Estimation of the genetic variation in complement activity of common carp (Cyprinus carpio L.)

Veterinary Immunology and Immunopalhology, Elsevier Science Publishers B.V., Amsterdam

37 ( 1993 ) 309-3 19

309

Estimation of the genetic variation in complement activity of common carp ( Cyprinus carpio L. ) G.F. Wiegertjes”,T. Yanoband W.B. van Muiswinkel” ‘Departtnen~ of EsperitnenIal Animal Morphology and Cell Biology, Agricultural University, PO Box 338. 6700 AH Wageningen, Nelherlands bLaboralory of Fisheries Chetnistry, Faculty OfAgriculture, Kyushu University, 6-1 O-l Hakoraki, Fukuoka 812. Japan (Accepted I4 October 1992)

ABSTRACT Wiegertjes, G.F., Yano, T. and van Muiswinkel, W.B., 1993. Estimation of the genetic variation in complement activity of common carp (Cyprinus carpio L.) Vet. Imtnunol. Itntnunopathol., 37: 309319. The complement status of hybrid, laboratory raised carp was determined by an in vitro approach ofthe alternate complement activity (ACHJo) and total haemolytic activity (CH,,), and by measurement of serum C3 levels. The lysis of target sheep red blood cells (RBC) in the haemolytic assay for CHso activity depended, amongst others, on the haemolysin concentration in the assay. Rocket electrophoresis showed a mean serum C3 concentration of 0.95 mg ml-‘. The variation for both ACHSO and CHSo haemolytic activity was approximately 30%. The degree of genetic determination of the parameters was investigated by estimation oftheir repeatabilities, which were relatively high for CH,o (0.71 ) and ACHSo activity (0.72), but lower for C3 levels (0.54). Correlations between ACHSo values and C3 levels were significant, but moderate (0.54-0.58). ABBREVIATIONS ACP, alternate complement pathway; CCP, classical complement pathway; EGTA, ethylene glycolbistetra-acetate; PBS, phosphate buffered saline; RBC, red blood cells; VBS, veronal buffered saline.

INTRODUCTION

In mammals, complement takes part in the opsonisation, killing and elimination of antigens such as bacteria. It can be activated through the classical complement pathway (CCP ), which is mostly antigen-antibody-complex dependent, and through the alternate complement pathway ( ACP). The latter Correspondence lo: G.F. Wiegertjes, Department of Experimental Animal Morphology Biology, Agricultural University, PO Box 338, 6700 AH Wageningen, Netherlands.

@ I993 Elsevier Science Publishers

B.V. All rights reserved 0 165-2427/93/$06.00

and Cell

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can be triggered without the need of immune-complexes by lipopolysaccharides of bacterial cell walls. The ACP and the CCP activate C3, the key component in both pathways, via C3 convertases. The Ca’+ dependency of Cl activation at the start of the CCP allows the in vitro separation of the ACP from the total haemolytic activity by the use of buffers with or without Ca’+ (Osler, 1976). In fish, haemolytic systems showing clear similarities to the mammalian complement system, have been described for the nurse shark (Jensen et al., 198 1 ), rainbow trout (Nonaka et al., 198 1) and brown trout (Ingram, 1987 ). So far, the haemolytic tests for carp, used sensitized rabbit (Gushing, 1945: Day et al., 1970) or sheep RBC (Yano et al., 1984) to assay total haemolytic activity, and chicken (Kaastrup and Koch, 1983) or rabbit RBC (Matsuyama et al., 1988) to assay haemolytic activity by the ACP. The genetic regulation of complement activity in fish remains largely unknown. Genetic polymorphism of the C3 component of rainbow trout complement has recently been described using electrophoresis; three distinctive bands were found within the population studied (Bjerring Jensen and Koch, 199 1). Reed et al. ( 1990), reported genetic differences in complement activity, measured with a radial diffusion method, between families of rainbow trout. Heritability estimates varied from medium to relatively high, indicating a genetic influence. With this in mind, Fjalestad ( 1990) proposed to use complement haemolytic activity as an indirect marker to select for more disease resistant salmonids. Only recently, Reed et al. ( 1992) confirmed their observations in atlantic salmon. Mallard et al. ( 1989), after studying the influence of major histocompatibility genes on the haemolytic activity of miniature swine serum, suggested that monitoring critical complement components like C3 could be a more effective way of assessing the complement status. In the present study, complement status was determined by an in vitro approach of the ACHS,, the total haemolytic activity CH5(), and by electrophoretie measurement of C3 in serum. The variation in haemolytic activity was studied in a hybrid population of common carp. The degrees of genetic determination of these parameters were examined by estimation of repeatability, investigating a genetic influence on complement activity in carp. MATERIALS

