- Email: [email protected]
The genetic influence on serum haemolytic activity in rainbow trout
Aquaculture, 85 (1990) 109-l 17 Elsevier Science Publishers B.V., Amsterdam
109 -
Printed
in The Netherlands
The Genetic Influence on Serum Haemolytic Activity in Rainbow Trout K.H. RBED’, E. BRUN’, H.J. LARSEN’
and T. REFSTIE3
‘Department of Animal Genetics, Norwegian College of Veterinary Medicine/National Veterinary Institute, Postbox 8156, Dep. N-0033 Oslo 1 (Norway) ‘Department of Microbiology and Immunology, Norwegian College of Veterinary Medicine (Norway) 3fnstitute of Aquaculture Research, Sunndalsora (Norwayj
ABSTRACT Reed, K.H., Brun, E., Larsen, H.J. and Refstie, T., 1990. The genetic influence on serum haemolytic activity in rainbow trout. Aquaculture, 85: 109-l 17. Haemolytic activity was analysed in sera from 202 rainbow trout (Salmo gairdneri). The material consisted of 20 full-sib groups within nine paternal half-sib groups. The fish were bloodsampled and individually carlin-tagged. Repeat blood-samples were taken 2 and 8 weeks after the first sampling. An increase in the antibody-dependent haemolytic activity and decrease in the non-specific haemolytic activity, from the first to the second sampling, indicated different activation patterns associated with these responses in rainbow trout. Both the antibody-dependent and the non-specific haemolytic activity showed statistically significant variation between families. The magnitude of the estimated her&abilities ranged from medium to relatively high.
INTRODUCTION
The possible use of immunological markers in future aquaculture breeding programmes as indirect measures for increased disease resistance is being explored. If this is to be achieved, the potential markers must show genetic variation, be easily determined and be correlated to resistance. The haemolytic activity in fish serum against heterologous red blood cells is considered to reflect a significant component of the natural and immunological defence mechanisms against invasion of pathogens and foreign agents (Sakai, 1981; Rijkers, 1982; Ourth and Bachinsky, 1987). Two kinds of haemolytic activity against heterologous red blood cells are usually present in normal fish sera. These are a specific (antibody-dependent) and a non-specific (natural) haemolytic activity. The latter is characterized by a spontaneous haemolysis (Nonaka et al., 1981). It has been suggested that the antibody-dependent and the non-specific hae-
0044~8486/90/$03.50
0 1990 Elsevier Science Publishers
B.V.
110
K.H. RBED ET AL.
molytic activities arise via the classical and the alternate pathways, respectively, of the complement system in rainbow trout (Sakai, 1981, 1983). The complement system comprises a multi-enzyme reaction chain which interacts in a cascade of reactions, uniting in a final common pathway which can terminate in lysis of a target cell. The classical pathway is usually triggered by antigen-antibody complexes, while the alternate pathway may be activated on the surface of bacteria in the absence of specific antibodies. Both activities are known to be sensitive to heat treatments and divalent cations. The present study was carried out to measure possible genetic variation in serum haemolytic activity in a family material of rainbow trout, as determined in three repeat samples. MATERIALANDMETHODS Haemolysin production
Twenty one-year-old rainbow trout, ranging from about 200 to 500 g body weight, were reared in fresh water at a temperature of approximately 9°C for production of haemolysin against sheep red blood cells (SRBC ). SRBC were washed three times with physiological saline. Each fish was injected four times intraperitoneally at 6-week intervals with 1 ml of a SRBC suspension containing 1 x lo8 cells/ml. The fish were bled 6 weeks after the last immunization. Assay of huemolytic activity
Haemolytic activity was measured by a plate method modified according to Lachman and Hobart (1979). A 0.094 M veronal/sucrose-Verona1 buffer containing 3 x 10T4 M Ca2+ and 1 x lo-” M Mg2+ at pH = 7.4 was used both for washing SRBC, and also as diluent in the haemolytic activity test. A volume of 15 ml 1% agarose with 0.25% packed SRBC was poured on 10x lo-cm defatted glass slides coated with agarose. Non-specific haemolytic activity and total haemolytic activity, with haemolysin added to the SRBC, were determined separately. When measuring the total haemolytic activity, the optimal dilution of the haemolysin used for sensitization of the SRBC was first determined by titration of the haemolysin in the test system. As given in Fig. 1, the haemolytic activity decreased when the haemolysin was diluted by more than 1: 100. Based on these tests, a dilution of 1: 50 was used for the total haemolytic activity tests described below. Sample wells of 4 mm in diameter were filled with 12 ~1 serum, the plates then being incubated at 22°C for 20 h, fixed with formalin and dried (Lie et al., 1986). The diameters of the lysed zones were measured by means of a socalled Measuring-Viewer. Two-fold dilutions of a rainbow trout serum sample with high haemolytic activity served as a standard. The haemolytic activities were quantified and expressed as percent of total haemolytic activity in the standard.
