Fish & Shellfish Immunology 21 (2006) 404e413 www.elsevier.com/locate/fsi
Genetic differences in natural antibody levels in common carp (Cyprinus carpio L.) Neli M. Kachamakova a,c, Ilgiz Irnazarow a, Henk K. Parmentier b, Huub F.J. Savelkoul c, Andrzej Pilarczyk a, Geert F. Wiegertjes c,* a Polish Academy of Sciences, Institute of Ichthyobiology and Aquaculture, Gołysz, 43-520 Chybie, Poland Adaptation Physiology Group, Wageningen Institute of Animal Sciences, Wageningen University, Marijkeweg 40, P.O. Box 338, 6709 PG Wageningen, The Netherlands c Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, Marijkeweg 40, P.O. Box 338, 6709 PG Wageningen, The Netherlands b
Received 22 November 2005; revised 17 January 2006; accepted 19 January 2006 Available online 20 March 2006
Abstract In mammals, natural antibodies (Nabs) are mostly of the IgM isotype and can bind to a particular antigen or pathogen even if the host has never been exposed. Despite their early detection and abundance, the exact role and genetic control of Nabs remain unclear. We have used an indirect ELISA with three different antigens (keyhole limpet haemocyanin, chicken ovalbumin and bovine serum albumin) to demonstrate the ubiquitous presence of Nabs in common carp. Serum levels of Nabs increased with age, i.e. 10-monthold fish showed higher levels than 4-month-old fish. Also, fish grown in earth ponds showed higher levels of Nabs than fish grown in a clean environment of UV-treated water. Furthermore, we show that Nabs are present in different levels in the serum of carp lines with a different genetic background, suggestive of a genetic control. These genetic differences were independent of antigen, age and environment. Genetic differences in levels of Nabs could not unequivocally be related to differences in survival under farmed conditions. The possibilities for using levels of Nabs as marker criterion for selection for genetic disease resistance are discussed. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Natural antibodies; Fish; Disease resistance
1. Introduction In mammals [1], birds [2] and fish [3] immunoglobulins (Igs) that can bind to a particular antigen or pathogen, even if the host has never been exposed to it, can be detected. These Igs are of low affinity, mostly of the IgM isotype, polyreactive and found at high concentration in the adult serum [4,5]. What triggers the formation and release of these Igs, however, remains an enigma [6]. So far, there is no consensus terminology for these antibodies, and they have been
* Corresponding author. Tel.: þ31 317 48 29 32; fax: þ31 317 48 39 55. E-mail address:
[email protected] (G.F. Wiegertjes). 1050-4648/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2006.01.005
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named residual, background, natural, pre-existing, innate, or naturally acquired antibodies. In the present study we will address these Igs as natural antibodies (Nabs). Although their existence has been known for a long time, the exact role of Nabs is not clear. Functions proposed for Nabs include clearance of damaged self-components [5] and ‘‘idiotype’’ network interactions within the immune system [7]. They are also connected with direct neutralization of bacteria in rats [8] and viruses present in the blood circulation of mice or fish [9,10]. Trapping of different pathogens in the marginal splenic zone, thus enhancing the antigen immunogenicity, is also suspected [11]. Also, natural IgM, being a major recognition molecule for activation of the classical pathway of complement, can indirectly neutralize pathogens [12,13]. Nabs in mammals are mostly of the IgM isotype and tend to have rather low antigen-binding affinities compensated for, to some extent, by the pentameric nature of secreted IgM [5]. In contrast to the pentameric IgM in mammals and sharks the teleost IgM is tetrameric, although dimeric and monomeric forms can be found as well [14,15]. Clear genetic differences have been reported to influence Nab titres in mammals and birds. In mice, different inbred strains showed natural vesicular stomatitis virus (VSV) specific neutralizing antibody titres, differing up to four times [9]. In chicken, genetic linkage between the capacity of different chicken lines to produce antigen-specific antibodies and the level of Nab present in plasma was proposed [16]. In fish, few reports have addressed the presence of Nabs directly. Genetic differences in total IgM concentration in the sera of Atlantic salmon strains suggested a genetic influence on the levels of total Ig [17]. Also in goldfish, genetic correlation between the production of specific Aeromonas salmonicida anti-A-protein antibodies and Nab titres was reported [3]. Clearly, more investigations are needed that address levels of Nabs in fish directly. Both specific and natural antibody levels can be assessed by enzyme-linked immunosorbent assay (ELISA) [18], which are potentially related to disease resistance and could be used as selective markers for genetic disease resistance. In the present study, Nab levels in serum from eight genetically different carp lines were measured, using an indirect ELISA with three different antigens (keyhole limpet haemocyanin, chicken ovalbumin and bovine serum albumin) to demonstrate the ubiquitous presence of Nabs in common carp. Total serum levels of Nabs were dependent on age and environment. Nabs were present in different levels in the serum of carp lines with a different genetic background, suggestive of a genetic control. These genetic differences were independent of antigen, age and environment.
