C-reactive protein (CRP) has been shown to be an important part of acute phase reaction in domestic mammals. We investigated the presence of CRP in Slovenian sheep in order to obtain a better understanding of the distribution of CRP in ovine species. The reference sample comprised fifty-two clinically healthy female sheep (14 Solča breed and 38 Bovško breed, aged between six months and four years). Assay for CRP was carried out in blood serum using a turbidimetric immunoassay (TIA) with rabbit polyclonal antibodies against human CRP molecule. Values for CRP ranged between 0.000 and 13.600 mg/l, while concentration of CRP (overall mean±s.e.m.) was 6.280±0.429 mg/l. Sample reference limits (95% confidence interval) were between 0.086 and 12.474 mg/l. Mean CRP concentrations for both breeds were 5.235±0.429 for Bovško sheep and 9.115±0.647 mg/l for Solča sheep, respectively (P<0.001). It can be concluded that CRP serum levels represent the result of compromised metabolic and immunological activity. This study was thought to be helpful in distinguishing between normal and pathological serum values for CRP in sheep.C reaktivni protein (CRP) spada među važnije komponente akutne upalne reakcije u domaćih sisavaca. U ovom radu smo istraživali prisutnost CRP u slovenskih ovaca radi boljeg poznavanja njegove raspodjele. Kao referentni uzorak su poslužile 52 ovce (14 solčavske i 38 bovške pasmine) klinički zdrave, u dobi između šest mjeseci i četiri godine. Vrijednost CRP smo utvrđivali u krvnom serumu pomoću turbidimetrijskog imuno testa (TIA) s poliklonskim protutijelima kunića za humani CRP. Sve vrijednosti CRP su bile između 0,000 i 13,600 mg/l dok je prosječna koncentracija bila 6,280±0,429 mg/l. Referentne vrijednosti CRP (na razini 95% značajnosti) za uzorak smo ocijenili između 0,086 i 12,474 mg/l. Prosječne vrijednosti CRP za pojedine pasmine su bile 5,235±0,429 za bovšku i 9,115±0,647 mg/l za solčavsku ovcu (P<0,001). Ocjenjujemo da su vrijednosti CRP rezultat zajedničkog djelovanja metaboličkog i imunosnog sustava. Ovo istraživanje je vrijedno kao pomoć u razlikovanju fizioloških i patoloških vrijednosti CRP u ovaca

Igor Vojtic

Stanislav Krajnc

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VETERINARSKI ARHIV 70 (3), 151-157, 2000Determination of C-reactive protein byturbidimetric immunoassay (TIA) in sheepIgor Vojtic* and Stanislav KrajncBiotechnology Research Centre, Veterinary Hospital, Maribor, SloveniaVOJTIC, I., S. KRAJNC: Determination of C-reactive protein by turbidimetricimmunoassay (TIA) in sheep. Vet. arhiv 70, 151-157, 2000.ABSTRACTC-reactive protein (CRP) has been shown to be an important part of acute phasereaction in domestic mammals. We investigated the presence of CRP in Slovenian sheep inorder to obtain a better understanding of the distribution of CRP in ovine species. Thereference sample comprised fifty-two clinically healthy female sheep (14 Sol~a breed and 38Bov{ko breed, aged between six months and four years). Assay for CRP was carried out inblood serum using a turbidimetric immunoassay (TIA) with rabbit polyclonal antibodiesagainst human CRP molecule. Values for CRP ranged between 0.000 and 13.600 mg/l, whileconcentration of CRP (overall mean±s.e.m.) was 6.280±0.429 mg/l. Sample reference limits(95% confidence interval) were between 0.086 and 12.474 mg/l. Mean CRP concentrationsfor both breeds were 5.235±0.429 for Bov{ko sheep and 9.115±0.647 mg/l for Sol~a sheep,respectively (P<0.001). It can be concluded that CRP serum levels represent the result ofcompromised metabolic and immunological activity. This study was thought to be helpful indistinguishing between normal and pathological serum values for CRP in sheep.Key words: turbidimetric immunoassay, C-reactive protein, reference limits, sheepIntroductionThe acute phase reaction is a very old element of natural immunity.Besides non-immune factors, the natural killer cells, T- and B-lymphocytes, as well as some serum proteins, are able to recognizeantigene epitopes and activate