GENETIC INFLUENCE ON SERUM HAEMOLYTIC
DILUTION
ACTIVITY IN RAINBOW TROUT
111
OF HAEMOLYSIN
Fig. 1. Haemolytic activity in rainbow trout serum at different dilutions of haemolysin. The haemolytic activity is expressed as percent increase of haemolytic activity.
Influence of temperature and Ca*+ and Mg*+ on the huemolytic activity The effect of temperature on inactivation of the haemolytic activity was tested by heating the serum at 20, 30, 34, 36, 38, 40, 42, 45, and 50°C for 20 min at each temperature. The reduction in activity from the different heat treatments is given in Fig. 2. As shown, the rainbow trout serum was found to have been completely inactivated by heating at 40” C for 20 min. In further analyses of total haemolytic activity, a procedure in which the antiserum was heated to 42’ C for 20 min was adopted. To test for Ca*+/M$+ dependency of the haemolytic activity, blood samples with EDTA as anticoagulant were analysed with and without the addition of Ca*+ and Mg2+ to the test system. Although the EDTA-plasma showed no haemolytic activity when the divalent cations were absent from the test system, the same plasma showed normal haemolytic activity when the divalent cations were added. The genetic material and sampling procedure Both total and non-specific haemolytic activity were determined in serum from three repeat samples in a family material of 202 one-and-a-half-year-old rainbow trout. The material consisted of 20 full-sib groups within nine paternal half-sib groups. The different full-sib groups had been kept separately in indoor tanks (1 m3) prior to the first sampling. At this first sampling, the fish were individually carlin-tagged and thereafter all sib groups were kept in one single tank (2.5 m3). The fish were then sampled 2 and 8 weeks after the first
K.H.RBEDETAL
TEMPERATURE
(“C )
Fig. 2. Haemolytic activity in serum from rainbow trout after heating at different temperatures. The haemolytic activity is expressed as percent of the total haemolytic activity in the standard.
sampling. About 30 fish died during the experimental period, most deaths occurring in conjunction with, or just after, the taking of blood samples. Some of the fish which died after the second sampling showed subcutaneous haemorrhages possibly indicative of an infectious agent. We were unable, however, to define any causal factors, except stress, which might have explained the observed mortality. Blood samples were kept at room temperature for about 4 h before being centrifuged at 4’ C for 10 min. The sera were stored at - 70’ C until analysed.
