AND A. E. ELLIS
Table 4. Percentage infection of Atlantic salmon parr (P) and post-smolts (PS) with PD following passive immunisation with antiserum taken from fish eight weeks after i.p. infection with PD. Ten fish were sampled per group per week. Parr were held in fresh water at 14)C, post-smolts in sea water at 12)C. Antiserum was injected from three days prior to three days after injection of PD-infective kidney homogenate Percentage PD, weeks post injection Treatment
Immune serum Day of injection relative to infection: "3 "2 "1 0 +1 +2 +3 Control serum Day of injection relative to infection: "3 "2 "1 0 +1 +2 +3 PBS
1
2
3
4
P
PS
P
PS
P
PS
P
PS
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
50E 20E 90E 90E 90E 50E 40E 100E
30E 30E 60E 50E 60E 20E 50E 30E
60 80 100 100 100 80 100 100
50 40 60 80 60 30 50 80
80 80 100 100 100 50 80 90
50 50 100 60 80 60 60 80
50 60 100 100 100 80 60 100
20 60 40 80 90 30 30 50
E=early PD.
against pancreas disease with no pathology developing (Table 4). This was in contrast to the control groups injected with PBS or control serum where levels of PD pathology of up to 100% occurred.
IV. Discussion In an earlier paper (Houghton, 1994), it was clearly demonstrated that Atlantic salmon respond immunologically to an experimental infection with pancreas disease developing a solid, long lasting protection to the disease. The exact mechanism of the protection was not investigated, although the author suggested that neutralising antibodies may play a role. The present results show that Atlantic salmon do produce neutralising antibodies following an experimental infection with the disease with relatively high levels of neutralisation occurring. These neutralising antibodies were maintained for at least 15 weeks post-infection. In addition, as well as antibodies being produced following intraperitoneal injection of PD infective
PANCREAS DISEASE IN ATLANTIC SALMON
471
kidney homogenate, fish which were infected following cohabitation with these fish and thus becoming infected via the natural route, also produced neutralising antibodies. The production of neutralising antibodies appears to occur quickly following infection. Sera taken from fish four weeks following infection by injection completely neutralised the PD agent. Sera from fish four weeks following infection by cohabitation did not fully neutralise the infectious inoculum but did so after eight weeks. This most probably reflects the lag phase involved with infection by cohabitation as the fish infected by injection developed acute PD after two weeks while the pathology did not develop in the cohabiting fish until week 3. Convalescent antisera taken from fish eight weeks following infection by injection appeared to have a high titre of neutralising antibody since complete neutralisation of the PD inoculum was achieved with antiserum diluted 1:100. An apparent partial neutralisation, indicated by delayed onset of PD pathology, occurred with antiserum diluted 1:1000. Although the production of neutralising antibodies has been clearly demonstrated, it still does not indicate whether they play a role in protection to the disease. Passive immunisation is a method where the protective nature of antibodies to pancreas disease can be demonstrated. From the present results, passive immunisation with primary antiserum taken eight weeks postinfection, protected fish against an i.p. challenge with PD-infective kidney homogenate. Protection levels of 100% occurred at all the times that fish were injected with the antiserum from three days before infection to three days post-infection. These results indicate that neutralising antibodies can remain active in the fish at least three days prior to infection and can still neutralise the virus when given three days post-infection at a stage when it is known that the PD agent is already within the blood system (Houghton, 1995). The previous finding (Houghton, 1994) that, following experimental infection, Atlantic salmon are resistant to subsequent challenges for at least nine months, suggests that neutralising antibodies to PD may remain in circulation for at least this length of time or that exposure of the fish to the virus induces a protective secondary response. In the present study, antiserum taken 15 weeks after infection fully neutralised the infective inoculum, and the complete protection a#orded by week 8 antiserum, even at dilutions of 1:100 indicate that the humoral immune response of Atlantic salmon to PD plays an important role in protection. The fact that salmon do respond by producing neutralising antibodies is encouraging for the development of a successful vaccine. The authors thank P. MacLachlan, S. Clements and F. Reid for assistance in sampling and maintaining the fish. The work was fund aided by the Scottish Salmon Growers Association and the Crown Estate Commissioners.