the immune system. A sudden rise incytokines, especially IL-1, IL-6 and TNF alpha, is followed by productionof several acute phase proteins such as fibrinogen, haptoglobin, serum* Contact address:Doc. dr. Igor Vojtic, Biotechnology Research Centre, Maribor Veterinary Hospital, Šentiljska c. 109, SL-2000 Maribor, Slovenia. Phone: 386 62 228 3700; Fax: 386 62 228 3701; E-mail: igor.vojtic@siol.netISSN 0372-5480Printed in Croatia151amyloid, ceruloplasmin and C-reactive protein, the concentrations ofwhich increase up to 1,000-fold (reviewed by BERCZI et al., 1998).Seventy years ago, C-reactive protein (CRP) was the first acute phaseprotein determined in serum from humans with Pneumococci infection.Until now the chemical structure of CRP has been well recognized. TheCRP protein belongs to pentraxins (BOTTAZZI et al., 1997). Pentraxinsare a family of pentameric proteins, examined by electron microscopyforming typically pentameric discoid subunits (SHRIVE et al., 1996) with amolecular mass of 20,000 Daltons each. CRP was named according to itsin vitro ability to bind to the C-polysaccharide of Streptococci bacteria(BEAUFORT et al., 1997). The in vivo functions of CRP still remainunclear. According to in vitro experiments it can be concluded that thesefunctions include the ability to activate complement cascade by classicalpathway (ABERNATHY et al., 1996; WOLBINK et al., 1996). Otherfunctions assigned to CRP are modulation of activity of blood platelets(CHERYK et al., 1996), erythrocyte aggregation (WENG et al., 1996),opsonization of killed (but not live) bacteria (BEAUFORT et al., 1997), andopsonization of necrotic tissue and cellular debris (ECKERSALL andCONNER, 1988). All CRP's studied have the capacity for bindingphosphorylcholyn that is mediated through calcium ions (NORDIN et al.,1996; SHRIVE et al., 1996). However, some differences among domesticspecies are recognized. For example, unlike human CRP, which is notglycoprotein, canine CRP is glycolsylated on two of five pentamer subunits(ECKERSALL and CONNER, 1988). Human CRP agglutinates lecithinmicelles, while bovine CRP cannot because of its low affinity to thephosphorylcholine (MORIMATSU et al., 1991). Although MORIMATSU etal. (1991) expressed some reservations about the use of antibodies onhuman to bovine CRP, there is a strong similarity of amino acidscomposition and pentameric configuration for both pentraxins. Moreover,CHERYK et al. (1996) make the point that anti-human CRP antibodies arecross-reactive with bovine CRP.As recently reported (ZHANG et al., 1996), signal transducers andactivators of transcriptions (STAT3) family members are mediators of IL-6 induced CRP gene activation. There is only one gene in the highervertebrates that controls synthesis of CRP protein in the liver. In contrast,animal species whose immune system operates without immunoglobulinsproduce more than one CRP with similar functions but individual specifity(LIU et al., 1987). Hence, no polymorphism or deficiency state for CRP inhumans is known. The sequence homology of CRP gene organisationindicates that they arose from an ancestral gene that has been highly152 Vet. arhiv 70 (3), 151-157, 2000I. Vojtic and S. Krajnc: Determination of C-reactive proteinby turbidimetric immunoassay in sheepconserved throughout 500 million years of evolution (LIU et al., 1987;LUND and OLAFSEN, 1998).Studies in several animal species provide evidence that the C-reactiveprotein plays an important role in physiology research and diagnostics(BURTON et al., 1994; TOUSSAINT et al., 1995; ALAVA et al., 1997;ZIMMERMANN et al., 1998). Until now, the role of CRP in sheep has notbeen well elucidated. Hence, we investigated the presence of CRP in aSlovenian sheep population in order to obtain a better understanding ofthe distribution of CRP in ovine species.Materials and methodsExperimental animals comprised fifty-two female sheep, clinicallyhealthy, aged between six months and four years, and were drawn fromtwo of the dominant Slovenian sheep population. The