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Serum iron levels in farmed Atlantic salmon: family variation and associations with disease resistance
ELSEVIER
Aquaculture 125 (1994) 3745
Serum iron levels in farmed Atlantic salmon: family variation and associations with disease resistance Jarle Ravndal”,“, Tone Lgvold”, Hans Bernhard Bentsena, Knut Hkon R@edb,Trygve Gjedrem”, Kjell-Axne R@rvika “AKVAFORSK, Institute of Aquaculture Research AS, As, Norway bDepartment of Morphology, Generics and Aquatic Biology, Norwegian College of Veterinary Medicine, Oslo, Norway Accepted 27
February 1994
Abstract Serum samples from previous experiments were utilized to study the variation between sib families in serum iron concentration, and the association between serum iron concentration in the sib group and survival in challenge tests with vibriosis, furunculosis, cold water vibriosis or bacterial kidney disease (BKD) in farmed Atlantic salmon. In the first experiment, fish that died from vibriosis had significantly higher serum iron concentration prior to the challenge test than surviving fish. No significant variation was found in serum iron concentration between 34 full-sib families or between 12 half-sib families, but a non-significant suggestion of negative correlations (r = - 0.25, P = 0.15; r= -0.35, P= 0.27), was observed between least square means of serum iron concentration and survival rates from vibriosis in the sib families. Sexually maturing fish had significantly higher serum iron concentrations and lower survival rates than immatures. In the second experiment, 23 full-sib families showed a significant variation in serum iron concentration, and a non-significant suggestion of a negative correlation ( r = - 0.34, P = 0.12) of about the same magnitude as in the first experiment was observed between full-sib least-square means for serum iron concentration and the survival rate of parallel full-sib families in a challenge test with cold water vibriosis. The corresponding correlations between serum iron levels and survival rates in challenge tests with fumnculosis and BKD were both close to zero. The results from the two experiments taken together suggests that families with increased levels of serum iron may be more suceptible for Vzhio infections.
1. Introduction Vibriosis, furunculosis, cold water vibriosis and bacterial kidney disease (BKD) are serious bacterial diseases in Norwegian salmon farming. Thus, improved natural resistance *Correspondingauthor, present address:FelleskjepetHavbruk AS, Box 655 Krossen,4301 Sandnes, Notway. 0044~8486/94/$07.00 0 1994 SSDI0044-8486(94)00049-T
Elsevier Science B.V. All rights reserved
38
J. Ravndal et al. /Aquaculture
125 (1994) 37-45
against such diseases may reduce the costs of disease outbreaks and the environmental impact of extensive use of antibiotics. One possible way to improve natural disease resistance is by selection. Indirect selection on immunological or physiological marker traits has several advantages. However, indirect selection requires that the marker traits show genetic variation, are easy to determine on a large scale, and, of course, are genetically correlated with resistance to disease (Fjalestad et al., 1993). Iron plays a key role in the pathology of bacterial infections by enhancing the multiplication and virulence of the invading micro-organisms (Weinberg, 1984). Therefore, depriving the pathogens of iron may serve as a non-specific protective mechanism against infection (Kluger and Bullen, 1987; Weinberg, 1989). In serum, iron is normally bound to and transported by serum transferrin. Bacteria have several mechanisms to overcome the ability of the host to restrict iron availability. The best studied bacterial mechanism is that of iron chelators, called siderophores, which compete with the iron binding ability of transferrin (Griffiths, 1987; Otto et al., 1992). Differences in disease susceptibility of salmonids possessing different transfenin genotypes have been shown during infections with BKD (Renibacterium salmoninarum; Suzumoto et al., 1977)) vibriosis ( Vibrio anguillarum; Pratschner, 1978) and furunculosis (Aeromonas salmonicida; Pratschner, 1978). The present study was based on serum samples collected in two previous experiments involving challenge tests of a family material of farmed Atlantic salmon. The main objective of the study was to obtain estimates of family variation in serum iron concentration and to investigate the association between serum iron concentration and mortality in challenge tests with vibriosis, furunculosis, cold water vibriosis or BKD. If a persistent association can be shown over time and between parallel sib groups, serum iron should be further investigated as a marker trait for indirect selection for disease resistance.
2. Materials and methods Experiment I The iron concentrations in serum from a family material of 824 Atlantic salmon (Salmo salar, L.) were determined (see below). The individually tagged fish, which consisted of 34 full-sib groups within 12 paternal half-sib groups (average weight of 780 g) , were kept and sampled as described in Raed et al. ( 1993). Six weeks after serum sampling, 728 fish that were still available were recorded for stage of sexual maturation (immature or maturing) , and intraperitoneally injected with live Vibrio anguillarum ( lo8 bacterium per fish). Fish surviving the subsequent outbreak of vibriosis were recorded 3 weeks later (Raed et al., 1993). Survival in the full-sib families ranged from 29 to 74%. Experiment 2 In early 1991, 90 l-year-old fingerlings from each of 81 full-sib families of Atlantic salmon ( 1990 year-class) were challenged indirectly by introduction (into the tank) of fish which were infected with the furunculosis bacteria (Aeromonas salmonicida), or directly by intraperitoneal injection either with cold water vibriosis bacteria (Vibrio salmonicida) or with BKD bacteria (Renibacterium salmoninarum) in 3 independent tests (30 fish per
J. Ravndal et al. /Aquaculture
125 (1994) 37-45
39
B Susceptible
q Resistant
0 Furunculosis
BKD
C.W.
vibriosls
Fig. 1. Total survival rates of two groups of 11 susceptible full-sib families and of 12 resistant full-sib families in a challenge test with furunculosis, and total survival rates of the same groups of families in a challenge test with BKD and cold water vibriosis (c.w. vibriosis). Standard deviations of full-sib group survival rates are indicated.