Statistical
analyses
Least-squares analysis was performed to study the genetic influence on the haemolytic activity in rainbow trout. The model used was Yijk=/lU Si+ d,+
eijk
where is the observation on the kth progeny of the jth dam (d) mated to the ith sire (s). p is the least square mean. Heritabilities were estimated using both the sire and the dam (within sire) components of variation. Standard error of heritability was estimated according to Becker (1967), Yijk
GENETIC INFLUEKE
ON SERUM HAEMOLYTIC ACTIVIm
IX RAI?;BOW TROCT
113
RESULTS
Mean values of the non-specific and total haemolytic activity in the genetic material at the three samplings are given in Table 1. The antibody-dependent component of the total haemolytic activity is also given, being calculated as the difference between the total and the non-specific haemolytic activity. A statistically significant (t= 4.2, P< 0.01) increase in total haemolytic activity between the first and the second sampling was detected. While this trait showed no statistically significant difference between the first and third sampling, a statistically significant decrease (t= 3.2, P < 0.01)was detected between the second and the third sampling. The non-specific haemolytic activity decreased significantly both from first to second (t=4.5, PcO.01) and from second to third (t=4.1, P-cO.01)time of samplin g. Consequently, a highly significant increase in the antibody-dependent part of the total haemolytic activity was detected from first to second sampling ( t = 5.77, P < 0.001). A statistically significant (P < 0.001)positive phenotypic correlation was recorded between total and non-specific haemolytic activity at the first (r=0.46), second (r=0.51), and third (r=0.53) sampling. No significant correlation was recorded between the non-specific and the antibody-dependent part of the total haemolytic activity at the first sampling, while this correlation was significantly positive at the second (r= 0.24) and third sampling (r= 0.19). Fig. 3 shows the mean values of total and non-specific haemolytic activity in offspring of the different sires as determined in the three repeat serum samples. The least-squares analyses for total and non-specific haemolytic activities, and the differences between these are shown in Table 2. Statistically significant (P < 0.01)effects of the model were recorded for all traits at all three samplings. The effects of the different variance components were also significant, among which the effects of dam were significant (P < 0.001) for all three traits at first sampling, and significant (P-c 0.01)for total and non-specific activity at the third sampling. The effects of sire on total and antibody-dependent component of total haemolytic activity were significant (P-c 0.05) at the TABLE 1 Mean values with standard deviation (SD) of total haemolytic activity (THA), non-specific haemolytic activity (NHA ), and the differences between these, in serum from rainbow trout, sampled on three different occasions. The haemolytic activities are expressed as percent of the total haemolytic activity in the standard Sampling
THA
NHA
THA-NHA
1 2 3
79.4 (28.1) 97.0 (60.6) 85.2 (38.7)
27.3 ( 15.9) 22.1 (17.8) 17.8 (13.9)
52.4 (2.5.1) 74.9 (53.8) 67.4 (33.6)
K.H. RBED ET AL
114 SAMPLE
150
-
100
-
50
t-
1
SAMPI
LE
0
TOTAL
m
NON-SPECIFIC
g Ih
::!
o-
L
1
2: ::: . ..
2
::: i: .
.
3
0
4
SAMPLE
150
ACTlVlTY
i
ii I,1,
:: ii . lh
ACTIVITY
3
. :: LL r-l
50
5. 1000 i 1
5
6
SIRE Fig. 3. Mean values of total and non-specific serum haemolytic activity in offspring of different rainbow trout sires, sampled on three different occasions. The haemolytic activity is expressed as percent of the total haemolytic activity in the standard.
115
GENETIC INFLUENCE ON SERUM HABMOLYTIC ACTIVITY IN RAINBOW TROUT
TABLE 2 Level of statistical significance (P) of the variance components analyses, and he&abilities (h’) with standard error (SE) of total haemolytic activities (THA), non-specific haemolytic activity (NHA), and difference between these, in serum from rainbow trout, sampled on three different occasions Effects
THA P
NHA
THA-NHA h*
SE
0.33 < 0.001
0.13 0.41
0.32 0.18
0.55 0.28
0.003 0.092 0.21
0.33 0.23
0.40 0.22
0.08 0.40
h*
SE
First sampling Total” < 0.001 Sire 0.099 Dam < 0.001
0.73 0.89
0.63 0.33
Second sampling Total”
0.96 0.59
Third sampling Total”
0.34 0.47
P
P
h*
SE
0.12
0.71 0.97
0.67 0.35
0.27 0.16
< 0.001 0.015 0.099
0.80 0.50
0.48 0.25
0.33 0.20
0.30 0.28
0.30 0.18
< 0.001
< 0.001
“Sire+dam.