AND METHODS

Fish Common carp (Cyprinus carpio L. ) were raised and kept at 23 -t 2°C in recirculation systems of filtered tap water, and were fed pelleted food (Trouvit K30, Trouw, Putten, Netherlands) at a daily ration of 1.5% body weight. Two times 50 carp of the same hybrid cross, between a single female parent of Israelian origin (A4.1) and a male parent of Dutch origin (W 1) were used

to study variation in haemolytic activity. Measurements were done at 12 and 15 months of age (n= 50, mean weight 247 g; n= 50, mean weight 346 g, respectively). Twenty carp of a cross between a single male and a single female of Polish (R3) origin, were individually marked for estimation of repeatabilities. Fish were 13 and 14 months old with a mean weight of 285 g at 13 months of age. Three fish were used for the production of haemolysin. They were 3.5 years old and weighed approximately 2 kg. Complement

and red blood cells

Blood was taken by caudal venipuncture from TMS (Tricdine Methane Sulfonate, Crescent Res. Chem., Phoenix, AZ) anaesthetized fish (0.02% w/ v ), and allowed to clot at room temperature for 1 h. The clot was removed by centrifugation and serum complement was immediately frozen at - 80°C. Haemolysin (carp antiserum against sheep RBC) was produced by injections (i.m.) of 10” RBC in PBS (0.15M phosphate-buffered saline, pH 7.2). Four weeks after a priming, a booster injection ( 10”) was given. Two weeks later, fish were bled for haemolysin. Control fish were injected with PBS only. Complement activity of haemolysin was inactivated by heating at 50°C for 30 min. Haemolysin was stored at - 20°C. Sheep blood was stored at 4°C in Alsever’s solution ( 1 : 1) for 1 week before use. Heparinized rabbit blood was 2 days old at most. RBC were washed in VBS (veronal-buffered saline, pH 7.6; Mayer, I96 1 ) three times before use in a haemolytic assay and six times before injection as antigen for haemolysin production. Haemolvtic

assay

Rabbit RBC were used for determination of lysis by the ACP. Sheep RBC, sensitized (60 mitt, 37°C) with pooled and diluted ( 1:25) carp haemolysin of known haemagglutination titre (32-64), were used for determination of CH,,. Buffer for the ACP was VBS+-EGTA (VBS, 1OmM Mg’+, 1OmM EGTA (ethylene glycolbistetra-acetate), pH 7.6) and for determination of total haemolytic activity VBS+ + (VBS, 1OmM Mg’+, 1mM Ca’+, pH 7.6). Tests were done in round bottomed 96-well microtiter plates (Greiner, Alphen aan de Rijn, Netherlands). One hundred ,~l serum was two-fold serially diluted in the appropriate buffer as complement source, subsequently 50 ~1 0.5% (v/v) RBC in VBS were added. A 100% haemolysed reference, a blank; no serum added, a CP control sensitization of sheep RBC with non-immune pooled carp serum and a standard complement sample (serum pool) for correction of plate differences were included. The plate was shaken for 10 s, incubated for 30 min at 15 “C, shaken again and incubated for another 30 min at 15 ‘C. Plates were centrifuged (La-