2. Materials and methods 2.1. Fish 2.1.1. Genetic effect The live gene bank at the Institute of Ichthyobiology and Aquaculture in Go1ysz, Poland is in possession of 19 lines of common carp (Cyprinus carpio L.) of various geographical origins [19]. Eight carp lines (Table 1), part of the live gene bank, were reproduced in the summer of 2002 and each carp line was divided over two treatment groups and each raised under different environmental conditions. Table 1 Origin (code, geographical background) and survival (average percentage SD) after the first rearing season and first winter, of eight carp lines part of the live gene bank used in the current study Line
Origin
Average survival (%) autumn of 2002
Average survival (%) winter of 2002e2003
R7 N R2 R0 R8 K D Ur
Hungarian German Polish Hungarian Hungarian Polish Israeli Ukrainian
34.2 23.0 24.5 7.6 24.9 13.3 11.6 7.4 12.7 9.1 28.8 11.5 19.8 13.4 23.6 9.4
46.4 14.2 59.8 1.1 58.2 4.9 31.5 27.4 29.6 20.0 61.4 37.4 40.2 15.2 70.1 14.1
Each carp line was grown in quadruplicate ponds at a stocking density of 3000 individuals/pond.
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For establishing natural survival, one group was raised in experimental ponds of 632 m2 surface, in quadruplicate (n ¼ 4 3000). From these fish, at 24 months of age approximately, serum samples from randomly chosen individuals were collected for establishment of the Nab levels in pond-raised carp (n ¼ 15 fish/line). Fish weighed 400e800 g at sampling. The other group was raised under controlled environmental conditions in triplicate (n ¼ 3 100) 70 l aquaria with UV-treated recirculating water, and fed pelleted dry food (ALLER AQUA e Denmark). From these fish, at 12 months of age approximately, serum samples from randomly taken individuals were collected (n ¼ 10 fish/line). Fish weighed 200e250 g at sampling. Serum samples from the two environmental groups were used in an indirect ELISA to study genetic differences in Nab levels. 2.1.2. Effect of environment As a part of a larger experiment, two of the above-mentioned carp lines (D and K) were again reproduced in the summer of 2004, and each carp line was divided over two treatment groups as above. The pond group was again raised in quadruplicate and the aquarium group in triplicate. Fish weighed 30e40 g (pond group) or 40e50 g (aquarium group) at sampling. From the fish grown in ponds and aquaria, at the same age (4 months approximately), representative serum samples were collected (n ¼ 15 fish/line). Serum samples were used in an indirect ELISA to study environmental effects on Nab levels. 2.1.3. Effect of age Two carp lines (D and K), reproduced as a part of a larger experiment in the summer of 2004 and grown in aquaria, were sampled at 4 and 10 months of age approximately. Fish weighed 40e50 g (4-month-old group) or 100e120 g (10-month-old group) at sampling. Serum samples from randomly chosen individuals were collected (n ¼ 15 fish/line) and used in an indirect ELISA to study age effects on Nab levels. 2.2. Serum sampling Blood was taken from the caudal vein of anaesthetized fish (0.3 g/l Tricaine Methane Sulphonate, Crescent Research, USA) and kept at 4 C for 24 h. After centrifugation, serum was collected and frozen at 20 C until used. 2.3. Enzyme-linked immunosorbent assay (ELISA) for detection of natural antibodies Natural antibodies to keyhole limpet haemocyanin (KLH; MP Biomedicals), egg white ovalbumin (OVA; Sigma) and bovine serum albumin (BSA; Sigma) were determined in individual serum samples using an indirect ELISA procedure, essentially as described for chicken [16]. In short, 96-well plates (Greiner) were coated with either 4 mg (OVA, BSA) or 1 mg (KLH) protein per ml. Based on pilot studies, starting serum dilution was 1:80, followed by twofold serial dilutions. Binding of natural antibodies to the proteins was detected using mouse-anti-carp IgM monoclonal antibody WCI 12 (1:500) [15], followed by goat-anti-mouse IgG conjugated with horse-radish peroxidase (HRP; Bio-Rad) at 1:2000 dilution. The colour was developed with 3,30 ,5,50 -tetramethyl benzidine (TMB; Sigma) for 10 min and the reaction stopped with 2.5 N H2SO4. Extinction at 450 nm was measured with a Multiskan (Flow, Irvine, UK) spectrophotometer. 2.4. Total protein content Total protein content of the different sera was determined using a standard Bradford assay (Sigma) and the absorbance read at 595 nm. The total protein content was calculated from a standard curve with BSA, fraction V (Sigma). 2.5. Statistical analysis Differences in extinctions in serially diluted sera were analyzed by linear regression analysis [20]. Regression equations were based on a general linear model assuming y ¼ b0 þ bx, where y ¼ extinction, x ¼ serum dilution,