second sampling. The effects of sire were also significant at 0.05 < P < 0.1 for total haemolytic activity at the first sampling and also for non-specific activity at the second sampling. The estimates of heritabilities (Table 2) were, in general, relatively high. The standard errors of these estimates were, however, also relatively high. DISCUSSION
The present study revealed considerable individual and family variation in rainbow trout with regard to total and non-specific serum haemolytic activity, and the difference between these. The marked influence of the dams is likely to include, besides the additive genetic variation, maternal and common environmental effects and non-additive genetic variation. However, the detected effects of sire suggest the presence of significant additive genetic variation in the serum haemolytic activity in rainbow trout. The estimated heritabilities for the two traits studied were medium to relatively high. The high standard error of these estimates means, however, that the detected heritability estimates should be considered as being only of a provisional nature. Considerably more extensive material is necessary for proper estimation of the heritability of the serum haemolytic activity in rainbow trout.
116
K.H. R0ED ET AL.
The sensitivity of both total and non-specific serum haemolytic activity to heat treatment and divalent cations is consistent with a model of complementmediated activation. Inactivation of the haemolytic activity in rainbow trout at 40°C is in agreement with other reports (Dorson et al., 1979; Sakai, 1981). The antibody-dependent haemolytic activity increased, and the non-specific activity decreased considerably from first to second sampling. This indicates that these two types of haemolytic activity mainly reflect different activation patterns. This may thus give support to the suggestion that the antibody-dependent and non-specific haemolytic activity in rainbow trout are mediated through the classical and alternate pathways, respectively, of the complement system (Sakai, 1981). Sakai (1983) has shown that fresh sera of salmonids (rainbow trout, masu salmon and coho salmon) have non-specific killing activity against Aeromonm salmonicida. The bacterial activity was proportional to the non-specific haemolytic activity. The same study also reported that the non-specific haemolytic activity was influenced by starvation and state of infection (furunculosis and vibriosis) of the fish. The use of haemolytic response in serum was thus suggested as a practical parameter for fish health assessment. The detected genetic variation found in the present study also suggests that it may prove possible to influence this trait through selection programmes. ACKNOWLEDGEMENTS
We sincerely acknowledge the employees at Akvaforsk, Sundalsora, for their culture and collection of fishes. Thanks are especially due to Mr. G. Hjeldnes for his kind help and sampling of fishes. This investigation was supported by the Norwegian Agriculture Research Council.
REFERENCES Becker, W.A., 1967. Manual of Procedures in Quantitative Genetics (2nd edition). Washington State University, Pullman, WA, 130 pp. Dorson, M., Torchy, C. and Michel, C., 1979. Rainbow trout complement fixation used for titration of antibodies against several pathogens. Ann. Rech. Vet., 10: 529-534. Lachman, P.d. and Hobart, M.-J., 1979. Complement technology. In: D.M. Weir (Editor), Handbook of Experimental Immunology. Vol. 1. Immunochemistry. Blackwell Scientific Publications, Oxford, pp. 5A. I-5A. 23. Lie, O., Syed, M. and Solbu, H., 1986. Improved agar plate assays of bovine lysozyme and haemolytic complement activity. Acta Vet. Stand., 27: 23-32. Nonaka, M., Yamaguchi, N., Natsuume-Sakai, S. and Takahashi, M., 1981. The complement system of rainbow trout (Salmo gairdneri). 1. Identification of the serum lytic homologous to mammalian complement. J. Immunol., 126: 1489-1494. Ourth. D.D. and Bachinsky, L.M., 1987. Bactericidal response ofchannel catfish (fcfaiuruspunc-
GENETIC INFLUENCE ON SERUM HAEMOLYTIC ACTIVITY IN RAINBOW TROUT
117
tutus) by the classical and alternative complement pathways against bacterial pathogens. J. Appl. Ichthyol., 3: 42-45. Rijkers. G.T., 1982. Non-lyrnphoid defence mechanisms in fish. Dev. Comp. Immunol., 6: 1-13. Sakai, D.K., 1981. Spontaneous and antibody-dependent hemolysis activities of fish sera and inapplicability of mammalian complements to the immune hemolysis reaction of fishes. Bull. Jpn. Sot. Sci. Fish., 47: 979-991. Sakai, D.K., 1983. Lytic and bactericidal properties of salmon sera. J. Fish Biol., 23: 457-466.