References Falk, K. & Dannevig, B. H. (1995). Demonstration of a protective immune response in infectious salmon anaemia (ISA)-infected Atlantic salmon Salmo salar. Diseases of Aquatic Organisms 21, 1–5.
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Hedrick, R. P. & McDowell, T. (1987). Passive transfer of sera with antivirus neutralising activity from adult channel catfish protects juveniles from channel catfish virus disease. Transactions of the American Fisheries Society 116, 277–281. Houghton, G. (1994). Acquired protection in Atlantic salmon Salmo salar parr and post-smolts against pancreas disease. Diseases of Aquatic Organisms 18, 109–118. Houghton, G. (1995). Kinetics of infection of plasma, blood leucocytes and lymphoid tissue from Atlantic salmon Salmo salar experimentally infected with pancreas disease. Diseases of Aquatic Organisms 22, 193–198. LaPatra, S. E., Turner, T., Lauda, K. A., Jones, G. R. & Walker, S. (1993). Characterization of the humoral response of rainbow trout to infectious haematopoietic necrosis virus. Journal of Aquatic Animal Health 5, 165–171. LaPatra, S. E., Lauda, K. A., Jones, G. R., Walker, S. C. & Shewmaker, W. D. (1994). Development of passive immunotherapy for control of infectious haematopoietic necrosis. Diseases of Aquatic Organisms 20, 1–6. McVicar, A. H. (1987). Pancreas disease of farmed Atlantic salmon, Salmo salar, in Scotland: epidemiology and early pathology. Aquaculture 67, 71–78. McVicar, A. H. (1990). Infection as a primary cause of pancreas disease in farmed Atlantic salmon. Bulletin of the European Association of Fish Pathologists 10, 84–87. Munro, A. L. S., Ellis, A. E., McVicar, A. H. & McLay, H. A. (1984). An exocrine pancreas disease of farmed Atlantic salmon in Scotland. Helgoländer Meeresunters 37, 571–586. Nelson, R. T., McLoughlin, M. F., Rowley, H. M., Platten, M. A. & McCormick, J. I. (1995). Isolation of a toga-like virus from farmed Atlantic salmon Salmo salar with pancreas disease. Diseases of Aquatic Organisms 22, 25–32. Olesen, N. J. & Vestergård-Jørgensen, P. E. (1986). Detection of neutralising antibody to Egtved virus in rainbow trout (Salmo gairdneri) by plaque neutralization test with complement addition. Journal of Applied Ichthyology 2, 33–41. Raynard, R. S. & Houghton, G. (1993). Development towards an experimental protocol for the transmission of pancreas disease in Atlantic salmon Salmo salar. Diseases of Aquatic Organisms 15, 123–128. Vestergård-Jørgensen, P. E., Olesen, N. J., Lorenzen, N., Winton, J. R. & Ristow, S. S. (1991). Infectious haematopoietic necrosis (IHN): detection of trout antibodies to the causative viruses by means of plaque neutralization, immunofluorescence and enzyme-linked immunosorbent assay. Journal of Aquatic Animal Health 3, 100–108.
Pancreas disease in Atlantic salmon: serum neutralisation and passive immunisation G. HOUGHTON
AND
A. E. ELLIS*
SOAEFD Marine Laboratory, PO Box 101, Victoria Road, Aberdeen, Scotland, U.K. (Received 5 March 1996, accepted in revised form 19 May 1996) Antisera raised in fish following experimental infection with pancreas disease either by intraperitoneal injection of infective kidney homogenate or by cohabitation with infected fish was found to give up to 100% neutralisation when incubated with PD infective kidney homogenate and then injected into fish. Complete neutralisation occurred with antisera obtained four, eight and 15 weeks after infection by injection and eight and 15 weeks after infection by cohabitation. When antisera, taken from fish eight weeks following infection by injection, was diluted up to 1:100, 100% neutralisation occurred. Significant levels of neutralisation still occurred at a 1:1000 dilution. Following passive immunisation of Atlantic salmon parr and post-smolts with week 8 antisera either one, two or three days before or at the same time as or one, two or three days after an i.p. injection of pancreas disease infective kidney homogenate, fish were 100% protected against the disease. ? 1996 Academic Press Limited
Key words:
pancreas disease, Atlantic salmon, serum neutralisation, passive immunisation.