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bofuge II, Heraeus Christ, Osterode am Harz, Germany) for 5 min at 500 g to spin down remaining RBC. The supematant in each well was carefully pipeted into flat bottomed 96-well microtiter plates (La Fontaine, Karlsruhe, Germany ). Haemolysis was read with a microtiter plate photospectrometer (Easy reader EAR-400, Groding, Salzburg, Austria) at 414 nm (reference wavelength 6 19 nm ). All titrations were done in duplicate. The complement dilution factor X was plotted in logarithmic form against the percentagehaemolysis Y using the Von Krogh ( 1916) equation as log X versus log Y/ ( 1- Y). The dilution corresponding with 50% haemolysis ml- ’ was expressedas CHSofor total haemolytic activity and asACHso for the ACP (Mayer, 1961; Platts-Mills and Ishizaka, 1974). Variation in haemolytic activity The phenotypic variation in ACHSOor CHSoactivity, determined by serial dilutions of complement in the haemolytic assay,wasestimated by measuring the complement activity of two groups of 50 animals of the A4.1 x W 1 hybrid cross, at 12 and 15 months of age.The distributions of the ACHso and CHjo values were tested for normality (Statistical Analysis Systems (SAS) Institute, 1990). Differences in mean ACHso or CHSObetweenthe two groups were analyzed with Students’ t-test (SAS Institute, 1990)) accepting a 5% error. The dependencyof the total haemolytic activity ( CHSo) on the haemolysin concentration in the assaywas studied by diluting the haemolysin ( 10,25, 50 and 1OO-fold). Serum C3 levels Serum C3 levels were determined by rocket electrophoresis (Axelsen and Bock, 1983) in an agarose gel containing monospecitic rabbit antiserum againstpurified carp C3 (Nakao et al., 1989). Electrophoresis was performed with 20 ml 1.5% (w/v) agarose(Type VII, Sigma Chemicals St. Louis, MO) in Gelman buffer, pH 8.8 (Gelman Science,Ann Arbor, MI) containing 0.25% (v/v) antiserum on 20 cmx6 cm glass plates. Serum samples were carbamylated with 2M KCNO ( I : 3) overnight at room temperature and diluted in PBS to 1: 9 final dilution. Three ~1duplicate samples were applied onto the gel in punched wells. Each glassplate contained serial dilutions of a reference serum sample with known C3 concentration to correct for plate differences. The length of the precipitation rockets after overnight electrophoresis (Vmax, 20 mA) and staining with CoomassieBrilliant Blue (CBB G250; BioRad Lab., Richmond, CA) was used to quantify the C3 levels.

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C.4RP

Repeatability estimates Repeatabilities, or intra-class correlations, were based on replicate measurements on the same individuals, and estimated asthe ratio of the betweenindividuals variance and total phenotypic variance (Falconer, 1989)) determined by analysis of variance (SAS Institute, 1990). The repeatability of ACHSo and CHso activity and of serum C3 levels was estimated by two replicate measurementson 20 individually marked carp (R3 line). Differences in mean ACHSO,CH50 or serum C3 levels between replicate measurementswere analyzed with Student’s t-test (SAS Institute, 1990), accepting a 5% error. Putative correlation(s) between ACH,,, CHjo and/or serum C3 levels, measured on these individuals, were considered significant when the probability was greaterthan 5% (SAS Institute, 1990). RESULTS

Variation in haemolvtic activity Figure 1 shows that the in vitro CHso haemolysis was dependent on the haemolysin concentration which was used for sensitization of the target sheep RBC in the haemolytic assay. The normal frequency distributions of the ACHjo and CH,(, values for both groups of 50 hybrid fish are visualized in Figs. 2(a) and 2(b), respectively. The two groups differed in age, but also in weight (data not shown). The mean (A) CHso values (Table 1) were not significantly different. An approx-

a

10 log

(dllutlon

32 factor

G-1 complement)

Fig. I. Relation bctwcen the total haemolytic activity and the haemolysin concentration used to scnsitizc target sheep RBC in an in vitro hacmolytic assay (dilution IOX ( + ). 25X (A ). 50X (0)or IOOX (0)).

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(a)

ib\

40

Fig. 3. Frequency and 1 ( n ) wcrc

80

120

distribution of the sanlc

160

200

CH50

I

of (a) carp lint

240

280

320

ml.