109 -
Printed
in The Netherlands
The Genetic Influence on Serum Haemolytic Activity in Rainbow Trout K.H. RBED’, E. BRUN’, H.J. LARSEN’
and T. REFSTIE3
‘Department of Animal Genetics, Norwegian College of Veterinary Medicine/National Veterinary Institute, Postbox 8156, Dep. N-0033 Oslo 1 (Norway) ‘Department of Microbiology and Immunology, Norwegian College of Veterinary Medicine (Norway) 3fnstitute of Aquaculture Research, Sunndalsora (Norwayj
ABSTRACT Reed, K.H., Brun, E., Larsen, H.J. and Refstie, T., 1990. The genetic influence on serum haemolytic activity in rainbow trout. Aquaculture, 85: 109-l 17. Haemolytic activity was analysed in sera from 202 rainbow trout (Salmo gairdneri). The material consisted of 20 full-sib groups within nine paternal half-sib groups. The fish were bloodsampled and individually carlin-tagged. Repeat blood-samples were taken 2 and 8 weeks after the first sampling. An increase in the antibody-dependent haemolytic activity and decrease in the non-specific haemolytic activity, from the first to the second sampling, indicated different activation patterns associated with these responses in rainbow trout. Both the antibody-dependent and the non-specific haemolytic activity showed statistically significant variation between families. The magnitude of the estimated her&abilities ranged from medium to relatively high.
INTRODUCTION
The possible use of immunological markers in future aquaculture breeding programmes as indirect measures for increased disease resistance is being explored. If this is to be achieved, the potential markers must show genetic variation, be easily determined and be correlated to resistance. The haemolytic activity in fish serum against heterologous red blood cells is considered to reflect a significant component of the natural and immunological defence mechanisms against invasion of pathogens and foreign agents (Sakai, 1981; Rijkers, 1982; Ourth and Bachinsky, 1987). Two kinds of haemolytic activity against heterologous red blood cells are usually present in normal fish sera. These are a specific (antibody-dependent) and a non-specific (natural) haemolytic activity. The latter is characterized by a spontaneous haemolysis (Nonaka et al., 1981). It has been suggested that the antibody-dependent and the non-specific hae-
0044~8486/90/$03.50
0 1990 Elsevier Science Publishers
B.V.
110
K.H. RBED ET AL.