family per test) at Vikan AkvaVet, Namsos (Gjedrem and Gjoen, 1994). In addition, a parallel group of 1054 fish from the same 8 1 full-sib families were later (May 1991) stocked in one cage at AKVAFORSK’s sea site at Veiset, about 13 fish from each full-sib family. All fish were fed a commercial feed mixture. Based on survival data for furunculosis in the challenge test, 23 extreme full-sib families were identified: in 11 highly susceptible families the survival rate ranged from 0 to 7%, and in 12 resistant families the survival rate ranged from 52 to 81%. The survival rates for the same 23 full-sib families in the challenge tests with cold water vibriosis and BKD ranged from 67 to lOO%,and from 0 to 90%, respectively (Fig. 1) . About 9 months after the challenge test (January 1992)) serum samples were collected at Veiset from 285 full sibs of the 23 extreme families (average weight of 1080 g), and iron concentrations in serum were determined. Chemical analysis
Total iron concentration in serum was chosen as a possible marker trait in the present study, since a proper method of determining the iron saturation of serum transferrin in Atlantic salmon has not yet been developed. Studies in our laboratories (not published) have shown that salmon serum transferrin has a much stronger affinity for iron than human serum transferrin. Hence, iron and salmon transferrin are not completely dissociated even at pH = 2.0. In both experiments, blood was drawn from the caudal vein into evacuated bloodcollection tubes without anticoagulants (Venoject@) . The samples were incubated at room temperature for 3 h and centrifuged. Serum was collected in microtiter plate wells (Experiment 1) or Eppendorf tubes (Experiment 2) and stored at - 70°C until assay. In the analysis, which involves destruction of organic material at 160°C small samples of serum were used; 150 ~1 in Experiment 1, and 250 ~1 in Experiment 2. During heating, the samples were treated with 500 ~1 nitric acid (HN03, 65%) for 45 min, and then with 500 ~1
40
J. Ravndalet al. /Aquaculture 125 (1994) 3745
hydrogen peroxide ( H,Oz, 30%) for 30 min. The treated samples were diluted by a solution of 4% HN03 and 1% H202 to 1.5 ml. The iron concentrations were determined with a Perkin Elmer model 3 100 (flame absorption spectrophotometer) (Experiment 1) or a GBC 906AA model (Experiment 2). Impact bead was used to increase absorbency. Optical density (OD) at 540 nm for each sample was checked and samples with OD above 0.22 were excluded to avoid errors due to hemolysis. Seronorm standards (Nycomed and Serco AS) were analyzed as described above every day during the analysis period. Out of 113 standards analyzed in Experiment 1, and 46 standards analyzed in Experiment 2, no significant differences (P > 0.05) between days were observed. Statistical analysis The material in Experiment 1 was a complete set of progeny groups from a nested mating design. A preliminary hierarchical analysis of variance did not show any significant effects of sire or dam within sire on serum iron concentration. A simplified general linear model was then applied to test for random effects of all family-associated sources of variation and to estimate least-square means:
where Yijkl=serum iron concentration of the Zthindividual, p = overall mean, x = random effect of the ith family (i= 1,2,...35 for full-sib and i= 1, 2,...12 for half-sib families), pj = fixed effect of the jth microtiter plate (j = 1,2,... 13)) mk = fixed effect of stage of sexual maturation (k = immature or maturing), and eiju = random error of the Zthindividual. Serum samples were randomly assigned to microtiter plates. A significant effect of microtiter plates is consequently expected to reflect variable laboratory conditions during the analysis of the different plates, and should be corrected for in the model. In Experiment 1, individual records of survival (dead or alive) were also analyzed as a dependent variable according to Model 1 after excluding the effects of microtiter plates from the model. The survival rate of the families is expected to be approximately normally distributed, since the number of individuals within each family was as large as 20 fish per full-sib group, and the mean survival rate in the families was intermediate (29-74%). A similar model was used to analyze serum iron concentrations as a dependent variable in Experiment 2: &jk
=
p
+A +
dj
+
eijk
where serum iron of the kth individual, p = overall mea&J = random effect of the ith full-sib family (i= 1, 2,...23), dj =fixed effect of the jth day of sample (j= 1, 2, 3), and eijk=random error of the kth individual. The serum iron concentrations in groups of dead and live fish (Experiment 1), and in groups of susceptible and resistant families (Experiment 2)) were analyzed according to Model 3 and Model 4, respectively: Yijk
=
YijH= /_& + gj +pj + mk + eijH
(Model 3)
Yijk=
(Model 4)
cL+gi
+dj
+eijk
where gi = fixed effect of the ith group ( i = dead or alive in Model 3 (Experiment 1) , and i = susceptible or resistant families in Model 4 (Experiment 2) ) .
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41
The correlation coefficients between least-square means (LSM) of survival rates and LSM of serum iron concentrations of the full-sib or the half-sib families (Experiment 1) , and the correlation coefficients between LSM of serum iron concentrations and the survival rates of parallel full-sib groups (Experiment 2) were estimated. The sib group correlations will approach the genetic correlations between the traits when the number of individuals in each family increases, assuming that the common environmental effects on the traits and the non-additive genetic correlations are zero.
