Gloor, Marianne

Gottstein, Bruno

Hässig, Michael

Maley, Stephen

Sager, Heinz

Tenter, Astrid

Bern Open Repository and Information System (BORIS)

ORIGINAL PAPERImmunodiagnosis of primary Toxoplasma gondii infection in sheepby the use of a P30 IgG avidity ELISAReceived: 16 June 2003 / Accepted: 1 July 2003 / Published online: 16 August 2003 Springer-Verlag 2003Abstract Toxoplasma gondii is a protozoan parasitewith a worldwide distribution. In both sheep and hu-mans, if the parasite is encountered during pregnancy,fetal infection and abortion can occur. Therefore,Toxoplasma infection in sheep has a major economicimpact upon sheep farming. Clinically, there is a need todistinguish recent (acute) infections from longstanding(chronic) infections. However, current serological tech-niques, such as detection of anti-T. gondii IgG, cannotdiscriminate between acute and chronic infections.Increasing immunoglobulin avidity is a good determin-ing factor of how recent an infection is. In this study, wedescribe the application and validation of a T. gondiiIgG avidity ELISA, based on the use of an aﬃnity-puriﬁed, native T. gondii P30 antigen. The assay wasused to examine sera from eight sheep experimentallyinfected with T. gondii and found that all seroconvertedwithin 21 days post-infection (p.i.), beginning withavidities that were initially low but that increased overtime, with all sheep reaching high IgG avidity within10 weeks p.i. In addition, sera from clinically healthybut T. gondii-seropositive lambs and ewes and seropos-itive ewes with a history of abortion were also subjectedto a preliminary serological investigation. High IgGavidities were found in 80% of the seropositive lambs, in90% of the clinically healthy ewes and in 97% of theewes with abortion problems. These ﬁndings indicatethat the animals had most likely contacted the parasite alonger time ago.IntroductionToxoplasma gondii is a protozoan parasite that can in-fect many warm-blooded animals, including sheep andhumans. The parasite has a worldwide distribution andin most cases infection does not cause severe illness.However, severe clinical disease can result when a pri-mary infection is acquired during pregnancy or when thehost immune response is compromised, such as withtransplant patients undergoing immunosuppressivetherapies or those infected with the human immunode-ﬁciency virus (Joynson and Wreghitt 2001).In most cases, immunocompetent individuals areprotected by both cell-mediated and humoral immunemechanisms. The production of speciﬁc antibodies mayhelp protect the host from subsequent re-infection.In sheep, current serological assays are used to detectT. gondii-speciﬁc antibodies after abortion. However,they do not always indicate whether the infection isacute or chronic (Hill and Dubey 2002), although theydo provide valuable epidemiological information (See-feldt et al. 1989; Wastling et al. 1995; Werre et al. 2002).Recent work investigated the maturation of speciﬁcantibodies to determine when infection was acquired.Jenum and colleagues evaluated a T. gondii-speciﬁc IgGavidity enzyme immunoassay (EIA) and demonstratedits usefulness in obstetrics (Jenum et al. 1997); and anIgG avidity ELISA has been used in serological studiesof neosporosis in cattle (Bjo¨rkman et al. 1999; Sageret al. 2003). However, limited information is availableconcerning the serodiagnosis of acute toxoplasmosis insheep. The T. gondii antigen used in the present studywas P30 (also called SAG1), a dominant membraneprotein of T. gondii (Lekutis et al. 2001), which has beenused in the serodiagnosis of congenital toxoplasmosis inhumans (Aubert et al. 2000; Villavedra et al. 2001), dogs(Silva et al. 2002) and camels (Abu-Zeid 2002). In theParasitol Res (2003) 91: 171–174DOI 10.1007/s00436-003-0964-9Heinz Sager Æ Marianne Gloor Æ Astrid TenterStephen Maley Æ Michael Ha¨ssig Æ Bruno GottsteinH. Sager Æ M. Gloor Æ B. Gottstein (&)Institute of Parasitology, University of Berne,La¨nggass-Strasse 122, 3012 Berne, SwitzerlandE-mail: bruno.gottstein@ipa.unibe.chFax: +41-31-6312622A. TenterInstitute of Parasitology,Tiera¨rztliche Hochschule, Hanover, GermanyS. MaleyMoredun Research Institute, Edinburgh, UKM. Ha¨ssigSection for Herd Health, Department for VeterinaryReproduction, University of Zu¨rich, Zu¨rich, Switzerlandpresent study, the development and ﬁrst validation stepsof a P30 IgG avidity ELISA for sheep is described.Materials and methodsA commercially available native, aﬃnity-puriﬁed P30 antigen(Z.I.SR2B, Avrille, France) was compared with a soluble