molytic activities arise via the classical and the alternate pathways, respectively, of the complement system in rainbow trout (Sakai, 1981, 1983). The complement system comprises a multi-enzyme reaction chain which interacts in a cascade of reactions, uniting in a final common pathway which can terminate in lysis of a target cell. The classical pathway is usually triggered by antigen-antibody complexes, while the alternate pathway may be activated on the surface of bacteria in the absence of specific antibodies. Both activities are known to be sensitive to heat treatments and divalent cations. The present study was carried out to measure possible genetic variation in serum haemolytic activity in a family material of rainbow trout, as determined in three repeat samples. MATERIALANDMETHODS Haemolysin production
Twenty one-year-old rainbow trout, ranging from about 200 to 500 g body weight, were reared in fresh water at a temperature of approximately 9°C for production of haemolysin against sheep red blood cells (SRBC ). SRBC were washed three times with physiological saline. Each fish was injected four times intraperitoneally at 6-week intervals with 1 ml of a SRBC suspension containing 1 x lo8 cells/ml. The fish were bled 6 weeks after the last immunization. Assay of huemolytic activity
Haemolytic activity was measured by a plate method modified according to Lachman and Hobart (1979). A 0.094 M veronal/sucrose-Verona1 buffer containing 3 x 10T4 M Ca2+ and 1 x lo-” M Mg2+ at pH = 7.4 was used both for washing SRBC, and also as diluent in the haemolytic activity test. A volume of 15 ml 1% agarose with 0.25% packed SRBC was poured on 10x lo-cm defatted glass slides coated with agarose. Non-specific haemolytic activity and total haemolytic activity, with haemolysin added to the SRBC, were determined separately. When measuring the total haemolytic activity, the optimal dilution of the haemolysin used for sensitization of the SRBC was first determined by titration of the haemolysin in the test system. As given in Fig. 1, the haemolytic activity decreased when the haemolysin was diluted by more than 1: 100. Based on these tests, a dilution of 1: 50 was used for the total haemolytic activity tests described below. Sample wells of 4 mm in diameter were filled with 12 ~1 serum, the plates then being incubated at 22°C for 20 h, fixed with formalin and dried (Lie et al., 1986). The diameters of the lysed zones were measured by means of a socalled Measuring-Viewer. Two-fold dilutions of a rainbow trout serum sample with high haemolytic activity served as a standard. The haemolytic activities were quantified and expressed as percent of total haemolytic activity in the standard.
GENETIC INFLUENCE ON SERUM HAEMOLYTIC
DILUTION
ACTIVITY IN RAINBOW TROUT
111
OF HAEMOLYSIN
Fig. 1. Haemolytic activity in rainbow trout serum at different dilutions of haemolysin. The haemolytic activity is expressed as percent increase of haemolytic activity.
Influence of temperature and Ca*+ and Mg*+ on the huemolytic activity The effect of temperature on inactivation of the haemolytic activity was tested by heating the serum at 20, 30, 34, 36, 38, 40, 42, 45, and 50°C for 20 min at each temperature. The reduction in activity from the different heat treatments is given in Fig. 2. As shown, the rainbow trout serum was found to have been completely inactivated by heating at 40” C for 20 min. In further analyses of total haemolytic activity, a procedure in which the antiserum was heated to 42’ C for 20 min was adopted. To test for Ca*+/M$+ dependency of the haemolytic activity, blood samples with EDTA as anticoagulant were analysed with and without the addition of Ca*+ and Mg2+ to the test system. Although the EDTA-plasma showed no haemolytic activity when the divalent cations were absent from the test system, the same plasma showed normal haemolytic activity when the divalent cations were added. The genetic material and sampling procedure Both total and non-specific haemolytic activity were determined in serum from three repeat samples in a family material of 202 one-and-a-half-year-old rainbow trout. The material consisted of 20 full-sib groups within nine paternal half-sib groups. The different full-sib groups had been kept separately in indoor tanks (1 m3) prior to the first sampling. At this first sampling, the fish were individually carlin-tagged and thereafter all sib groups were kept in one single tank (2.5 m3). The fish were then sampled 2 and 8 weeks after the first
K.H.RBEDETAL
TEMPERATURE
(“C )
Fig. 2. Haemolytic activity in serum from rainbow trout after heating at different temperatures. The haemolytic activity is expressed as percent of the total haemolytic activity in the standard.
sampling. About 30 fish died during the experimental period, most deaths occurring in conjunction with, or just after, the taking of blood samples. Some of the fish which died after the second sampling showed subcutaneous haemorrhages possibly indicative of an infectious agent. We were unable, however, to define any causal factors, except stress, which might have explained the observed mortality. Blood samples were kept at room temperature for about 4 h before being centrifuged at 4’ C for 10 min. The sera were stored at - 70’ C until analysed.