I. Introduction Pancreas disease (PD) is a disease of farmed Atlantic salmon (Salmo salar) and leads to a total necrosis of the acinar cells of the exocrine pancreas (Munro et al., 1984; McVicar, 1987). McVicar (1990) demonstrated that the disease was caused by an infectious agent of possible viral aetiology. This was confirmed by Raynard & Houghton (1993) who developed a standardised method for experimental transmission of the disease. In a study on the kinetics of infection of fish experimentally injected with pancreas disease, Houghton (1995) demonstrated that plasma, blood leucocytes and lymphoid tissue were all involved in the dissemination of the PD virus through the fish. Recently, the causative agent of PD has been isolated in tissue culture and identified as a toga-like virus (Nelson et al., 1995). Houghton (1994) provided evidence that Atlantic salmon which had recovered from an experimental infection of pancreas disease developed a very strong resistance to the disease which was maintained for at least nine months post-infection. The exact nature of the response was not known but it was *Author to whom correspondence should be addressed. 465 1050–4648/96/060465+08 $18.00/0
? 1996 Academic Press Limited
466
G. HOUGHTON AND A. E. ELLIS
suggested that antibody may play a role. Neutralising antibodies have already been shown to play an important protective role in other viral diseases of fish such as to viral haemorrhagic septicaemia (VHS) in rainbow trout (Oncorhynchus mykiss) (Olesen & Vestergård-Jørgensen, 1986), infectious haematopoietic necrosis (HN) in rainbow trout (Vestergård-Jørgensen et al., 1991; LaPatra et al., 1993) and infectious salmon anaemia (ISA) in Atlantic salmon (Falk & Dannevig, 1995). In addition, passive immunisation of fish with antisera to fish viruses such as channel catfish virus (CCV) (Hedrick & McDowell, 1987), IHN (LaPatra et al., 1994) and ISA (Falk & Dannevig, 1995) have clearly shown a protective role of antibody to these viruses. The aims of the present work were to investigate whether Atlantic salmon experimentally infected with PD produced serum neutralising antibodies and, whether any antibodies produced could protect against the disease in passive immunisation trials. II. Materials and Methods FISH
Atlantic salmon were reared and maintained at the Fish Cultivation Unit of the Marine Laboratory, Aultbea, Wester Ross, Scotland. The unit has a history of being free of PD and IPN. Fish which were used to raise antisera were post-smolts maintained in sea water at 12) C in 2 m2 tanks at a flow rate of 20 l min "1. For the serum neutralisation experiments, salmon parr were used which were maintained in fresh water at 14) C in 1 m diameter tanks at a flow rate of 5 l min "1. For the passive immunisation experiments, both parr and post-smolts were used. Parr were maintained in fresh water and post-smolts were maintained in sea water as described above. Fish were fed a standard commercial diet by automatic feeders to satiation (Mainstream diets, BP nutrition). Fish were acclimated in tanks 2–4 weeks prior to experimentation. Prior to injection, freshwater fish were anaesthetised in ethyl-4-aminobenzoate (benzocaine, Sigma, UK) dissolved in ethanol, and sea water fish were anaesthetised in 3-aminobenzoic acid ethyl ester (MS222, Sigma, U.K.). HISTOLOGY
For histological examination of the pancreas, pyloric caeca with pancreas were fixed in 20% bu#ered formol saline, embedded in para$n wax and 5 ìm sections were cut and stained with haematoxylin and eosin. Assessment of PD pathology was carried out according to Raynard & Houghton (1993). PREPARATION OF PD INFECTIVE AND CONTROL KIDNEY HOMOGENATES
Pools of PD-infective and control non-infective kidney homogenates for use in experiments were prepared as described by Raynard & Houghton (1993). The infective and non-infective pools were homogenised, diluted in phosphate bu#ered saline (PBS, pH 7·2, w/o Ca2, Mg2 Gibco, U.K.), filtered (120 ìm and then 22 ìm, Millipore) and stored in liquid nitrogen before use.