3. Results Genetic variation in serum iron concentrations In Experiment 1, the least-square means (LSM) concentrations of serum iron of the fullsib and half-sib families ranged from 1.51 to 2.10 pg/ml, and from I.67 to 1.91 pg/ml, respectively. The variation in serum iron between the full-sib or half-sib families was not significant (Table 1) , but a significant (P < 0.01) effect of both microtiter plate and stage of sexual maturation was observed. The effect of microtiter plate is assumed to be caused by variable laboratory conditions during the analysis of the different plates. The mean value and the phenotypic variation of serum iron concentration in fish from Experiment 2 were much lower than in Experiment 1 (Table 1) . The LSM concentrations of serum iron of the full-sib families ranged from 0.69 to 1.14 pg/ml. A significant variation in serum iron concentrations was detected between the full-sib groups (Table 1) . No significant effect was found of day of sample (P > 0.05)) indicating that the number of days the fish were exposed to handling before the serum sample was collected did not affect the serum iron levels. Association between serum iron and survival rate In Experiment 1, fish that died from vibriosis had a significantly (P < 0.05) higher level of serum iron 6 weeks before challenge than surviving fish (Fig. 2, 1.81 vs. 1.68 pg Fe/ ml). No significant difference in serum iron level was detected between the full-sibs of susceptible sib groups and resistant sib groups in the challenge tests in Experiment 2 (Fig. 2). A significant variation in survival rates between half-sib families (but not between fullsib families) was detected (P < 0.05) in Experiment 1; the survival rates ranged from 34 Table 1 Serum iron concentrations (pg/ml) and associated statistics: Number (N) of individuals, full-sib groups and half-sib groups, standard deviations (s.d.) of phenotypic observations (mean) and of full-sib and half-sib leastsquare means (LSM) for serum iron ( pg/ml) , and the significance ( P) of the marginal (type III) contributions of families according to Model 1 (Experiment 1) and Model 2 (Experiment 2)
Experiment 1 Experiment 2
Phenotypic
Full-sib LSM
Half-sib LSM
N
Mean f s.d.
N
s.d.
P
N
s.d.
P
824 285
1.70 f 0.67 0.89 f 0.32
34 23
0.13 0.13
0.66 0.01
12
0.07
0.79
42
J. Ravndal et al. /Aquaculture 125 (1994) 37-45 20
18
16
14
1 2
10
08
06
04
02
oc IExperiment
Experiment
1
2
Fig. 2. Least-square mean estimates of serum iron concentrations (pg/mi) of fish that died or survived in a challenge test with vibriosis in Experiment 1 (Model 3), and full sibs of families that showed susceptibility or resistance in a challenge test with furunculosis in Experiment 2 (Model 3). Standard errors are indicated.
to 65%, and 29 to 74%, respectively. No significant correlation was detected between LSM of serum iron concentrations of the families and survival rates, either after challenge with vibriosis in Experiment 1 or after challenge with furunculosis, cold water vibriosis or BKD in Experiment 2 (Table 2). However, a consistent tendency was observed across the two experiments, indicating a possible association between reduced serum iron levels and increased survival in challenge tests with Vibrio ssp. and to some extent also with cold water vibriosis. In Experiment 1, sexually maturing fish had significantly higher serum iron concentrations and mortality rates than immature fish (Table 3). Within the two groups, fish that died showed a non-significant tendency (P = 0.07) towards higher concentrations of serum iron than surviving fish. Table 2 Correlation coefficients (r) and significance (P) for the association between full-sib or half-sib families leastsquare means (LSM) for serum iron according to Models 1 and 2, and survival rates in the same material (Experiment I), or in parallel full-sib groups (Experiment 2) in challenge tests with different diseases Full-sib LSM
Half-sib LSM
r
P
r
P
-0.25
0.15
- 0.35
0.27
0.12 -0.34 0.10
0.60 0.12 0.66
Experiment I
Vibriosis Experiment 2
Furunculosis Cold water vibriosis Bacterial kidney disease
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et al. /Aquaculture
125 (1994) 3745
43
Table 3 Number (N) of immature and sexually maturing individuals in Experiment 1, survival rates in a challenge test with vibriosis, and least-square means of serum iron prior to the test according to Model 1
N
Survival (%) Serum iron @g/ml)
Immature
Sexually maturing
P
613 66 1.67
115 32 1.86
‘Co.01
The significance (P) of the marginal (type III) contributions were computed from the same model.
4. Discussion The significant association between high concentrations of serum iron and mortality for individuals of vibriosis-infected farmed Atlantic salmon in Experiment 1 (Fig. 2) is consistent with previous studies in mammals, showing that bacterial growth, multiplication and virulence are enhanced by increased iron availability and total serum iron concentrations (Blumberg et al., 1981; Sawatzki, 1987; Cox, 1989; Weinberg, 1990; Bullen et al., 1991). The lack of a significant full sib correlation between LSM serum iron concentrations and mortality rates caused by furunculosis, cold water vibriosis and BKD in Experiment 2 may have several explanations. Firstly, the serum samples in this experiment were collected from parallel full-sib groups of the families that were challenge tested, and about 9 months later than the challenge test. In Experiment 1, the serum iron concentrations were determined from the same fish that were used in the challenge test, and the sera were collected 6 weeks prior to the infection. If serum iron level is a variable (e.g. time- or age-dependent) genetic property of the individual, the correlations in Experiment 2 are expected to be more affected by fluctuations in serum iron concentrations. Secondly, the lower serum iron concentrations observed in Experiment 2 may result in lower transferrin saturation. The presence of unsaturated transferrin in plasma seems to be an important bacteriostatic factor in mammals (Huebers and Finch, 1987; Bullen et al., 1990). Differences between families in iron saturation of serum transferrin would then be expected to affect bacterial growth more at high serum iron concentrations (Experiment 1) than at low serum iron concentrations (Experiment 2). The differences in average serum iron concentrations between the present experiments may be caused by non-genetic factors, and did not exceed the range observed in other studies carried out by AKVAFORSK. Studies by Wolf and Crosa (1986) and by Biosca and Amaro (1991) have shown that both siderophores and related outer membrane proteins in V. anguikzrum are potential pathogenic factors in fish, probably because of the improved ability of the bacteria to secure a sufficient iron supply. Similar iron-sequestering mechanisms are also found in typical strains of A. salmonicidu (Trust et al., 1983; Kay et al., 1985; Hirst et al., 1991). Our experiments may indicate that the iron sequestering mechanisms ofA. sulmonicidu are more efficient than those of V. unguillurum and V. sulmonicidu. Little is known about the iron competition mechanisms of R. salmoninurum. Rorvik et al. ( 1992) suggest that the availability of iron in the gastro-intestinal tract may be more important than the serum iron level for the growth and multiplication of A. sulmonicidu. They observed a highly significant and positive correlation between mortality in
44
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a natural outbreak of furunculosis and concentrations of dietary iron, but no correlation with serum iron concentrations. Sexually maturing fish had higher concentrations of iron in serum than immature fish. This may in part explain the significantly increased mortality from vibriosis in the maturing group in Experiment 1. Serum iron may be rapidly and inexpensively determined. The present indications of a significant variation in serum iron concentrations between salmon full-sib families (Experiment 2), and of higher serum iron concentrations in the fish susceptible to infection by Vibrio ssp. and to some extent by cold water vibriosis, may suggest that serum iron concentration should be further investigated as a marker for indirect selection to improve disease resistance in farmed Atlantic salmon.