first (N=38) wasthe Bov{ko breed, which is a small sheep from the Alpine region. Thesecond, (N=14) Sol~a breed, is the Slovenian breed most frequentlyreared in all of the Slovenian regions. During the experiment the sheepwere stall housed. Hay, grass silage and vitamin-mineral mix were offeredregularly.Blood samples were collected by jugular venipuncture into theVacuette system (Greiner, Austria) with a clot activator. Serum CRPconcentrations were determined by turbidimetric immunoassay (TIA)using liquid phase immunoprecipitation reaction with polyclonalantibodies obtained by rabbit immunisation with human CRP (Protiline,BioMerieux). The formation of insoluble antigen-antibody complexes wasmonitored at 340 nm by the increase in turbidity, which is proportional tothe concentration of CRP in the sample. Analytical performances of TIAconcerning human CRP were 2.5% and 4.0% for intra-series and inter-series variation, respectively, at medium levels of CRP. An automaticcentrifugal analyser (Hycell, France) was used in this experiment.With the aim of establishing the reference range for CRP, the datafrom the reference sample were fitted to a Normal (Gaussian)distribution. The goodness-of-fit was checked using Kolmogorov-Smirnovtest, according to the IFCC reference method and flow charts (JONES andPAYNE, 1997). To establish a reference interval we included the central95% fraction of the reference distribution (mean±2s.d.). The differencebetween groups was tested using Student's T-test.Vet. arhiv 70 (3), 151-157, 2000 153I. Vojtic and S. Krajnc: Determination of C-reactive proteinby turbidimetric immunoassay in sheepResultsThe overall mean±SEM concentration of CRP for experimentalsheep, irrespective of breed, was 6.280±0.429 mg/l. Minimal and maximalvalues measured were between 0.000 and 13.600 mg/l of serum,respectively. Data for both breeds indicates that it matches the patternanticipated to be drawn from a population with a normal distribution (P=0.227). The estimated lower and upper values for overall mean rangedbetween 5.480 and 7.142 mg/l of CRP at the 95 % confidence level. Thereference limits for CRP between 0.086 and 12.474 mg/l were at the samelevel.The results for a particular breed were substantially different. Themean CRP concentrations were 5.235±0.429 for Bov{ko sheep and9.115±0.647 mg/l for Sol~a sheep, respectively. The difference in themeans (t value= 4.794) of these two groups was significant (P<0.001).DiscussionThe present study confirms that ovine CRP cross-react withpolyclonal rabbit antibodies against human CRP. The results of thisexperiments also showed the potential (although non-specific) of TIA inelucidation of physiological changes and their possible relationship tobetween breed variations. The reference range was assessed on a sufficientnumber of animals according to IFCC flow chart (JONES and PAYNE,154 Vet. arhiv 70 (3), 151-157, 2000I. Vojtic and S. Krajnc: Determination of C-reactive proteinby turbidimetric immunoassay in sheepTable 1. Descriptive statistics and comparison of CRP values for sheepbreeds. Serum CRP concentrations were determined by turbidimetricimmunoassay (TIA) using liquid phase immunoprecipitationreaction with polyclonal antibodies obtained by rabbitimmunisation with human CRP (Protiline, BioMerieux)CRP mg/l Bov{ko sheep Sol~a sheepN 38 14Minimum 0.000 5.270Maximum 13.000 13.600Mean 5.235*** 9.115***SD 2.645 2.24195% CI for mean 4.366-6.104 7.717-10.513***P<0.001; CI=confidence interval1997). Thus, our results are useful for specific diagnostic procedures inanimals with symptoms of diseases.The differences observed between breeds were unexpected, due tostrong genetic homogeneity for CRP protein among species and withinspecies, also (LIU et al., 1987; LUND and OLAFSEN, 1998). Thus, thegenetic background of these two breeds is somewhat different. The wildancestor of Sol~a sheep is Ovis vignei var. arkal, and of Bov{ko sheep Ovisammon var. mussimon.It seems reasonable to postulate that some other reasons areresponsible for