somaticantigen ELISA (SA-ELISA) for its potential use in sheep. TheToxoplasma gondii SA was obtained from an in vitro-cultivated RHstrain, as described for Neospora caninum by Sager et al. (2003).Additionally, a traditional endozoite antigen (TEA) and a re-combinant H11 antigen were comparatively assessed in the sameway. The preparation of the SA, the coating of the ELISA platesand all subsequent test steps were done as described by Gottsteinet al. (1998) and Sager et al. (2003).For comparing the conventional ELISAs with diﬀerent anti-gens, 92 sera were taken from randomly selected healthy sheep atslaughter (Wyss et al. 2000). The measured absorption values wereexpressed as percentages of a positive control (antibody units; AU)and the cut-oﬀ was calculated from negative controls, as deﬁned bythe 3-fold standard deviation of a cluster of negative sera. Samplesabove the cut-oﬀ were considered as positive and those below asnegative. The SA-ELISA was used as the ‘‘gold standard’’ (pro-viding reference values) and the results obtained were comparedwith those of the three other antigens (P30, TEA, H11).For the IgG avidity ELISA, only the P30 antigen was used. Foreach serum, two 4-fold dilution series (starting at a dilution of 1:25)were loaded into the appropriate plate wells. Washing was per-formed with phosphate-buﬀered saline containing 0.3% Tween 20(PBS/Tween) and with PBS/Tween containing 6 M urea for thetwo dilution series, respectively. The detailed avidity ELISA pro-cedure was as described by Sager et al. (2003). The calculation ofavidity (expressed as a percentage) was done according to Jenumet al. (1997). Four ewes were experimentally inoculated per os with2,000 sporulated M3 T. gondii oocysts; and serum samples weretaken at weekly intervals (week 0, 1, 2, 3, 4, 6, 8, 12post-infection;p.i.) throughout pregnancy for the primary test validation. Anotherfour ewes were tested serologically, either at 1, 4, 6, 8 and 12 weeksp.i. (n=2), or at 2, 4, 7, 10, 12 and 14 weeks p.i. (n=2). Addi-tionally, sera from seropositive but healthy ewes (n=41) and lambs(n=35) at slaughter and T. gondii-positive sera from sheep origi-nating from a herd with an abortion history (n=114) were tested inthe P30 IgG avidity ELISA.ResultsSerological analysis of 92 healthy sheep at slaughter bydiﬀerent Toxoplasma gondii ELISAs revealed a highcorrelation of more than 95% between SA and P30(Fig. 1) and between SA and TEA, respectively (datanot shown). The soluble SA antigen was used as the‘‘gold standard’’ for calculating sensitivity and speciﬁc-ity. Both P30 and TEA antigens exhibited a relativesensitivity of 96% and a relative speciﬁcity of 100%. Incontrast, the corresponding sensitivity of the H11 ELI-SA was only 34% and the speciﬁcity 89% (Table 1).With the anti-T. gondii P30 IgG avidity ELISA, allexperimentally infected ewes seroconverted within3 weeks p.i. IgG avidities were determined 2 weeks p.i.and a time-dependent increase was observed. At10 weeks p.i., avidity values were above 35% in allanimals (Fig. 2). Based on these results, avidities up to25% were classiﬁed as low, those between 26% and 35%as intermediate and those above 35% as high.Fig. 1 Comparative dot-plot of values obtained with 92 sheep seratested in the Toxoplasma gondii SA- and P30-ELISA (see Materialsand methods). The values are expressed as antibody units (AU).The dashed lines indicate the negative/positive thresholds deter-mined for each antigenTable 1 Comparison of results from three diﬀerent Toxoplasmagondii ELISAs (see Materials and methods) with sera from92 clinically healthy sheep, sampled at slaughterSomatic antigen ELISAPositive NegativeP30-ELISA Positive 54 0Negative 2 36TEA-ELISA Positive 54 0Negative 2 36H11-ELISA Positive 19 4Negative 37 32Fig. 2 Avidities obtained from eight sheep experimentally infectedwith T. gondii. The dashed lines mark the area borders of low (up to25%), intermediate (26–35%) and high avidity (more than 35%).dpi Days post-infection172When naturally infected lambs and sheep wereinvestigated using the avidity ELISA, it was found that90% of sera from adult sheep (without any abortionhistory) had high avidities, as did 80% of the sera fromseropositive lambs (Table 2). The diﬀerence, however,was not signiﬁcant, as determined by the v2 test(P=0.38). Sheep from a farm with an abortion historyshowed a high avidity in 97.4% of animals. The v2 testdid not reveal a signiﬁcant diﬀerence, in comparison withthe healthy ewes at slaughter (P=0.07). Additionally, aStudent¢s t-test was performed on the avidities of sheepwith