PANCREAS DISEASE IN ATLANTIC SALMON
467
The protein concentration was determined following procedure 690-A (Sigma, U.K.).
PRODUCTION OF SALMON ANTISERA TO PANCREAS DISEASE FOLLOWING INFECTION BY I.P. INFECTION OR COHABITATION
Sibling Atlantic salmon post-smolts (mean wt 189·7&33·5 g), held at 12) C, were infected by i.p. injection of PD infective kidney homogenate (N=180) with controls receiving non-infective kidney homogenate (N=60), at a dose of 10 ìg protein g "1 body wt. At the same time, non-injected fish were dye marked using a panjet (Wright Dental Health Group, U.K.) and added to the same tanks as the i.p. infected fish at a ratio of injected to non-injected of 3:1. These fish therefore became infected by cohabitation. Non-infected controls were kept in a separate tank. Samples (N=10) of pyloric caeca and pancreas were taken at weeks 1, 2, 3 and 4 following injection or cohabitation to assess pathology. Fish (N=10) were bled at weeks 4, 8 and 15, and sera from each sample time were pooled and stored at "80) C until used.
SERUM NEUTRALISATION
Undiluted antiserum, or antiserum diluted 1:10, 1:50, 1:100 and 1:1000 in PBS, and non-immune control sera were mixed with an equal volume of PD infective or non-infective control kidney homogenate and incubated at room temperature (20) C) for 1·5 h. This was then injected i.p. into salmon parr (mean 21·5 g, N=50) at a dose of 6 ìg kidney protein g "1 body wt which was twice the minimum infective dose (Raynard & Houghton, 1993). Fish were kept at 14) C to test for infectivity. Controls consisted of PD infective homogenate incubated with non-immune control serum or PBS and non-infective homogenate incubated with antiserum, non-immune control serum or PBS. Samples of pyloric caeca and pancreas were taken from 10 fish within each group at weekly intervals for up to five weeks.
PASSIVE IMMUNISATION
Fish were passively immunised with antiserum taken from fish eight weeks following an i.p. injection of PD-infective kidney homogenate. Salmon parr (N=40 for each group, mean weight 25 g) and post-smolts (N=40 for each group, mean weight 39 g) were injected with week 8 antiserum on days 3, 2 and 1 before, or at the same time as, or one, two or three days after an i.p. injection of 0·1 ml of PD infective kidney homogenate at a dose of 6 ìg protein g "1 body wt. The volume of serum (undiluted) injected into the parr was 0·1 ml and 0·3 ml for the post-smolts. Controls (N=40 parr and 40 post-smolts for each group at each time) consisted of fish injected with PBS or non-immune control serum at the same intervals as for the antiserum. Fish (N=10 for each group) were sampled for pyloric caeca and pancreas at weekly intervals to assess pancreas pathology.
G. HOUGHTON AND A. E. ELLIS
468
Table 1. Percentage of Atlantic salmon post-smolts showing pancreas disease pathology following experimental infection by i.p. injection of infective kidney homogenate or following infection by cohabitation (cohab). Ten fish were sampled per group per week. Temperature 12)C Percentage PD, weeks post-infection Group
Control i.p. Control cohab PD i.p. PD cohab
1
2
3
4
0 0 90E 0
0 0 80 50E
0 0 80 50
0 0 80 90
E=early PD.