such a difference. Using artificial selection in sheepbreeding, the frequencies of particular genes were modulated, especiallyfrom an economical point of view. With every new generation the genepool is removed from its natural relations and from its biological optimumat the same time. In general, this deviation reflects itself through differentendocrinological, immunological and metabolic shifts, as suggested byELSASSER et al. (1997) and NONNECKE et al. (1997). Until now,selection-dependent changes concerning immunological fitness of thesheep population have not been documented. The different CRP levelsobserved can be attributed to the differences in terms of productivitybetween breeds. The Bov{ko sheep is a milk-purpose breed, whereas theSol~a sheep is a meat-wool purpose breed. In agreement with thishypothesis, MÜLLER et al. (1998) found a significant correlation betweenCRP and thyroxin (r=0.35) and CRP and cholesterol concentrations(r=0.52) in breeding bulls. If we advance this concept to ovine species itcan be concluded that CRP serum levels represents the result ofcompromised metabolic and immunological activity. Very similar effectshave previously been seen in dairy cows (MORIMATSU et al., 1991;ZIMMERMMAN et al., 1998).This study contributes to a better understanding of the distribution ofCRP in ovine species. The reference values presented here could beuseful in physiological research and diagnostics. The question that remainsis this: what if the CRP concentrations are elevated through disease orother tissue damage. To answer this, further research is needed.ReferencesABERNATHY, V. J., R. O. WEBSTER, T. E. DAHMS (1996): C-reactive protein inhibitsincreased pulmonary vascular permeability induced by FMPL in isolated rabbit lungs.Am. J. Physiol. 40, H507-H513.ALAVA, M. A., N. GONZALES-RAMON, P. HEEGAARD, S. GUZYLACK, M. J. M. TOUSSAINT, C.LIPPERHEIDE, F. MADEC, E. GRUYS, P. D. ECKERSALL, F. LAMPREAVE, A. PINEIRO(1997): Pig-MAP, porcine acute phase proteins and standardisation of assays inEurope. Comp. Haematol. Int. 7, 208-213.Vet. arhiv 70 (3), 151-157, 2000 155I. Vojtic and S. Krajnc: Determination of C-reactive proteinby turbidimetric immunoassay in sheepBEAUFORT, DE A. J., J. A. M. LANGERMANS, A. M. MATZE-VAN DER LANS, P. S. HIEMSTRA,J. M. VOSSEN, R. VAN FURTH (1997): Difference in binding of killed and liveStreptococcus pneumoniae serotypes by C-reactive protein. Scand. J. Immunol. 46, 597-600.BERCZI, I., D. A. CHOW, E. R. SABBADINI (1998): Neuroimmunoregulation and naturalimmunity. Dom. Anim. Endocrinol. 15, 273-281.BOTTAZZI, B., V. VOURET-CRAVIARI, A. BASTONE, L. DE GIOLA, C. MATTEUCCI, G. PERI, F.SPREAFICO, M. PAUSA, C. D'ETTORRE, E. GIANAZZA, A. TAGLIABUE, M. SALMONA, F.TEDESCO, M. INTRONA, A. MANTOVANI (1997): Multimer formation and ligandrecognition by the long pentraxin PTX3 - Similarities and differences with the shortpentraxins C-reactive protein and serum amyloid P component. J. Biol. Chem. 272,32817-32823.BURTON, S. A., D. J. HONOR, A. L. MACKENZIE, P. D. ECKERSAL, R. J. MARKHAM, B. S.HORNEY (1994): C-reactive protein concentration in dogs with inflammatoryleukograms. Am. J. Vet. Res. 55, 613-618CHERYK, L. A., M. A. HAYES, P. A. GENTRY (1996): Modulation of bovine platelet functionby C-reactive protein. Vet. Immunol. Immunopathol. 52, 27-36.ECKERSALL, P. D., J. G. CONNER (1988): Bovine and canine acute phase proteins. Vet. Res.Comm. 12, 169-178.ELSASSER, T. H., S. KAHL, N. C. STEELE, T. S. RUMSEY (1997): Nutritional modulation ofsomatotropic axis-cytokine relationships in cattle: a brief review. Comp. Biochem.Physiol. A116, 209-221.JONES, R. G., R. B. PAYNE (1997): Clinical Investigation and Statistics in LaboratoryMedicine. ABC Venture Publ. London.LIU, T. Y., C. SYIN, N. Y. NGUYEN, A. SUZUKI, R. A. BOYKINS, K. J. LEI, N. GOLDMAN(1987): Comparison of protein structure and genomic structure of human, rabbit, andLimulus C-reactive proteins: possible implications for function and evolution. J. ProteinChem. 