ACHso and (b) CH,,) hacmolytic activity. Groups (.A4. I x W I ) ngcd I Z and I5 months rcspccti\,cly.

imate variation in (A) CHso activity Table 1) was observed in both groups.

of 20-30%

(coefficient

I (0

of variation:

Serum C3 levels Serum C3 levels were easily calculated

from the length of the rockets after

)

COMPLEMENT

TABLE

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CARP

315

I

Mean hacmolytic diffcrcnt ages

ACHSo

and

CHSo

activity

oflish

of the same

carp

lint

(A4.

No.’

Age (months)

ACHs,,

41 49

I, I5

20.6 + 6.9 32.4+6.7

33.8 20.6

CHS,,

48 49

I? 15

121.7 f 38.8 146.1 k47.8

31.6 32.7

TABLE

activily ml-‘)

af :!

l’aramctcr

‘Number ‘Cocfticicnt

Hacmolytic ((A) CHSo

I x W I ), measured

CV’

oflish. of variation 1

Mean hacmolytic ACHS,, and CH,, activity, strum C3 lcvcls and rcpcatability on rhc same individuals (R3 lint) by sampling ar IWO occasions with I month I’aramctcr

No.’

Age

Hacmolytic

activity

(months)

((A)

ml-‘)

ACHso

70 I8

13 I4

CHS,,

20 I9 I8

I3 I4 13

I9

14

c3

‘Number

CH,,

cstimatcs. in between

C3-lcvcl (mgm-‘)

58.5 ?I 14.7 51.3& 13.8 270.6 355.9

measured

R’

0.72

+ 98.3 + 70.6

0.71 0.78

f 0.24

1.12+0.29

0.54

of fish.

‘Rcpcatability.

staining with CBB. The mean serum level at 13 months of age was 0.78 mg ml- ‘, for the second measurements at 14 months of age 1.12 mg ml- ’ (Table 2 ). The mean levels were not significantly different.

Repeatability estimates The repeatabilities for (A)CHSO activity and serum C3 levels, based on serial dilutions of complement and rocket immuno-electrophoresis, are presented in Table 2. Repeatability was high for both CHso (0.7 1) and ACHSo (0.72 ). Repeatability was lower (0.54) for serum C3 levels. No significant differences between mean ACHSO, CHso values or mean serum C3 levels of both replicate measurements were found. Correlations between ACH=,,, values and serum C3 levels were significant, but moderate (Fig. 3), with calculated correlation coefficients of 0.54 and 0.58 for the replicates at 13 and 14 months of age, respectively. No significant

316

C.F. WIEGERTJES 1.60

El-AL.

,

Fig. 3. Correlation between ACHSo and serum C3 levels. Rcplicatcs mcasurcd on samples from the same individuals (R3 lint).

I (0 ) and 2 (A ) wrc

correlations between ACP activity and total haemolytic activity, or between CHso and serum C3 levels, were found. DISCUSSION

The choice of target RBC is one of the parameters strongly influencing the in vitro haemolytic activity. Kaastrup and Koch ( 1983), for instance, using chicken RBC, were not able to differentiate clearly between an ACP and CCP in crucian carp. In this study we used rabbit RBC to demonstrate lysis by the ACP (Platts-Mills and Ishizaka, 1974; Matsuyama et al., 1988), and sheep RBC for measurement of total haemolytic activity (Mayer, 1961; Yano et al., 1984). Unlike Cushing ( 1945) and Day et al. ( 1970), who used carp natural antibodies, carp haemolysin was used to sensitize target RBC in our study. Lysis of these sensitized sheepRBC was shown to depend on the concentration of haemolysin used (Fig. 1). Rocket immuno-electrophoresis provided a fast and reliable method of measuring serum concentrations of complement factor C3. To our knowledge no other reports so far have mentioned serum C3 concentrations in fish. The mean C3 concentration of 0.95 mg ml-’ was comparable with human serum concentrations of approximately 1 mg ml- ’ (Osler, 1976). An extensive, normally distributed, phenotypic variation in haemolytic activity characterized the hybrid offspring. The environmental variance, contributing to the phenotypic variance, was limited by the use of laboratory animals with a common and highly standardized environment from hatch. Rijkers et al. ( 1980) previously reported non-responding carp as complement donor in a haemolytic plaque assay.Ingram ( 1987) reported non-re-