III. Results PD PATHOLOGY OF FISH USED FOR RAISING ANTISERA
The incidence of PD in the fish infected by i.p. and cohabitation is shown in Table 1. I.p. infected fish showed acute PD by week 2 following infection while the cohabiting fish showed pathology one week later. Infection levels of 80–90% were recorded in both groups. SERUM NEUTRALISATION
Serum neutralisation with sera from fish infected by injection and cohabitation Undiluted sera obtained from fish four, eight and 15 weeks after injection of PD-infective kidney homogenate gave a 100% reduction in PD pathology (100% neutralisation) when incubated with PD infective kidney homogenate and tested by i.p. injection into salmon (Table 2). The week 4 sera obtained from fish infected by cohabitation produced no neutralisation, with PD pathology levels of 70–100% occurring, similar to those obtained in the PBS-controls and control serum. However, sera obtained from fish eight and 15 weeks following cohabitation infection gave a reduction in PD pathology of 100% (100% neutralisation) (Table 2). No PD pathology occurred in fish injected with non-infective control kidney homogenate incubated with PBS, control serum or antisera. Serum neutralisation following dilution of antisera A reduction of PD pathology of 100%, representing a neutralisation of 100%, occurred when week 8 antisera (from fish infected by i.p. injection), which was either undiluted or diluted in PBS to 1:10, 1:50, 1:100, was incubated with PD-infective kidney homogenate and tested by i.p. injection of salmon parr (Table 3). At a dilution of 1:1000, a reduction in pathology occurred although levels of neutralisation were less than 100%. At week 2 postinjection, where peak levels of PD pathology of 100% occurred in the PBS and control serum groups, only 20% PD pathology (neutralisation of 80%) was
PANCREAS DISEASE IN ATLANTIC SALMON
469
Table 2. Percentage infection of Atlantic salmon parr with pancreas disease following injection of PD-infective kidney homogenate which had been incubated with antisera taken at weeks 4, 8 and 15 from fish following their infection by injection or cohabitation. Ten fish sampled per group per week. Temperature 12)C Percentage PD weeks post-injection
Fish injected with PD-infective kidney homogenate incubated with:
1
2
3
4
5
Antisera from fish i.p. infected with PD taken after: wk 4 wk 8 wk 15
0 0 0
0 0 0
0 0 0
0 0 0
0 0 0
Antisera from fish infected with PD by cohabitation taken after: wk 4 wk 8 wk 15 PBS Control sera
0 0 0 0 0
70E 0 0 40E 70E
80 0 0 90 90
100 0 0 80 90
100 0 0 80 90
E=early PD.
Table 3. Percentage infection of Atlantic salmon parr with pancreas disease following incubation of PD-infective kidney homogenate with week 8 anti-serum. Ten fish sampled per group per week. Temperature 14)C. Percentage PD weeks post-injection Group 1 PD-infective kidney incubated with: Immune serum neat Immune serum 1:10 Immune serum 1:50 Immune serum 1:100 Immune serum 1:1000 Control serum neat PBS
0 0 0 0 0 10E 70E
2
3
4
0 0 0 0 20 100 100
0 0 0 0 40 90 90
0 0 0 0 70 90 90
E=early PD.
seen in the 1:1000 dilution group. By week 4 post-injection, levels of PD pathology had increased but this may just represent multiplication of non-neutralised virus in the fish. PASSIVE IMMUNISATION
Atlantic salmon parr and post-smolts that were injected with week 8 antisera at daily intervals ranging from three days before to three days following injection of PD-infective kidney homogenate were 100% protected
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G. HOUGHTON AND A. E. ELLIS
Table 4. Percentage infection of Atlantic salmon parr (P) and post-smolts (PS) with PD following passive immunisation with antiserum taken from fish eight weeks after i.p. infection with PD. Ten fish were sampled per group per week. Parr were held in fresh water at 14)C, post-smolts in sea water at 12)C. Antiserum was injected from three days prior to three days after injection of PD-infective kidney homogenate Percentage PD, weeks post injection Treatment
Immune serum Day of injection relative to infection: "3 "2 "1 0 +1 +2 +3 Control serum Day of injection relative to infection: "3 "2 "1 0 +1 +2 +3 PBS
1
2
3
4
P
PS
P
PS
P
PS
P
PS
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
0 0 0 0 0 0 0
50E 20E 90E 90E 90E 50E 40E 100E
30E 30E 60E 50E 60E 20E 50E 30E
60 80 100 100 100 80 100 100
50 40 60 80 60 30 50 80
80 80 100 100 100 50 80 90
50 50 100 60 80 60 60 80
50 60 100 100 100 80 60 100
20 60 40 80 90 30 30 50
E=early PD.