6, 263-271LUND, V., J. A. OLAFSEN (1998): Comparative study of pentraxin-like proteins in differentfish species. Develop. Comp. Immunol. 22, 185-194.MORIMATSU, M., A. WATANABE, K. YOSHIMATSU, T. FUJINAGA, M. OKOBO, M. NAIKI (1991):Elevation of bovine serum C-reactive protein and serum amyloid P component levelsby lactation. J. Dairy Res. 58, 257-261.MÜLLER, U., T. DALLE, G. LEUTHOLD, R. STAUFENBIEL, M. KRÜGER, C. KRETZSCHMAR, T.SEIDLER, M. F. G. SCHMIDT (1998): Züchtungsbiologische Charakterisierung derBeziechnungen zwischen Stoffwechselindikatoren und immunokompetenten Merkmalenbei hungerbelasteten Milchrindbullen. Arch. Tierz. Dummerstorf 41, 359-370.NONNECKE, B. J., L. J. BURTON, M. E. KEHRLI (1997): Associations between function andcomposition of blood mononuclear leukocyte populations from Holstein bulls treatedwith daxamethasone. J. Dairy Sci. 80, 2403-2410.NORDIN, G., I. SAMUELSSON, B. ANDERSSON, J. BORJESON (1996): C-reactive protein - Thedifference between quantitation in serum and EDTA plasma. Scand. J. Clin. Lab.Investig. 56, 123-127.SHRIVE, A. K., G. M. T. CHEETHAM, D. HOLDEN, D. A. A. MYLES, W. G. TURNELL, J. E.VOLANAKIS, M. B. PEPYS, A. C. BLOOMER, T. J. GREENHOUGH (1996): Threedimensional structure of human C-reactive protein. Nature Structural Biol. 3, 346-354.TOUSSAINT, M. J. M., A. M. VANEDEREN, E. GRUYS (1995): Implication of clinical pathologyin assessment of animal health and in animal production and meat inspection. Comp.Haematol. Int. 5, 149-157.WENG, X. D., G. CLOUTIER, R. BEAULIEU, G. O. ROEDERER (1996): Influence of acute phaseproteins on erythocyte aggregation. Am. J. Physiol. 40, H2346-H2352.156 Vet. arhiv 70 (3), 151-157, 2000I. Vojtic and S. Krajnc: Determination of C-reactive proteinby turbidimetric immunoassay in sheepWOLBINK, G. J., M. C. BROUWER, S. BUYSMANN, I. J. M. TENBERGE, C. E. HACK (1996):CRP-mediated activation of complement in vivo - assessment by measuring circulatingcomplement-C-reactive protein complexes. J. Immunol. 157, 473-479.ZHANG, D. X., M. SUN, D. SAMOLS, I. KUSHNER (1996): STAT3 participates in transcriptionalactivation of the C-reactive protein gene by interleukin-6. J. Biol. Chem. 271, 9503-9509.ZIMMERMANN, S., A. NEUMANN, M. KRÜGER, M. FÜRL, K. ELZE (1998): Die Eignung einerklinischen Puerperalkontrolle sowie von Stoffwechsleparametern, unter besondererBerücksichtigung des C-reaktiven Proteins, als Kriterium für das Konzeptsionsgeschenin folgenden Reproduktionszyklus des Rindes. Züchtungskunde 70, 261-281.Received: 14 May 1999Accepted: 8 May 2000VOJTIC, I., S. KRAJNC: Odre|ivanje C reaktivnog proteina pomo}uturbidimetri~nog imuno testa (TIA) u ovaca. Vet. arhiv 70, 151-157, 2000SA@ETAKC reaktivni protein (CRP) spada me|u va`nije komponente akutne upalne reakcije udoma}ih sisavaca. U ovom radu smo istra`ivali prisutnost CRP u slovenskih ovaca radi boljegpoznavanja njegove raspodjele. Kao referentni uzorak su poslu`ile 52 ovce (14 sol~avske i 38bov{ke pasmine) klini~ki zdrave, u dobi izme|u šest mjeseci i ~etiri godine. Vrijednost CRPsmo utvr|ivali u krvnom serumu pomo}u turbidimetrijskog imuno testa (TIA) s poliklonskimprotutijelima kuni}a za humani CRP. Sve vrijednosti CRP su bile izme|u 0,000 i 13,600 mg/ldok je prosje~na koncentracija bila 6,280±0,429 mg/l. Referentne vrijednosti CRP (na razini95% zna~ajnosti) za uzorak smo ocijenili izme|u 0,086 i 12,474 mg/l. Prosje~ne vrijednostiCRP za pojedine pasmine su bile 5,235±0,429 za bov{ku i 9,115±0,647 mg/l za sol~avskuovcu (P<0,001). Ocjenjujemo da su vrijednosti CRP rezultat zajedni~kog djelovanjametaboli~kog i imunosnog sustava. Ovo istra`ivanje je vrijedno kao pomo} u razlikovanjufizioloških i patoloških vrijednosti CRP u ovaca.Klju~ne rije~i: turbidimetrijski imuno test, C reaktivni protein, referentne vrijednosti,ovcaVet. arhiv 70 (3), 151-157, 2000 157I. Vojtic and S. Krajnc: Determination of C-reactive proteinby turbidimetric immunoassay in sheep

Određivanje C reaktivnog proteina pomoću turbidimetričnog imuno testa (TIA) u ovaca.

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