Acknowledgements
The authors wish to thank Dr. Ragnar Salte for constructive criticism of an earlier draft of the manuscript. This work was partly financed by the Norwegian Fisheries Research Council and by the Norwegian Agricultural Research Council.
References Biosca, E.G. and Amaro, C., 1991. Siderophores and related outer membrane proteins in Vibrio spp. which arc potential pathogens of fish and shellfish. J. Fish Dis., 14: 249-263. Blumberg, B.S., Lustbader, E.D. and Whiteford, P.L., 1981. Changes in serum iron levels due to infection with hepatitis B virus. Proc. Natl. Acad. Sci., 78(5): 3222-3224. Bullen, J.J., Ward, C.G. and Rogers, H.J., 1990. Iron, infection, and the role of bicarbonate. FEMS Microbial. Lett., 71: 27-30. Bullen, J.J., Ward, C.G. and Rogers, H.J., 1991. The critical role of iron in some clinical infections. Eur. J. Clin. Microbial. Infect. Dis., lO(8): 613-617. Cox, C.D., 1989. Importance of iron in bacterial virulence. In: T.J. Beveridge and R.J. Doyle (Editors), Metal Ions and Bacteria. John Wiley & Sons, Toronto, pp. 207-246. Fjalestad, K.T., Gjedmm, T. and Gjerde, B., 1993. Genetic improvement of disease resistance in fish: An overview. Aquaculture, 111:65-74. Gjedrem, T. and Gj@en, H.M., 1994. Susceptibility of Atlantic salmon to furunculosis, BKD and cold water vibriosis and genetic correlations between them. Aquacult. Fish Manage. (in press). Griffith% E., 1987. The iron uptake systems of pathogenic bacteria In: J.J. Bullen and E. Griffiths (Editors), Iron and Infection: Molecular, Physiological and Clinical Aspects. John Wiley & Sons, Chichester, pp. 69-137. Hirst, I.D., Ellis, A.E. and Hastings, TX, 1991. Utilization of transferrin by Aeromonas salmonicida. In: Diseases of Fish and Shellfish. Proceedings of Symposium, 25-29 August, Budapest, Hungary, pp. 52 (Abstract). Huebets, H.A. and Finch, CA., 1987. The physiology of transferrin and transfeirin receptors. Physiol. Rev., 67(2): 520-582. Kay, W.W., Phipps, B.M., Ishiguro, E.E. and Trust, T.J., 1985. Porphyrin binding by the surface army virulence protein of Aeromonas salmonicida. J. Bacterial., 164( 3): 1332-1336. KIuger, M.J. and Bullen, J.J., 1987. Clinical and physiological aspects. In: J.J. Bullen and E. Griffitbs (Editors), Iron and Infection: Molecular, Physiological and Clinical Aspects. John Wiley & Sons, Chichester, pp. 243282. Otto, B.R., Verweij-van Vught, A.M.J.J. and MacLaren, D.M., 1992. Transferrins and heme-compounds as iron sources for pathogenic bacteria. &it. Rev. Microbial., 18(3): 217-233.
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Pratschner, G.A., 1978. The relative resistance of six transferrin genotypes of Coho salmon (Oncorhynchus kisurch) to cytophagosis, furunculosis and vibriosis. M.Sc. Thesis, College of Fisheries, University of Washington, Seattle (unpubl. ) (7 1 pp. Reed, K.H., Fjalestad, K.T. and Stremsheim, A., 1993. Genetic variation in lysozyme activity and spontanous haemolytic activity in Atlantic salmon (salmo salar L.). Aquaculture, 114: 19-33. Rervik, K.A., Bentsen, H.B., Salte, R. and Thomassen, M., 1992. Synergetic effects between dietary iron and omega-3 unsaturated fatty acids in farmed Atlantic salmon. Internal AKVAFORSK report, 94 pp. Sawatzki, G., 1987. The role of iron binding proteins in bacterial infections. In: G. Winkelmann, D. van der Helm and J.B. Neilands (Editors), Iron Transport in Microbes, Plants and Animals. VHC Verlagsgesellschaft mbH, Weinheim, pp. 477490. Suzumoto, B.K., S&reck, C.B. and McIntyre, J.D., 1977. Relative resistance of three transferrin genotypes of coho salmon (Oncorhynchus kkutch) and their hematological responses to bacterial kidney disease. J. Fish. Res. Board Can., 34: l-8. Trust, T.J., Ishiguro, E.E., Chart, H. and Kay, W.W., 1983. Virulence properties of Aeromonas salmonicida. J. World Maricult. Sot., 14: 193-200. Weinberg, E.D., 1984. Iron withholding: A defense against infection and neoplasia. Physiol. Rev., 64( 1): 6% 102. Weinberg, E.D., 1989. Cellular regulation of iron assimilation. Q. Rev. Biol., 64(3): 261-290. Weinberg, E.D., 1990. Cellular iron metabolism in health and disease. Drug Metab. Rev., 22(5): 531-579. Wolf, M.K. and Crosa, J.H., 1986. Evidence for the role of a siderophore in promoting Vi&o anguillarum infections. J. Gen. Microbial., 132: 2949-2952.