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b0 ¼ value of each y when each x ¼ 0 and bx ¼ coefficient for each x. Statistically significant differences between slopes of regression lines were estimated on six dilution steps, and used to characterize relative affinities of the sera for all antigens tested. Statistically significant differences in total protein contents were estimated by Student’s t-test for dependent samples [21]. Differences with p < 0.05 were considered significant. 3. Results 3.1. Survival of the eight carp lines raised in earth ponds Survival of the fish under natural conditions is an important parameter in carp production. Therefore, part of the fish, reproduced in the summer of 2002, were grown in earth ponds to determine survival after the first rearing season and after first wintering (Table 1). The survival after first few months of rearing did not show significant differences between the carp lines: the lowest survival was seen for the Hungarian carp line R0 (11.6%) while the highest survival was noted for the Hungarian carp line R7 (34.2%). The second counting of the number of fish was done after the first wintering. Again, differences were not significant owing to high standard deviations. After wintering the lowest survival again was noted for the Hungarian carp line R0 (31.5%), while high survival was measured for several carp lines but not for the Hungarian carp line R7. 3.2. Effect of genetic background Genetic effects on Nab levels were studied in eight genetically different carp lines grown either in ponds or in aquaria. Nab levels against three different antigens (KLH, OVA and BSA) were determined by ELISA in serum samples from randomly taken individuals. Linear regression analysis of the Nab levels of fish grown in ponds showed statistically significant differences between the slopes of the dilution curves of the eight carp lines analysed, indicating different affinity characteristics (Table 2). For visualization of these differences we chose to depict only the data of four carp lines: the carp line with the highest extinction (K), the lowest extinction (N) and two carp lines showing average (Ur, R2) extinction (Fig. 1). The other four lines (D, R0, R7, R8) showed extinctions between the highest and the lowest line. Genetic differences were independent of the antigen used in the ELISA. The standard deviations were small, indicating that only minor differences existed between individuals. Linear regression analysis of the Nab levels of fish grown in aquaria showed similar statistically significant differences between the same carp lines (Table 3). Fig. 2 indicates the same carp lines which showed the highest (K) or lowest (N) extinction (K, Ur > N, p < 0.0001, slope p < 0.005; K, Ur > R2, p NS, slope p NS). Again, genetic differences were independent of the antigen used in the ELISA, and standard deviations were small. These data suggested that Nab levels are under genetic control, independent of the environment used to grow the fish. Also, apparently, the observed genetic differences were independent of age, since the fish grown in ponds and the fish grown in aquaria differed in age. However, to resolve the confounding effects of age and environment in this particular experiment, other experiments were performed to study the effect of environment and age, independently. Table 2 Regression equations, based on ELISA measurements to detect natural antibodies (Nabs) in serum samples from carp grown in ponds for 2 years Line
KLH
OVA
BSA
D K N R0 R2 R7 R8 Ur
y ¼ 1.16x þ 2.49 y ¼ L1.32x D 2.93 y ¼ L0.93x D 1.97 y ¼ 1.34x þ 2.93 y ¼ L1.05x D 2.28 y ¼ 0.95x þ 2.01 y ¼ 1.30x þ 2.88 y ¼ L1.25x D 2.71
y ¼ 0.88x þ 1.89 y ¼ L0.98x D 2.16 y ¼ L0.64x D 1.40 y ¼ 1.07x þ 2.27 y ¼ L0.86x D 1.85 y ¼ 0.98x þ 2.15 y ¼ 1.07x þ 2.32 y ¼ L0.95x D 2.07
y ¼ 0.87x þ 1.88 y ¼ L0.97x D 2.11 y ¼ L0.62x D 1.33 y ¼ 1.07x þ 2.28 y ¼ L0.81x D 1.77 y ¼ 0.95x þ 2.04 y ¼ 1.03x þ 2.22 y ¼ L0.93x D 2.02
Nabs were measured against keyhole limpet haemocyanin (KLH), ovalbumin (OVA) and bovine serum albumin (BSA). The regression lines for the carp lines K, N, R2 and Ur (bold) are depicted in Fig. 1.