Statistical
analyses
Least-squares analysis was performed to study the genetic influence on the haemolytic activity in rainbow trout. The model used was Yijk=/lU Si+ d,+
eijk
where is the observation on the kth progeny of the jth dam (d) mated to the ith sire (s). p is the least square mean. Heritabilities were estimated using both the sire and the dam (within sire) components of variation. Standard error of heritability was estimated according to Becker (1967), Yijk
GENETIC INFLUEKE
ON SERUM HAEMOLYTIC ACTIVIm
IX RAI?;BOW TROCT
113
RESULTS
Mean values of the non-specific and total haemolytic activity in the genetic material at the three samplings are given in Table 1. The antibody-dependent component of the total haemolytic activity is also given, being calculated as the difference between the total and the non-specific haemolytic activity. A statistically significant (t= 4.2, P< 0.01) increase in total haemolytic activity between the first and the second sampling was detected. While this trait showed no statistically significant difference between the first and third sampling, a statistically significant decrease (t= 3.2, P < 0.01)was detected between the second and the third sampling. The non-specific haemolytic activity decreased significantly both from first to second (t=4.5, PcO.01) and from second to third (t=4.1, P-cO.01)time of samplin g. Consequently, a highly significant increase in the antibody-dependent part of the total haemolytic activity was detected from first to second sampling ( t = 5.77, P < 0.001). A statistically significant (P < 0.001)positive phenotypic correlation was recorded between total and non-specific haemolytic activity at the first (r=0.46), second (r=0.51), and third (r=0.53) sampling. No significant correlation was recorded between the non-specific and the antibody-dependent part of the total haemolytic activity at the first sampling, while this correlation was significantly positive at the second (r= 0.24) and third sampling (r= 0.19). Fig. 3 shows the mean values of total and non-specific haemolytic activity in offspring of the different sires as determined in the three repeat serum samples. The least-squares analyses for total and non-specific haemolytic activities, and the differences between these are shown in Table 2. Statistically significant (P < 0.01)effects of the model were recorded for all traits at all three samplings. The effects of the different variance components were also significant, among which the effects of dam were significant (P < 0.001) for all three traits at first sampling, and significant (P-c 0.01)for total and non-specific activity at the third sampling. The effects of sire on total and antibody-dependent component of total haemolytic activity were significant (P-c 0.05) at the TABLE 1 Mean values with standard deviation (SD) of total haemolytic activity (THA), non-specific haemolytic activity (NHA ), and the differences between these, in serum from rainbow trout, sampled on three different occasions. The haemolytic activities are expressed as percent of the total haemolytic activity in the standard Sampling
THA
NHA
THA-NHA
1 2 3
79.4 (28.1) 97.0 (60.6) 85.2 (38.7)
27.3 ( 15.9) 22.1 (17.8) 17.8 (13.9)
52.4 (2.5.1) 74.9 (53.8) 67.4 (33.6)
K.H. RBED ET AL
114 SAMPLE
150
-
100
-
50
t-
1
SAMPI
LE
0
TOTAL
m
NON-SPECIFIC
g Ih
::!
o-
L
1
2: ::: . ..
2
::: i: .
.
3
0
4
SAMPLE
150
ACTlVlTY
i
ii I,1,
:: ii . lh
ACTIVITY
3
. :: LL r-l
50
5. 1000 i 1
5
6
SIRE Fig. 3. Mean values of total and non-specific serum haemolytic activity in offspring of different rainbow trout sires, sampled on three different occasions. The haemolytic activity is expressed as percent of the total haemolytic activity in the standard.