Aquaculture 125 (1994) 3745
Serum iron levels in farmed Atlantic salmon: family variation and associations with disease resistance Jarle Ravndal”,“, Tone Lgvold”, Hans Bernhard Bentsena, Knut Hkon R@edb,Trygve Gjedrem”, Kjell-Axne R@rvika “AKVAFORSK, Institute of Aquaculture Research AS, As, Norway bDepartment of Morphology, Generics and Aquatic Biology, Norwegian College of Veterinary Medicine, Oslo, Norway Accepted 27
February 1994
Abstract Serum samples from previous experiments were utilized to study the variation between sib families in serum iron concentration, and the association between serum iron concentration in the sib group and survival in challenge tests with vibriosis, furunculosis, cold water vibriosis or bacterial kidney disease (BKD) in farmed Atlantic salmon. In the first experiment, fish that died from vibriosis had significantly higher serum iron concentration prior to the challenge test than surviving fish. No significant variation was found in serum iron concentration between 34 full-sib families or between 12 half-sib families, but a non-significant suggestion of negative correlations (r = - 0.25, P = 0.15; r= -0.35, P= 0.27), was observed between least square means of serum iron concentration and survival rates from vibriosis in the sib families. Sexually maturing fish had significantly higher serum iron concentrations and lower survival rates than immatures. In the second experiment, 23 full-sib families showed a significant variation in serum iron concentration, and a non-significant suggestion of a negative correlation ( r = - 0.34, P = 0.12) of about the same magnitude as in the first experiment was observed between full-sib least-square means for serum iron concentration and the survival rate of parallel full-sib families in a challenge test with cold water vibriosis. The corresponding correlations between serum iron levels and survival rates in challenge tests with fumnculosis and BKD were both close to zero. The results from the two experiments taken together suggests that families with increased levels of serum iron may be more suceptible for Vzhio infections.
1. Introduction Vibriosis, furunculosis, cold water vibriosis and bacterial kidney disease (BKD) are serious bacterial diseases in Norwegian salmon farming. Thus, improved natural resistance *Correspondingauthor, present address:FelleskjepetHavbruk AS, Box 655 Krossen,4301 Sandnes, Notway. 0044~8486/94/$07.00 0 1994 SSDI0044-8486(94)00049-T
Elsevier Science B.V. All rights reserved
38
J. Ravndal et al. /Aquaculture
125 (1994) 37-45
against such diseases may reduce the costs of disease outbreaks and the environmental impact of extensive use of antibiotics. One possible way to improve natural disease resistance is by selection. Indirect selection on immunological or physiological marker traits has several advantages. However, indirect selection requires that the marker traits show genetic variation, are easy to determine on a large scale, and, of course, are genetically correlated with resistance to disease (Fjalestad et al., 1993). Iron plays a key role in the pathology of bacterial infections by enhancing the multiplication and virulence of the invading micro-organisms (Weinberg, 1984). Therefore, depriving the pathogens of iron may serve as a non-specific protective mechanism against infection (Kluger and Bullen, 1987; Weinberg, 1989). In serum, iron is normally bound to and transported by serum transferrin. Bacteria have several mechanisms to overcome the ability of the host to restrict iron availability. The best studied bacterial mechanism is that of iron chelators, called siderophores, which compete with the iron binding ability of transferrin (Griffiths, 1987; Otto et al., 1992). Differences in disease susceptibility of salmonids possessing different transfenin genotypes have been shown during infections with BKD (Renibacterium salmoninarum; Suzumoto et al., 1977)) vibriosis ( Vibrio anguillarum; Pratschner, 1978) and furunculosis (Aeromonas salmonicida; Pratschner, 1978). The present study was based on serum samples collected in two previous experiments involving challenge tests of a family material of farmed Atlantic salmon. The main objective of the study was to obtain estimates of family variation in serum iron concentration and to investigate the association between serum iron concentration and mortality in challenge tests with vibriosis, furunculosis, cold water vibriosis or BKD. If a persistent association can be shown over time and between parallel sib groups, serum iron should be further investigated as a marker trait for indirect selection for disease resistance.
2. Materials and methods Experiment I The iron concentrations in serum from a family material of 824 Atlantic salmon (Salmo salar, L.) were determined (see below). The individually tagged fish, which consisted of 34 full-sib groups within 12 paternal half-sib groups (average weight of 780 g) , were kept and sampled as described in Raed et al. ( 1993). Six weeks after serum sampling, 728 fish that were still available were recorded for stage of sexual maturation (immature or maturing) , and intraperitoneally injected with live Vibrio anguillarum ( lo8 bacterium per fish). Fish surviving the subsequent outbreak of vibriosis were recorded 3 weeks later (Raed et al., 1993). Survival in the full-sib families ranged from 29 to 74%. Experiment 2 In early 1991, 90 l-year-old fingerlings from each of 81 full-sib families of Atlantic salmon ( 1990 year-class) were challenged indirectly by introduction (into the tank) of fish which were infected with the furunculosis bacteria (Aeromonas salmonicida), or directly by intraperitoneal injection either with cold water vibriosis bacteria (Vibrio salmonicida) or with BKD bacteria (Renibacterium salmoninarum) in 3 independent tests (30 fish per
J. Ravndal et al. /Aquaculture
125 (1994) 37-45
39
B Susceptible
q Resistant
0 Furunculosis
BKD
C.W.
vibriosls
Fig. 1. Total survival rates of two groups of 11 susceptible full-sib families and of 12 resistant full-sib families in a challenge test with furunculosis, and total survival rates of the same groups of families in a challenge test with BKD and cold water vibriosis (c.w. vibriosis). Standard deviations of full-sib group survival rates are indicated.