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extinction (OD 450 nm)
a)
1,8 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0
extinction (OD 450nm)
b)
1,8 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0
extinction (OD 450nm)
c)
1,8 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0
1/80
1/160
1/320
1/640
1/1280
dilution of the serum Fig. 1. Natural antibodies from carp raised in earth ponds for approximately 2 years. Natural antibodies were detected by ELISA using three different antigens. Average OD values þ SD of 15 individuals per carp line from four carp lines (K e:e, N exe, Ur eBe, R2 e,e) are shown. Data are presented as extinction curves based on serial dilution of carp sera. (a) Binding to keyhole limpet haemocyanin (K, Ur > N, p < 0.0001, slope p < 0.0001; K, Ur > R2, p < 0.005, slope p < 0.005). (b) Binding to ovalbumin (K, Ur > N, p < 0.0001, slope p < 0.0001; K, Ur > R2, p < 0.05, slope p NS). (c) Binding to bovine serum albumin (K, Ur > N, p < 0.0001, slope p < 0.0001; K, Ur > R2, p < 0.05, slope p < 0.05).
3.3. Effect of environment To study the effect of environment (ponds versus aquaria), a selection of two carp lines (D and K) was sampled at the same age (4 months). Since genetic differences in levels of Nabs were detected independent of the antigen used in the ELISA, we chose to show the results for one antigen (BSA) only. Natural antibody levels against BSA were determined by ELISA in serum samples from randomly taken individuals. The results clearly show significantly higher levels of Nabs in fish grown in ponds, compared with the same carp lines raised in aquaria (Fig. 3a). This difference was independent of the genetic background, although differences
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Table 3 Regression equations, based on ELISA measurements to detect natural antibodies (Nabs) in serum samples from carp grown under controlled conditions (aquaria) for 1 year Line
KLH
OVA
BSA
D K N R0 R2 R7 R8 Ur
y ¼ 1.43x þ 3.21 y ¼ L1.44x D 3.45 y ¼ L1.38x D 3.11 y ¼ 1.43x þ 3.32 y ¼ L1.39x D 3.16 y ¼ 1.34x þ 3.02 y ¼ 1.38x þ 3.14 y ¼ L1.44x D 3.35
y ¼ 1.35x þ 2.94 y ¼ L1.47x D 3.27 y ¼ L1.21x D 2.63 y ¼ 1.40x þ 3.10 y ¼ L1.29x D 2.85 y ¼ 1.23x þ 2.65 y ¼ 1.32x þ 2.92 y ¼ L1.37x D 3.03
y ¼ 1.27x þ 2.78 y ¼ L1.35x D 3.05 y ¼ L1.16x D 2.53 y ¼ 1.27x þ 2.88 y ¼ L1.18x D 2.61 y ¼ 1.12x þ 2.41 y ¼ 1.25x þ 2.79 y ¼ L1.30x D 2.89
Nabs were measured against keyhole limpet haemocyanin (KLH), ovalbumin (OVA) and bovine serum albumin (BSA). The regression lines for the carp lines K, N, R2 and Ur (bold) are depicted in Fig. 2.