115
GENETIC INFLUENCE ON SERUM HABMOLYTIC ACTIVITY IN RAINBOW TROUT
TABLE 2 Level of statistical significance (P) of the variance components analyses, and he&abilities (h’) with standard error (SE) of total haemolytic activities (THA), non-specific haemolytic activity (NHA), and difference between these, in serum from rainbow trout, sampled on three different occasions Effects
THA P
NHA
THA-NHA h*
SE
0.33 < 0.001
0.13 0.41
0.32 0.18
0.55 0.28
0.003 0.092 0.21
0.33 0.23
0.40 0.22
0.08 0.40
h*
SE
First sampling Total” < 0.001 Sire 0.099 Dam < 0.001
0.73 0.89
0.63 0.33
Second sampling Total”
0.96 0.59
Third sampling Total”
0.34 0.47
P
P
h*
SE
0.12
0.71 0.97
0.67 0.35
0.27 0.16
< 0.001 0.015 0.099
0.80 0.50
0.48 0.25
0.33 0.20
0.30 0.28
0.30 0.18
< 0.001
< 0.001
“Sire+dam.
second sampling. The effects of sire were also significant at 0.05 < P < 0.1 for total haemolytic activity at the first sampling and also for non-specific activity at the second sampling. The estimates of heritabilities (Table 2) were, in general, relatively high. The standard errors of these estimates were, however, also relatively high. DISCUSSION
The present study revealed considerable individual and family variation in rainbow trout with regard to total and non-specific serum haemolytic activity, and the difference between these. The marked influence of the dams is likely to include, besides the additive genetic variation, maternal and common environmental effects and non-additive genetic variation. However, the detected effects of sire suggest the presence of significant additive genetic variation in the serum haemolytic activity in rainbow trout. The estimated heritabilities for the two traits studied were medium to relatively high. The high standard error of these estimates means, however, that the detected heritability estimates should be considered as being only of a provisional nature. Considerably more extensive material is necessary for proper estimation of the heritability of the serum haemolytic activity in rainbow trout.
116
K.H. R0ED ET AL.
The sensitivity of both total and non-specific serum haemolytic activity to heat treatment and divalent cations is consistent with a model of complementmediated activation. Inactivation of the haemolytic activity in rainbow trout at 40°C is in agreement with other reports (Dorson et al., 1979; Sakai, 1981). The antibody-dependent haemolytic activity increased, and the non-specific activity decreased considerably from first to second sampling. This indicates that these two types of haemolytic activity mainly reflect different activation patterns. This may thus give support to the suggestion that the antibody-dependent and non-specific haemolytic activity in rainbow trout are mediated through the classical and alternate pathways, respectively, of the complement system (Sakai, 1981). Sakai (1983) has shown that fresh sera of salmonids (rainbow trout, masu salmon and coho salmon) have non-specific killing activity against Aeromonm salmonicida. The bacterial activity was proportional to the non-specific haemolytic activity. The same study also reported that the non-specific haemolytic activity was influenced by starvation and state of infection (furunculosis and vibriosis) of the fish. The use of haemolytic response in serum was thus suggested as a practical parameter for fish health assessment. The detected genetic variation found in the present study also suggests that it may prove possible to influence this trait through selection programmes. ACKNOWLEDGEMENTS
We sincerely acknowledge the employees at Akvaforsk, Sundalsora, for their culture and collection of fishes. Thanks are especially due to Mr. G. Hjeldnes for his kind help and sampling of fishes. This investigation was supported by the Norwegian Agriculture Research Council.
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tutus) by the classical and alternative complement pathways against bacterial pathogens. J. Appl. Ichthyol., 3: 42-45. Rijkers. G.T., 1982. Non-lyrnphoid defence mechanisms in fish. Dev. Comp. Immunol., 6: 1-13. Sakai, D.K., 1981. Spontaneous and antibody-dependent hemolysis activities of fish sera and inapplicability of mammalian complements to the immune hemolysis reaction of fishes. Bull. Jpn. Sot. Sci. Fish., 47: 979-991. Sakai, D.K., 1983. Lytic and bactericidal properties of salmon sera. J. Fish Biol., 23: 457-466.