family per test) at Vikan AkvaVet, Namsos (Gjedrem and Gjoen, 1994). In addition, a parallel group of 1054 fish from the same 8 1 full-sib families were later (May 1991) stocked in one cage at AKVAFORSK’s sea site at Veiset, about 13 fish from each full-sib family. All fish were fed a commercial feed mixture. Based on survival data for furunculosis in the challenge test, 23 extreme full-sib families were identified: in 11 highly susceptible families the survival rate ranged from 0 to 7%, and in 12 resistant families the survival rate ranged from 52 to 81%. The survival rates for the same 23 full-sib families in the challenge tests with cold water vibriosis and BKD ranged from 67 to lOO%,and from 0 to 90%, respectively (Fig. 1) . About 9 months after the challenge test (January 1992)) serum samples were collected at Veiset from 285 full sibs of the 23 extreme families (average weight of 1080 g), and iron concentrations in serum were determined. Chemical analysis
Total iron concentration in serum was chosen as a possible marker trait in the present study, since a proper method of determining the iron saturation of serum transferrin in Atlantic salmon has not yet been developed. Studies in our laboratories (not published) have shown that salmon serum transferrin has a much stronger affinity for iron than human serum transferrin. Hence, iron and salmon transferrin are not completely dissociated even at pH = 2.0. In both experiments, blood was drawn from the caudal vein into evacuated bloodcollection tubes without anticoagulants (Venoject@) . The samples were incubated at room temperature for 3 h and centrifuged. Serum was collected in microtiter plate wells (Experiment 1) or Eppendorf tubes (Experiment 2) and stored at - 70°C until assay. In the analysis, which involves destruction of organic material at 160°C small samples of serum were used; 150 ~1 in Experiment 1, and 250 ~1 in Experiment 2. During heating, the samples were treated with 500 ~1 nitric acid (HN03, 65%) for 45 min, and then with 500 ~1
40
J. Ravndalet al. /Aquaculture 125 (1994) 3745
hydrogen peroxide ( H,Oz, 30%) for 30 min. The treated samples were diluted by a solution of 4% HN03 and 1% H202 to 1.5 ml. The iron concentrations were determined with a Perkin Elmer model 3 100 (flame absorption spectrophotometer) (Experiment 1) or a GBC 906AA model (Experiment 2). Impact bead was used to increase absorbency. Optical density (OD) at 540 nm for each sample was checked and samples with OD above 0.22 were excluded to avoid errors due to hemolysis. Seronorm standards (Nycomed and Serco AS) were analyzed as described above every day during the analysis period. Out of 113 standards analyzed in Experiment 1, and 46 standards analyzed in Experiment 2, no significant differences (P > 0.05) between days were observed. Statistical analysis The material in Experiment 1 was a complete set of progeny groups from a nested mating design. A preliminary hierarchical analysis of variance did not show any significant effects of sire or dam within sire on serum iron concentration. A simplified general linear model was then applied to test for random effects of all family-associated sources of variation and to estimate least-square means:
where Yijkl=serum iron concentration of the Zthindividual, p = overall mean, x = random effect of the ith family (i= 1,2,...35 for full-sib and i= 1, 2,...12 for half-sib families), pj = fixed effect of the jth microtiter plate (j = 1,2,... 13)) mk = fixed effect of stage of sexual maturation (k = immature or maturing), and eiju = random error of the Zthindividual. Serum samples were randomly assigned to microtiter plates. A significant effect of microtiter plates is consequently expected to reflect variable laboratory conditions during the analysis of the different plates, and should be corrected for in the model. In Experiment 1, individual records of survival (dead or alive) were also analyzed as a dependent variable according to Model 1 after excluding the effects of microtiter plates from the model. The survival rate of the families is expected to be approximately normally distributed, since the number of individuals within each family was as large as 20 fish per full-sib group, and the mean survival rate in the families was intermediate (29-74%). A similar model was used to analyze serum iron concentrations as a dependent variable in Experiment 2: &jk
=
p
+A +
dj
+
eijk
where serum iron of the kth individual, p = overall mea&J = random effect of the ith full-sib family (i= 1, 2,...23), dj =fixed effect of the jth day of sample (j= 1, 2, 3), and eijk=random error of the kth individual. The serum iron concentrations in groups of dead and live fish (Experiment 1), and in groups of susceptible and resistant families (Experiment 2)) were analyzed according to Model 3 and Model 4, respectively: Yijk
=
YijH= /_& + gj +pj + mk + eijH
(Model 3)
Yijk=
(Model 4)
cL+gi
+dj
+eijk
where gi = fixed effect of the ith group ( i = dead or alive in Model 3 (Experiment 1) , and i = susceptible or resistant families in Model 4 (Experiment 2) ) .
J. Ravndal et al. /Aquaculiure
125 (1994) 37-45
41
The correlation coefficients between least-square means (LSM) of survival rates and LSM of serum iron concentrations of the full-sib or the half-sib families (Experiment 1) , and the correlation coefficients between LSM of serum iron concentrations and the survival rates of parallel full-sib groups (Experiment 2) were estimated. The sib group correlations will approach the genetic correlations between the traits when the number of individuals in each family increases, assuming that the common environmental effects on the traits and the non-additive genetic correlations are zero.