against pancreas disease with no pathology developing (Table 4). This was in contrast to the control groups injected with PBS or control serum where levels of PD pathology of up to 100% occurred.
IV. Discussion In an earlier paper (Houghton, 1994), it was clearly demonstrated that Atlantic salmon respond immunologically to an experimental infection with pancreas disease developing a solid, long lasting protection to the disease. The exact mechanism of the protection was not investigated, although the author suggested that neutralising antibodies may play a role. The present results show that Atlantic salmon do produce neutralising antibodies following an experimental infection with the disease with relatively high levels of neutralisation occurring. These neutralising antibodies were maintained for at least 15 weeks post-infection. In addition, as well as antibodies being produced following intraperitoneal injection of PD infective
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kidney homogenate, fish which were infected following cohabitation with these fish and thus becoming infected via the natural route, also produced neutralising antibodies. The production of neutralising antibodies appears to occur quickly following infection. Sera taken from fish four weeks following infection by injection completely neutralised the PD agent. Sera from fish four weeks following infection by cohabitation did not fully neutralise the infectious inoculum but did so after eight weeks. This most probably reflects the lag phase involved with infection by cohabitation as the fish infected by injection developed acute PD after two weeks while the pathology did not develop in the cohabiting fish until week 3. Convalescent antisera taken from fish eight weeks following infection by injection appeared to have a high titre of neutralising antibody since complete neutralisation of the PD inoculum was achieved with antiserum diluted 1:100. An apparent partial neutralisation, indicated by delayed onset of PD pathology, occurred with antiserum diluted 1:1000. Although the production of neutralising antibodies has been clearly demonstrated, it still does not indicate whether they play a role in protection to the disease. Passive immunisation is a method where the protective nature of antibodies to pancreas disease can be demonstrated. From the present results, passive immunisation with primary antiserum taken eight weeks postinfection, protected fish against an i.p. challenge with PD-infective kidney homogenate. Protection levels of 100% occurred at all the times that fish were injected with the antiserum from three days before infection to three days post-infection. These results indicate that neutralising antibodies can remain active in the fish at least three days prior to infection and can still neutralise the virus when given three days post-infection at a stage when it is known that the PD agent is already within the blood system (Houghton, 1995). The previous finding (Houghton, 1994) that, following experimental infection, Atlantic salmon are resistant to subsequent challenges for at least nine months, suggests that neutralising antibodies to PD may remain in circulation for at least this length of time or that exposure of the fish to the virus induces a protective secondary response. In the present study, antiserum taken 15 weeks after infection fully neutralised the infective inoculum, and the complete protection a#orded by week 8 antiserum, even at dilutions of 1:100 indicate that the humoral immune response of Atlantic salmon to PD plays an important role in protection. The fact that salmon do respond by producing neutralising antibodies is encouraging for the development of a successful vaccine. The authors thank P. MacLachlan, S. Clements and F. Reid for assistance in sampling and maintaining the fish. The work was fund aided by the Scottish Salmon Growers Association and the Crown Estate Commissioners.
References Falk, K. & Dannevig, B. H. (1995). Demonstration of a protective immune response in infectious salmon anaemia (ISA)-infected Atlantic salmon Salmo salar. Diseases of Aquatic Organisms 21, 1–5.
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