COMPLEMENT

ACTIVITY

OF COMMON

317

CRRP

sponding rainbow trout in complement assays,both authors indicating a variation in complement activity. A moderate but significant correlation between ACHso activity and serum C3 levels was observed, possibly indicating a direct quantitative relationship between C3, the key component in the complement cascade,and ACP activity (Fig. 3). The degreeof genetic determination of a parameter can be estimated by the repeatability, which sets an upper limit to the heritability. The number of measurements neededfor an accurate estimation dependson the repeatability; the higher the repeatability, the lower the gain from multiple measurements on the same individual (Falconer, 1989). Relatively high degreesof genetic determination were found for both lysis by the ACP as for total haemolytic activity (Table 2). A slightly lower repeatability was found for serum C3 levels. These high values not only indicate that merely a moderate gain in accuracy would have been reachedwith more than two measurements, but also that ACHsOand CH50 activity may be suitable parameters for selection. A similar conclusion was drawn by R0ed et al. ( 1990, 1992)) who measured the haemolytic activity of salmonid fish. We feel that the observed variation and estimated degreeof genetic determination of haemolytic activity justify a future investigation of the individual’s genetic determination for high or low haemolytic responsiveness,following an approachaccordingto Biozzi (Biozzi et al., 1979). Such individuals could subsequentlybe cloned by gynogenetictechniques (Komen et al., 1991) to obtain homozygous lines of carp. Previous observations (Wiegertjes et al., 1991) led us to conclude that the hybrid cross between carp of Polish (R3) and Hungarian (R8) background constitutes a genetic basis suitable for selection of such individuals. The haemolytic activity of R3 x R8 individuals is currently under investigation. ACKNOWLEDGEMENTS

The authors wish to thank S. Gamage and J.M. Karczewski for their technical support, S.H. Leenstra and W. Heijmen for taking excellent care of the animal husbandry, J.W.M. Haas and M.G.B. Nieuwland for the RBC supply and the Fish Culture Exp. Station Golysz, Polish Academy of Sciencesand the Fish and Aquaculture ResearchStation-Dor, Israel for providing the different carp lines. We also wish to thank Dr. R.J.M. Stet for valuable suggestions concerning the manuscript. REFERENCES Axelsen. N.H. and Bock. E.. 1983. Electroimmunoassay Stand. J. Immunol.. 17: 103-106.

(Rocket

Immunoclectrophoresis).

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ET AL.