3. Results Genetic variation in serum iron concentrations In Experiment 1, the least-square means (LSM) concentrations of serum iron of the fullsib and half-sib families ranged from 1.51 to 2.10 pg/ml, and from I.67 to 1.91 pg/ml, respectively. The variation in serum iron between the full-sib or half-sib families was not significant (Table 1) , but a significant (P < 0.01) effect of both microtiter plate and stage of sexual maturation was observed. The effect of microtiter plate is assumed to be caused by variable laboratory conditions during the analysis of the different plates. The mean value and the phenotypic variation of serum iron concentration in fish from Experiment 2 were much lower than in Experiment 1 (Table 1) . The LSM concentrations of serum iron of the full-sib families ranged from 0.69 to 1.14 pg/ml. A significant variation in serum iron concentrations was detected between the full-sib groups (Table 1) . No significant effect was found of day of sample (P > 0.05)) indicating that the number of days the fish were exposed to handling before the serum sample was collected did not affect the serum iron levels. Association between serum iron and survival rate In Experiment 1, fish that died from vibriosis had a significantly (P < 0.05) higher level of serum iron 6 weeks before challenge than surviving fish (Fig. 2, 1.81 vs. 1.68 pg Fe/ ml). No significant difference in serum iron level was detected between the full-sibs of susceptible sib groups and resistant sib groups in the challenge tests in Experiment 2 (Fig. 2). A significant variation in survival rates between half-sib families (but not between fullsib families) was detected (P < 0.05) in Experiment 1; the survival rates ranged from 34 Table 1 Serum iron concentrations (pg/ml) and associated statistics: Number (N) of individuals, full-sib groups and half-sib groups, standard deviations (s.d.) of phenotypic observations (mean) and of full-sib and half-sib leastsquare means (LSM) for serum iron ( pg/ml) , and the significance ( P) of the marginal (type III) contributions of families according to Model 1 (Experiment 1) and Model 2 (Experiment 2)
Experiment 1 Experiment 2
Phenotypic
Full-sib LSM
Half-sib LSM
N
Mean f s.d.
N
s.d.
P
N
s.d.
P
824 285
1.70 f 0.67 0.89 f 0.32
34 23
0.13 0.13
0.66 0.01
12
0.07
0.79
42
J. Ravndal et al. /Aquaculture 125 (1994) 37-45 20
18
16
14
1 2
10
08
06
04
02
oc IExperiment
Experiment
1
2
Fig. 2. Least-square mean estimates of serum iron concentrations (pg/mi) of fish that died or survived in a challenge test with vibriosis in Experiment 1 (Model 3), and full sibs of families that showed susceptibility or resistance in a challenge test with furunculosis in Experiment 2 (Model 3). Standard errors are indicated.
to 65%, and 29 to 74%, respectively. No significant correlation was detected between LSM of serum iron concentrations of the families and survival rates, either after challenge with vibriosis in Experiment 1 or after challenge with furunculosis, cold water vibriosis or BKD in Experiment 2 (Table 2). However, a consistent tendency was observed across the two experiments, indicating a possible association between reduced serum iron levels and increased survival in challenge tests with Vibrio ssp. and to some extent also with cold water vibriosis. In Experiment 1, sexually maturing fish had significantly higher serum iron concentrations and mortality rates than immature fish (Table 3). Within the two groups, fish that died showed a non-significant tendency (P = 0.07) towards higher concentrations of serum iron than surviving fish. Table 2 Correlation coefficients (r) and significance (P) for the association between full-sib or half-sib families leastsquare means (LSM) for serum iron according to Models 1 and 2, and survival rates in the same material (Experiment I), or in parallel full-sib groups (Experiment 2) in challenge tests with different diseases Full-sib LSM
Half-sib LSM
r
P
r
P
-0.25
0.15
- 0.35
0.27
0.12 -0.34 0.10
0.60 0.12 0.66
Experiment I
Vibriosis Experiment 2
Furunculosis Cold water vibriosis Bacterial kidney disease
J. Raw&l
et al. /Aquaculture
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43
Table 3 Number (N) of immature and sexually maturing individuals in Experiment 1, survival rates in a challenge test with vibriosis, and least-square means of serum iron prior to the test according to Model 1
N
Survival (%) Serum iron @g/ml)
Immature
Sexually maturing
P
613 66 1.67
115 32 1.86
‘Co.01
The significance (P) of the marginal (type III) contributions were computed from the same model.
4. Discussion The significant association between high concentrations of serum iron and mortality for individuals of vibriosis-infected farmed Atlantic salmon in Experiment 1 (Fig. 2) is consistent with previous studies in mammals, showing that bacterial growth, multiplication and virulence are enhanced by increased iron availability and total serum iron concentrations (Blumberg et al., 1981; Sawatzki, 1987; Cox, 1989; Weinberg, 1990; Bullen et al., 1991). The lack of a significant full sib correlation between LSM serum iron concentrations and mortality rates caused by furunculosis, cold water vibriosis and BKD in Experiment 2 may have several explanations. Firstly, the serum samples in this experiment were collected from parallel full-sib groups of the families that were challenge tested, and about 9 months later than the challenge test. In Experiment 1, the serum iron concentrations were determined from the same fish that were used in the challenge test, and the sera were collected 6 weeks prior to the infection. If serum iron level is a variable (e.g. time- or age-dependent) genetic property of the individual, the correlations in Experiment 2 are expected to be more affected by fluctuations in serum iron concentrations. Secondly, the lower serum iron concentrations observed in Experiment 2 may result in lower transferrin saturation. The presence of unsaturated transferrin in plasma seems to be an important bacteriostatic factor in mammals (Huebers and Finch, 1987; Bullen et al., 1990). Differences between families in iron saturation of serum transferrin would then be expected to affect bacterial growth more at high serum iron concentrations (Experiment 1) than at low serum iron concentrations (Experiment 2). The differences in average serum iron concentrations between the present experiments may be caused by non-genetic factors, and did not exceed the range observed in other studies carried out by AKVAFORSK. Studies by Wolf and Crosa (1986) and by Biosca and Amaro (1991) have shown that both siderophores and related outer membrane proteins in V. anguikzrum are potential pathogenic factors in fish, probably because of the improved ability of the bacteria to secure a sufficient iron supply. Similar iron-sequestering mechanisms are also found in typical strains of A. salmonicidu (Trust et al., 1983; Kay et al., 1985; Hirst et al., 1991). Our experiments may indicate that the iron sequestering mechanisms ofA. sulmonicidu are more efficient than those of V. unguillurum and V. sulmonicidu. Little is known about the iron competition mechanisms of R. salmoninurum. Rorvik et al. ( 1992) suggest that the availability of iron in the gastro-intestinal tract may be more important than the serum iron level for the growth and multiplication of A. sulmonicidu. They observed a highly significant and positive correlation between mortality in
44
J. Ravndal et al. /Aquaculture
I25 (1994) 3745
a natural outbreak of furunculosis and concentrations of dietary iron, but no correlation with serum iron concentrations. Sexually maturing fish had higher concentrations of iron in serum than immature fish. This may in part explain the significantly increased mortality from vibriosis in the maturing group in Experiment 1. Serum iron may be rapidly and inexpensively determined. The present indications of a significant variation in serum iron concentrations between salmon full-sib families (Experiment 2), and of higher serum iron concentrations in the fish susceptible to infection by Vibrio ssp. and to some extent by cold water vibriosis, may suggest that serum iron concentration should be further investigated as a marker for indirect selection to improve disease resistance in farmed Atlantic salmon.