were most significantly detected in carp line D. Again, Nab levels were higher in carp line K than in carp line D, although this difference was statistically significant only for the aquaria group (Kaq > Daq, p < 0.0001, slope p < 0.0001). 3.4. Effect of age To study the effect of age (4 versus 10 months), a selection of two carp lines (D and K) was sampled under the same environmental conditions (aquaria). As above, we chose to show the results for one antigen (BSA) only. Natural antibody levels against BSA were determined by ELISA in serum samples from randomly taken individuals. The results clearly show significantly higher levels of Nabs in 10-month-old fish, compared with fish of 4 months of age (Fig. 3b), independent of the genetic background. Again, Nab levels were higher in carp line K than in carp line D, independently of the age (K 4 months > D 4 months, p < 0.0001, slope p < 0.0001; K 10 months > D 10 months, p < 0.001, slope p < 0.01). 3.5. Total protein levels Total protein levels in serum samples tested for Nabs were determined with a Bradford assay (Table 4). Total protein levels ranged from 18 to 29 mg/ml. No relation between total protein levels and Nab levels could be detected. In the first experiment, overall, sera collected from carp raised in ponds contained more total protein than sera collected from carp raised in aquaria. However, this could not be confirmed in the second experiment. Also, sera collected from fish at a later age did not always contain more protein. 4. Discussion This paper clearly shows a genetic influence on natural antibody (Nab) levels in common carp, which suggests a potential for Nab levels to serve as selective marker for genetic disease resistance. We measured levels of Nabs in eight carp lines with a different genetic background. Nab levels were clearly and consistently different between these carp lines, allowing for a classification of different lines as low-, medium- or high-Nab responder carp. Linear regression analysis of the ELISA data indicated that the Nabs showed similar affinity characteristics to all three antigens tested. Differences between carp lines, therefore, should be ascribed to quantitative rather than qualitative differences in Nabs. Our data suggest that the level of Nabs present in sera of different carp lines is under genetic control. These genetic differences were preserved independently of age and environment, although the latter parameters affected the titres of Nabs. In general, environmental conditions are considered important for the levels of Nabs in animals. We studied two carp lines of the same age, raised under different environmental conditions (pond versus aquaria). Indeed, higher levels of Nabs were found in the carp grown in ponds. It is thought that the Nab repertoire and levels may be either shaped by continuous polyclonal stimulation by exogenous microbes, initiating cross reactivity driven responses of
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a) 1,8
extinction (OD 450nm)
1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0
b)
extinction (OD 450 nm)
1,8 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0
c) extinction (OD 450nm)
1,8 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0
1/80
1/160
1/320
1/640
1/1280
serum dilution Fig. 2. Natural antibodies from carp raised in aquaria for approximately 1 year. Natural antibodies were detected by ELISA using three different antigens. The extinction curves from four carp lines (K e:e, N exe, Ur eBe, R2 e,e) are shown. Data are the average þ SD of 10 individuals per carp line. (a) Binding to keyhole limpet haemocyanin (K, Ur > N, p < 0.05, slope p NS; K, Ur > R2, p NS, slope p NS). (b) Binding to ovalbumin (K, Ur > N, p < 0.005, slope p < 0.05; K, Ur > R2, p NS, slope p NS). (c) Binding to bovine serum albumin (K, Ur > N, p < 0.005, slope p < 0.05; K, Ur > R2, p NS, slope p NS).
auto-reactive B cells, or corresponds with the secretion of naturally occurring auto-reactive B cell clones or both [16]. The fact that we found higher levels of Nabs in pond-raised fish could point to an effect of exogenous stimuli on fish Nab levels. As expected, older animals showed more Nabs than younger ones. We studied two carp lines, raised in the same environment, at a different age (4 versus 10 months). Indeed, higher levels of Nabs were found in the carp that were of higher age. We did not measure total IgM. In salmonid fish, total serum IgM concentrations were found to increase
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a)
411
2,5
extinction (OD 450nm)
2
1,5
1
0,5
0
extinction (OD 450nm)
b)
2,5 2 1,5 1 0,5 0
1/80
1/160
1/320
1/640
1/1280
serum dilution Fig. 3. Natural antibodies from carp, detected by ELISA using bovine serum albumin as detection antigen. (a) Levels of Nabs in two 4-month-old carp lines (K exeand D e:e) raised in earth ponds (smooth line) or in aquaria (broken line). Data are the average þ SD of 15 individuals per carp line. (Dp > Daq, p < 0.0001, slope p < 0.0001; Kp > Kaq, p < 0.05, slope p NS; Kaq > Daq, p < 0.0001, slope p < 0.0001; Kp > Dp, p NS, slope p NS). (b) Levels of Nabs in two carp lines (K exeand D e:e), both raised in aquaria but analysed at the different age of 4 (broken line) or 10 (smooth line) months. Data are the average þ SD of 15 individuals per carp line. (D 10 months > D 4 months, K 10 months > K 4 months, p < 0.0001, slope p < 0.0001.) There was also clear difference between the lines ((K 4 months > D 4 months, p < 0.0001, slope p < 0.0001; K 10 months > D 10 months, p < 0.001, slope p < 0.01).