observed abortions (n=11) and clinically healthyewes (n=103) from the ﬂock with an abortion history.No signiﬁcant diﬀerence could be found (P=0.49).DiscussionThe aﬃnity-puriﬁed, native Toxoplasma gondii P30antigen proved to be suitable as a diagnostic assay for thepresence of speciﬁc anti-T. gondii antibodies when com-pared with a reference SA-ELISA, as demonstrated by ahigh correlation of their respective results. Positive/neg-ative discrimination appeared to be more distinct withthe P30 ELISA. The P30 T. gondii IgG avidity ELISAwas subsequently validated with sera from experimen-tally infected sheep. Within the ﬁrst 4 weeks p.i., most ofthe sera remained below 35% avidity, whereas at10 weeks p.i., all sera showed avidities >35%. The cutoﬀline discriminating between higher and lower aviditieswas therefore positioned at this value.The P30 IgG avidity ELISA can be used to discrim-inate between acute and chronic T. gondii infection insheep. Thus, naturally infected healthy ewes at slaughtershowed high avidities in 90% of cases. As 80% of theseropositive lambs had high IgG avidities, we mayconclude that they were exposed to the parasite veryearly in life (Duncanson et al. 2001). The animals orig-inating from a ﬂock with an abortion problem showed ahigh seroprevalence (114 out of 117 were seropositive;i.e. 97%). Although this is indicative of a high exposureof the animals to T. gondii oocysts, no signiﬁcant dif-ference in the distribution of avidity could be observedwhen compared with randomly selected seropositiveewes. This may be due to the fact that primary exposureof the ﬂock to the parasite occurred years before, as theblood samples used in this study came from a ﬂockwhere the ﬁrst abortions occurred 4 years previously(M. Ha¨ssig, personal communication). However,abortions persisted in this ﬂock and continued afterblood sampling. The fact that T. gondii-seroposive eweswith high avidity underwent subsequent abortions indi-cates that the parasite was most likely not the causativeagent. Finally, sheep living persistently in high endemicareas should acquire the infection before their ﬁrstpregnancy and should thus be immune at conception.It is important to mention that the presented ﬁelddata are only preliminary, but they give a ﬁrst impres-sion about the suitability of such a test system for rou-tine diagnostics. There is evidence that the avidityELISA is able to discriminate between acute and chronicinfection. Once avidity has matured and reaches a highvalue (chronic stage), we can no longer obtain furtherinformation about the previous time-point of infection.In other words, the IgG avidity ELISA is very useful as adiagnostic tool over a relatively short time. We willtherefore focus our further investigations on animalsthat have recently aborted, to further study antibodymaturation at this important time, using either naturallyor experimentally infected ewes.Acknowledgements This work was supported by the Swiss FederalVeterinary Oﬃce (grant 1.01.02) and BBW grant C01.0122 withinthe framework of COST 854 ACTION. We are grateful to UrsulaMa¨usli for her technical assistance in the laboratory.ReferencesAbu-Zeid YA (2002) Protein G ELISA for detection of antibodiesagainst Toxoplasma SAG1 in dromedaries. 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VetParasitol 112:1–10Table 2 Anti-P30 avidities determined in sheep initially seroposi-tive in a T. gondii somatic antigen ELISAAvidity ‘‘Normal’’ewesLambs Ewes withabortion historyLow 1 3 2Intermediate 3 4 1High 37 28 111Total 41 35 114173Seefeldt SL, Kirkbride CA, Dubey JP (1989) Comparison of en-zyme-linked immunosorbent assay, indirect ﬂuorescent anti-body test, and direct agglutination test for detectingToxoplasma gondii antibodies in naturally aborted ovine fe-tuses. J Vet Diagn Invest 1:124–127Silva NM, Lourenc¸o EV, Silva DAO, Mineo JR (2002) Optimi-sation of cut-oﬀ titres in Toxoplasma gondii speciﬁc ELISA andIFAT in dog sera using immunoreactivity to SAG-1 antigen asa molecular marker of infection. Vet J 163:94–98Villavedra M, Battistoni JJ, Rossi S, Carol H, Nieto A (2001)Avidity analysis of the human immune response to a chitinbinding protein of Toxoplasma gondii. 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Immunodiagnosis of primary Toxoplasma gondii infection in sheep by the use of a P30 IgG avidity ELISA

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Immunodiagnosis of primary Toxoplasma gondii infection in sheep by the use of a P30 IgG avidity ELISA

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