Acknowledgements
The authors wish to thank Dr. Ragnar Salte for constructive criticism of an earlier draft of the manuscript. This work was partly financed by the Norwegian Fisheries Research Council and by the Norwegian Agricultural Research Council.
References Biosca, E.G. and Amaro, C., 1991. Siderophores and related outer membrane proteins in Vibrio spp. which arc potential pathogens of fish and shellfish. J. Fish Dis., 14: 249-263. Blumberg, B.S., Lustbader, E.D. and Whiteford, P.L., 1981. Changes in serum iron levels due to infection with hepatitis B virus. Proc. Natl. Acad. Sci., 78(5): 3222-3224. Bullen, J.J., Ward, C.G. and Rogers, H.J., 1990. Iron, infection, and the role of bicarbonate. FEMS Microbial. Lett., 71: 27-30. Bullen, J.J., Ward, C.G. and Rogers, H.J., 1991. The critical role of iron in some clinical infections. Eur. J. Clin. Microbial. Infect. Dis., lO(8): 613-617. Cox, C.D., 1989. Importance of iron in bacterial virulence. In: T.J. Beveridge and R.J. Doyle (Editors), Metal Ions and Bacteria. John Wiley & Sons, Toronto, pp. 207-246. Fjalestad, K.T., Gjedmm, T. and Gjerde, B., 1993. Genetic improvement of disease resistance in fish: An overview. Aquaculture, 111:65-74. Gjedrem, T. and Gj@en, H.M., 1994. Susceptibility of Atlantic salmon to furunculosis, BKD and cold water vibriosis and genetic correlations between them. Aquacult. Fish Manage. (in press). Griffith% E., 1987. The iron uptake systems of pathogenic bacteria In: J.J. Bullen and E. Griffiths (Editors), Iron and Infection: Molecular, Physiological and Clinical Aspects. John Wiley & Sons, Chichester, pp. 69-137. Hirst, I.D., Ellis, A.E. and Hastings, TX, 1991. Utilization of transferrin by Aeromonas salmonicida. In: Diseases of Fish and Shellfish. Proceedings of Symposium, 25-29 August, Budapest, Hungary, pp. 52 (Abstract). Huebets, H.A. and Finch, CA., 1987. The physiology of transferrin and transfeirin receptors. Physiol. Rev., 67(2): 520-582. Kay, W.W., Phipps, B.M., Ishiguro, E.E. and Trust, T.J., 1985. Porphyrin binding by the surface army virulence protein of Aeromonas salmonicida. J. Bacterial., 164( 3): 1332-1336. KIuger, M.J. and Bullen, J.J., 1987. Clinical and physiological aspects. In: J.J. Bullen and E. Griffitbs (Editors), Iron and Infection: Molecular, Physiological and Clinical Aspects. John Wiley & Sons, Chichester, pp. 243282. Otto, B.R., Verweij-van Vught, A.M.J.J. and MacLaren, D.M., 1992. Transferrins and heme-compounds as iron sources for pathogenic bacteria. &it. Rev. Microbial., 18(3): 217-233.
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Pratschner, G.A., 1978. The relative resistance of six transferrin genotypes of Coho salmon (Oncorhynchus kisurch) to cytophagosis, furunculosis and vibriosis. M.Sc. Thesis, College of Fisheries, University of Washington, Seattle (unpubl. ) (7 1 pp. Reed, K.H., Fjalestad, K.T. and Stremsheim, A., 1993. Genetic variation in lysozyme activity and spontanous haemolytic activity in Atlantic salmon (salmo salar L.). Aquaculture, 114: 19-33. Rervik, K.A., Bentsen, H.B., Salte, R. and Thomassen, M., 1992. Synergetic effects between dietary iron and omega-3 unsaturated fatty acids in farmed Atlantic salmon. Internal AKVAFORSK report, 94 pp. Sawatzki, G., 1987. The role of iron binding proteins in bacterial infections. In: G. Winkelmann, D. van der Helm and J.B. Neilands (Editors), Iron Transport in Microbes, Plants and Animals. VHC Verlagsgesellschaft mbH, Weinheim, pp. 477490. Suzumoto, B.K., S&reck, C.B. and McIntyre, J.D., 1977. Relative resistance of three transferrin genotypes of coho salmon (Oncorhynchus kkutch) and their hematological responses to bacterial kidney disease. J. Fish. Res. Board Can., 34: l-8. Trust, T.J., Ishiguro, E.E., Chart, H. and Kay, W.W., 1983. Virulence properties of Aeromonas salmonicida. J. World Maricult. Sot., 14: 193-200. Weinberg, E.D., 1984. Iron withholding: A defense against infection and neoplasia. Physiol. Rev., 64( 1): 6% 102. Weinberg, E.D., 1989. Cellular regulation of iron assimilation. Q. Rev. Biol., 64(3): 261-290. Weinberg, E.D., 1990. Cellular iron metabolism in health and disease. Drug Metab. Rev., 22(5): 531-579. Wolf, M.K. and Crosa, J.H., 1986. Evidence for the role of a siderophore in promoting Vi&o anguillarum infections. J. Gen. Microbial., 132: 2949-2952.