Table 4 Total proteins (mg/ml, average SD) detected in the serum of individuals, representing different carp lines, used in the current study Lines
D K N R0 R2 R7 R8 Ur
Fish from ponds
Fish from aquaria
2-Year-old (2002)
4-Month-old (2004)
1-Year-old (2002)
4-Month-old (2004)
10-Month-old (2004)
18.8 6.3 29.4 10.2 26.9 7.7 31.7 9.2 29.2 12.9 25.9 9.2 23.9 5.0 20.8 6.8
26.4 4.8 22.3 7.9
22.9 4.0 24.6 7.7 21.5 5.8 24.9 6.2 23.4 3.9 21.9 5.6 23.3 6.4 24.9 7.1
28.3 4.6 28.3 4.6*
27.2 4.7 27.1 4.5
Bovine serum albumin was used as a standard in the Bradford assay. A number of n ¼ 10 individuals was analysed for the fish reproduced in 2002, and n ¼ 15 for the fish reproduced in 2004. *Kp (4-month-old) < Kaq (4-month-old), P < 0.05.
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with increased size [22]. Similarly, in chickens, higher levels of Nabs were found in older individuals [16] and in mammals, the production of natural anti-mannan Abs in cattle increased in an age-dependent manner [23]. We studied Nab levels in eight carp lines with a different genetic background, part of a live gene bank. Nab levels were different between the carp lines, with the German carp line ‘N’ consistently showing the lowest level of Nabs, and the Polish line ‘K’ consistently showing the highest level of Nabs. We also determined the survival rate of the different carp lines grown in ponds. Survival, in fact, is one of the best criteria for determining the level of resistance because it reflects the cumulative effects of all hostepathogen interactions during production [24]. However, although clear differences in survival rate existed between the carp lines, there was no obvious relation between the Nab levels and survival under natural conditions. A positive correlation between high survival rate after wintering of carp lines ‘K’ and ‘Ur’ (60e70%) and high titres of Nabs could be made. However, a reverse relation was seen for the carp line ‘N’, where high survival rate (60% approximately) was connected with low Nab levels. Apparently, survival rate under natural conditions is a complex trait that cannot be easily explained by a single factor such as levels of Nabs. Likely, putative correlations of Nab levels with disease resistance are more easily detected in defined challenge experiments that do not suffer from the many confounding effects influencing survival rate in the natural environment [25]. Indeed, carp line K was found to show the highest survival among six other carp lines tested in a challenge experiment with Aeromonas hydrophila [26]. This suggests that the high levels of Nabs in this carp line could act as a first line of defence against microorganisms and could possibly be used as a selective marker for genetic breeding for disease resistance against this specific pathogen. Acknowledgements We thank the technical assistance of Ger De Vries Reilingh for assisting in the ELISA and also Bart Ducro for helping in the statistical proceeding of the data. This work was funded by the European Community’s Improving Human Potential Programme under contract HPRN-CT-2001-00214, PARITY, including a fellowship for Neli Kachamakova. References [1] Kaneko Y, Takashima Y, Xuaun X, Igarashi I, Nagasawa H, Mikami T, et al. Natural IgM antibodies in sera from various animals but not the cat kill Toxoplasma gondii by activating the classical complement pathway. Parasitology 2004;128:123e9. [2] Lammers A, Klomp MEV, Nieuwland MGB, Savelkoul HFJ, Parmentier HK. 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