Biozzi. G., Mouton. D.. Sant’Anna, 0.4.. Passes, H.C.. Gcnnari. M., Reis, M.H.. Fcrrcira. V.C..4.. Hcumann, A.M., Bouthillicr. Y.. Ibancz. GM.. Stiffcl. C. and Siqucira. M.. 1979. Gcnctics of immunorcsponsivencss to natural antigens in the mouse. Curr. Top. Microbial. Immunol., 85: 3 I-98. Bjerring Jensen, L. and Koch, C.. I99 I. Genetic polymorphism of component C3 of rainbow trout (Ol,c.orh!,,fc.hf,s ,rr,t,lii.ss) complcmcnt. Fish Shellfish Immunol.. I: 237-247. Cushing. Jr.. J.E., 1945. A comparative study of complcmcnt I. The spccilic inactivation of the components. J. Immunol., 50: 6 l-74. Day, N.K.. Good, R.A.. Finstad. J.. Johannscn. R.. Pickering. R.J. and Gcwurz, H.. 1970. Intcractions between endotoxic lipopolysaccharidcs and the complcmcnt system in the scra of lower vertebrates. Proc. Sot. Exp. Biol. Med., 133: 1397-140 I. Falconer. D.S., 1989. Introduction to quantitative gcnctics. 3rd Edn., Longman Scicntitic Tcchnical, New York, 438 pp. Fjalestad, K.T., 1990. Possibilities to include disease rcsistancc in the Norwegian breeding programme for salmonids. In: Proceedings of the Fourth Congress of Gcnctics Applied to Livcstock Production XVI, 23-27 July 1990. Edinburgh, Joyce Darling. Pcnicuik, pp. 461-464. Ingram. G.A.. 1987. Haemolytic activity in the serum of brown trout, SLI/IIIO o’I~(~cI.J. Fish Biol.. 31A: 9-17. Jensen, J.A., Festa. E., Smith, D.S. and Cayer, M., I98 I. The complement system of the nurse shark: Hemolytic and comparative characteristics. Science. 2 14: 566-569. Kaastrup. P. and Koch, C.. 1983. Complement in the carp fish. Dcv. Camp. Immunol.. 7: 7S I782. Komen. J.. Bongers, A.B.J.. Richter, C.J.J., van Muiswinkel. W.B. and Huisman. E.A., 199 I. Gynogenesis in common carp ( CJ~~~~~~II~~ cu~l~ic~ L.) II. The production of homozygous gynogenetic clones and FI hybrids. Aquaculture. 92: I27- 142. Mallard, B.A., Wilkie. B.N. and Kennedy, B.W.. 1989. lnllucncc of major histocompatibility gents on serum hemolytic complement activity in miniature swine. Am. J. Vet. Res.. 50: 359-363. Matsuyama. H.. Tanaka, K., Nakao, M. and Yano. T.. 1988. Characterization of the alternative complement pathway of carp. Dev. Comp. Immunol.. 12: 403-408. Mayer, M.M., 1961. Complement and complement fixation. In: E.A. Kabat and M.M. Mayer (Editors), Experimental Immunochemistry. Charles C. Thomas, Springfield, IL, pp. I33240. Nakao, M., Yano, T., Matsuyama, H. and Uemura, T., 1989. Isolation of the third component of complement (C3) from carp serum. Nippon Suisan Gakkaishi, 55: 202 I-3037. Nonaka, M., Yamaguchi, N.. Natsuume-Sakai, S. and Takahashi, M.. I98 I. The complcmcnt system of rainbow trout (Sulruo gai&oi) I. Identification of the serum lytic system homologous to mammalian complement. J. Immunol., 126: I489- 1494. Osler, A.G., 1976. Complement. Mechanisms and functions. Prentice-Hall. Englewood Cliffs, N.J., I95 pp. Platts-Mills, T.A.E. and Ishizaka, K., 1974. Activation of the alternate pathway of human complement by rabbit cells. J. Immunol., 113: 348-358. Rijkers. G.T., Frederix-Walters, E.M.H. and van Muiswinkel, W.B., 1980. The haemolytic plaque assay in carp (Cjlprinus carpio). J. Immunol. Methods, 33: 79-86. 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-I 17. Reed, K.H., Fjalestad K.. Larsen, H.J. and Midthjel, L.. 1992. Genetic variation in haemolytic activity in Atlantic salmon (Su/r~~osu/u~ L.). J. Fish Biol., 40: 739-750. Statistical Analysis Systems Institute, 1990. SAS User’s Guide: Statistics. Version 6, 4th Edition, Cary, NC. Von Krogh, M., 1916. Colloidal chemistry and immunology. J. Infect. Dis., 19: 452-477.

C‘OMI'LE~lENTr\CTIVITY01:COMMON

Ci\Kl'

Wicgcrtjcs. G.F.. Stct. R.J.M. and van Muiswinkcl. high and low rcspondcr clones of common carp nol.. 15: Suppl. I. S87. Yano. T.. Ando. H. and Nakao. M., 1984. Optimum plcmcnt titer of carp and seasonal variation of 91-101.

319 W.B.. 1991. Gcnctic basis for selection of (C~7~Yr1tr.sLYI).[IIOL.). Dcv. Comp. Irnmuconditions for the assay of hcmolytic comthe titers. J. Fat. Agric. Kyushu Univ.. 29: