Veterinary Parasitology 140 (2006) 273–280 www.elsevier.com/locate/vetpar
Neospora caninum IgG avidity tests: An interlaboratory comparison C. Bjo¨rkman a,*, G. Alvarez-Garcia b, F.J. Conraths e, J.G. Mattsson c, L.M. Ortega-Mora b, H. Sager d, G. Schares e a
Department of Clinical Sciences, Swedish University of Agricultural Sciences, P.O. Box 7054, SE-750 07 Uppsala, Sweden Universidad Complutense de Madrid, Departamento de Sanidad Animal, Facultad de Veterinaria, E-28040 Madrid, Spain c Department of Parasitology (SWEPAR), National Veterinary Institute and Swedish University of Agricultural Sciences, SE-751 89 Uppsala, Sweden d Institute of Parasitology, University of Berne, La¨nggass-Strasse 122, CH-3012 Berne, Switzerland e Institute of Epidemiology, Friedrich-Loeffler-Institut, Seestrasse 55, D-16868 Wusterhausen, Germany
b
Received 21 December 2005; received in revised form 11 April 2006; accepted 27 April 2006
Abstract Avidity tests can be used to discriminate between cattle that are acutely and chronically infected with the intracellular parasite Neospora caninum. The aim of this study was to compare the IgG avidity ELISA tests being used in four European laboratories. A coded panel of 200 bovine sera from well documented naturally and experimentally N. caninum infected animals were analysed at the participating laboratories by their respective assay systems and laboratory protocols. Comparing the numeric test results, the concordance correlation coefficients were between 0.479 and 0.776. The laboratories categorize the avidity results into the classes ‘‘low’’ and ‘‘high’’ which are considered indicative of recent and chronic infection, respectively. Three laboratories also use an ‘‘intermediate’’ class. When the categorized data were analysed by Kappa statistics there was moderate to substantial agreements between the laboratories. There was an overall better agreement for dichotomized results than when an intermediate class was also used. Taken together, this first ring test for N. caninum IgG avidity assays showed a moderate agreement between the assays used by the different laboratories to estimate the IgG avidity. Our experience suggests that avidity tests are sometimes less robust than conventional ELISAs. Therefore, it is essential that they are carefully standardised and their performance continuously evaluated. # 2006 Elsevier B.V. All rights reserved. Keywords: IgG avidity; Neospora caninum; Interlaboratory; Cattle; Antibody; ELISA
1. Introduction
* Corresponding author. Tel.: +46 18671778; fax: +46 18673545. E-mail address:
[email protected] (C. Bjo¨rkman).
The intracellular parasite Neospora caninum is an important cause of bovine abortion world-wide (Dubey, 2003). Abortion outbreaks in cattle herds
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have been connected with recent infection in the pregnant heifers and cows, whereas endemic abortion patterns are thought to be consistent with presence of chronically infected dams (Thurmond et al., 1997; McAllister et al., 2000; Schares et al., 2002; Dubey, 2003). Demonstration of specific antibodies in serum from an animal is indicative of N. caninum infection (Bjo¨rkman and Uggla, 1999; Dubey, 2003). However, because the antibody levels fluctuate during pregnancy in persistently infected animals (Stenlund et al., 1999), antibody levels or titres cannot be used to discriminate between acutely and chronically infected animals. For this purpose avidity ELISA tests, based on the fact that the first antibodies synthesized after an antigenic challenge have lower affinity for the antigen than those produced later, can be used. The first N. caninum IgG avidity test utilised membrane proteins incorporated into iscoms as antigen (Bjo¨rkman et al., 1999). It has later been accompanied by tests based on tachyzoite extracts (Maley et al., 2001; Sager et al., 2003; Aguado-Martinez et al., 2005) and the N. caninum membrane protein NcSRS2 (p38) (Schares et al., 2002) and an IgG avidity Western blot assay (AguadoMartinez et al., 2005). Avidity testing has proved a powerful tool for epidemiological investigations on bovine Neospora infection and related abortion problems (Dijkstra et al., 2002; Schares et al., 2002; Sager et al., 2003; Bjo¨rkman et al., 2005; Fro¨ssling et al., 2005). Although N. caninum avidity testing has been used for some years, no evaluation of the agreement between the different avidity assays has been done so far. This study was part of a larger European research project and aimed to compare the various N. caninum IgG avidity tests being used in our laboratories.
and aliquots were sent to the other participating laboratories. 2.2. Sera (a) One hundred and fifty two serum samples came from 11 naturally N. caninum infected dairy herds in Germany, The Netherlands and Sweden. Five of the herds had recently before sampling experienced epidemic abortions whereas five had had endemic abortion problems. In the remaining herd several of the animals had seroconverted during 1 year but no detrimental effects on reproduction were observed (Dijkstra et al., 2002). Initially, all sera collected in these herds were sent to SLU/ SVA Uppsala where they were analysed by IgG avidity iscom ELISA (Bjo¨rkman et al., 1999, 2003). Sera to be included in the study were selected to cover the whole range of possible avidity values. For two of the herds with epidemic abortion (Herd 3 and Herd 4 in Schares et al., 2002) more detailed information was available. Fourteen of the sera were collected from dams that recently aborted and 22 from dams without abortion. The animals had been sampled 17–31 days after the epidemic abortion started on these farms. (b) Forty-eight serum samples came from six experimentally N. caninum infected pregnant cows. Three of the cows had been given 600 N. caninum oocysts (Nc-Liv) orally at 70 days gestation (Trees et al., 2002) whereas three cows had been subcutaneously injected with 5 108 tachyzoites of the Nc-1 isolate 140 days into gestation (Innes et al., 2001). Samples collected from all cows before inoculation and 2, 4, 6, 8, 12, 16 and 20 weeks after inoculation were included in the avidity comparison.
2. Materials and methods 2.3. Avidity tests 2.1. Study design A coded panel of 200 bovine sera from well documented naturally and experimentally N. caninum infected cattle were tested in four laboratories (FLI Wusterhausen, UCM Madrid, IPB Berne and SLU/ SVA Uppsala) for IgG avidity by their respective assay systems. The sera were coded by SLU/SVA Uppsala
The samples were analysed at the participating laboratories using their respective assay systems and laboratory protocols (Table 1). The laboratories categorized the avidity results into the classes ‘‘low’’ and ‘‘high’’ which are considered indicative of recent and chronic infection, respectively. Three of the laboratories also used an ‘‘intermediate’’ class.
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Table 1 N. caninum IgG avidity tests used by the four participating different laboratories Laboratory
Antigen
Reference
Classification
FLI Wusterhausen
NcSRS2 (p38) (inhouse preparation)
Schares et al. (2002)
Low < 55 High 55
SLU/SVA Uppsala
Iscom antigen (inhouse preparation)
Bjo¨rkman et al. (1999, 2003)
Low < 35 Intermediate 35–50 High > 50
UCM Madrid
Tachyzoite sonicate lysate (CivtestTM Bovis Neospora Hipra)
Sager et al. (2003), Aguado-Martinez et al. (2005)
Low < 25 Intermediate 25–40 High > 40
IPB Berne
Tachyzoite sonicate lysate (inhouse preparation)
Gottstein et al. (1998), Sager et al. (2003)
Low 35
2.4. Data analysis The numeric test results were analysed for test agreement by calculation of concordance correlation coefficient (Lin, 1989), and by Bland and Altman’s statistics for limits-of-agreement (Bland and Altman, 1986). Categorical data were analysed by Kappa statistics using either 3 3 (not including FLI Wusterhausen) or 2 2 tables. Before the latter were applied, the intermediate results reported by three of the laboratories were reclassified as either low or high. x-Values of 0.21–0.40, 0.41–0.60 and 0.61– 0.80 indicated fair, moderate and substantial agreement, respectively (Thrusfield, 1997). The data analysis was performed using Stata Statistical Software release 8.1 (Stat Corp., College Station, TX, USA).
3. Results Out of the panel of 200 sera the various laboratories identified different numbers of samples as N. caninum positive and estimated avidity values for these positive sera (Table 2). The comparison of the avidity estimates by different laboratories showed variable degrees of concordance (Fig. 1). The concordance correlation coefficients ranged between 0.479 and 0.776 (Table 3). The lower values were found for test combinations including IPB Berne, and were mainly a result of lower bias correction factors. The IPB Berne test gave considerably lower avidity values than the
tests used by the other laboratories. The difference averages for IPB Berne as compared with the other tests were 13.5 to 15.8 (Table 3). When the categorized data were analysed by Kappa statistics (Table 3) there was a moderate to substantial agreement between most of the laboratories. The best agreement was found between FLI Wusterhausen and SLU/SVA Uppsala after the results from Uppsala had been dichotomized so that all intermediate avidities belonged to the low avidity class. There was an overall better agreement between the laboratories for dichotomized results than when an intermediate class was used as well. It is worth noting that the agreement was most improved when the intermediate avidities were considered low. In two herds with recent epidemic abortions for which the history of the individual animals was known, the laboratories obtained for all (FLI Wusterhausen, SLU/SVA Uppsala) or the majority (UCM Madrid, IPB Berne) of sera of aborting dams
Table 2 Number of N. caninum antibody positive/negative samples and number of samples for which avidity values were obtained by the different laboratories
FLI Wusterhausen SLU/SVA Uppsala UCM Madrid IPB Berne
Ab negative
Ab positive
Avidity value
11 9 16 19
189 191 184 181
189 181 184 181
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Fig. 1. Graphical display of Neospora caninum IgG avidity data from the four laboratories. Solid line: the line of perfect concordance; dashed line: the reduced major axis of the data.
low or intermediate avidity values (Table 4). For nonaborting dams all tests obtained a variable proportion of high avidity results. The results from the experimentally infected cows were extracted from the total data and are presented in
Fig. 2. All laboratories found an overall increase in avidity with time after tachyzoite infection in two of three animals, although there were also a few outlier results. In the remaining tachyzoite infected dam, cow 3, FLI Wusterhausen and SLU/SVA Uppsala observed
Table 3 Statistical values obtained by Lin’s concordance, Bland Altman’s limit-of-agreement and Kappa statistic methods for N. caninum IgG avidity measurements at four laboratories FLI Wusterhausen– SLU/SVA Uppsala Concordance corr coeff Bias corr factor Pearson corr coeff 95% limits of agreement Difference average Kappa values Intermediate = lowa Intermediate = highb a b
FLI Wusterhausen– UCM Madrid
FLI Wusterhausen– IPB Berne
SLU/SVA Uppsala– UCM Madrid
SLU/SVA Uppsala– IPB Berne
UCM Madrid– IPB Berne
0.776
0.700
0.484
0.738
0.488
0.479
0.998
0.978
0.791
0.978
0.768
0.811
0.777
0.715
0.612
0.755
0.635
0.590
30 to 28
37 to 38
21 to 50
32 to 38
20 to 51
29 to 56
15.8
13.5
0.8
1.1
14.6
2.7
0.721
0.607
0.566
0.478 0.606
0.503 0.611
0.358 0.464
0.526
0.466
0.392
0.564
0.491
0.432
Avidity values classified as intermediate in SLU/SVA Uppsala, UCM Madrid and IPB Berne were reclassified as low. Avidity values classified as intermediate in SLU/SVA Uppsala, UCM Madrid and IPB Berne were reclassified as high.
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Table 4 Avidity testing of sera of dams from herds with a recent N. caninum associated abortion outbreak Test
Abortion history
Avidity Low
Intermediate
High
No result
FLI Wusterhausen
Aborting Non-aborting
14 11
– –
0 11
0 0
SLU/SVA Uppsala
Aborting Non-aborting
11 9
3 6
0 7
0 0
UCM Madrid
Aborting Non-aborting
10 6
3 5
1 11
0 0
IPB Berne
Aborting Non-aborting
8 4
3 3
1 13
2 2
increasing avidities, whereas the results of UCM Madrid changed from initially high to intermediate with time after infection. At IPB Berne the avidity results for this animal was variable, changing from high to low two times (Fig. 2). In the three animals infected by oocysts the avidity indices increased either slowly or
remained at low or intermediate levels throughout the observation period depending either on the individual animal or on the laboratory or both (Fig. 2). IgG avidity could first be measured 2–4 weeks after injection with tachyzoites, whereas it took 4–8 weeks until results were obtained in the cows given oocysts.
Fig. 2. N. caninum IgG avidity in six experimentally N. canium infected pregnant cows measured in four laboratories: (A) FLI Wusterhausen, (B) SLU/SVA Uppsala, (C) UCM Madrid and (D) IPB Berne. Cows subcutaneously injected with 5 108 tachyzoites of the Nc-1 isolate 140 days into gestation: cow 1 (*––*), cow 2 (~––~) and cow 3 (&––&). Cows orally given 600 N. caninum oocysts (Nc-Liv) at 70 days gestation: cow 4 (*–*), cow 5 (&–&) and cow 6 (D–D).
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4. Discussion In this first ring test for N. caninum IgG avidity assays we compared the tests we use in our four laboratories. The tests are all modified indirect ELISAs in which the bonds between low affinity antibodies and the antigen are disrupted during an incubation step with the protein denaturating agent urea. The avidity is a measure of the antigen-binding strength of populations of antibodies in the test sera. The ELISA tests that were used are based on different antigen preparations and therefore most probably measure different subsets of antibodies, subsets that might diverge in affinity maturation. The iscoms in the SLU/SVA Uppsala test comprises a mixture of N. caninum membrane proteins including the NcSRS2 employed in FLI Wusterhausen (Schares et al., 2000; Bjo¨rkman and Hemphill, 1998), whereas the other two tests are based on tachyzoite sonicate lysates containing a mixture of intracellular and membrane antigens. Although Neospora avidity analysis is a new technique the methodology has been used for several years in human medicine for diagnosis of Toxoplasma gondii infection and it has been found that the antigen composition affects the avidity result (Marcolino et al., 2000; Beghetto et al., 2003). It is therefore not surprising that we found only a modest agreement between the avidity values measured in our laboratories but it was unforeseen that the tests used in UCM Madrid and IPB Berne that are based on the same kind of antigen preparations and follow the same protocols showed the lowest agreement. However, both the concentration of urea (Hedman et al., 1989) and the incubation time considerably affect the avidity results. Moreover, urea from different producers may give different test result. In this comparison, IPB Berne had consistently lower test results than the other laboratories. IPB Berne have earlier reported lower avidity results for some cows than SLU/SVA Uppsala in an investigation comprising a limited number of samples and only these two tests (Sager et al., 2003). Because of economic restraints and limited amount of each serum available, the samples were analysed in singular. Replicate testing should have given higher accuracy of the test results, and probably a somewhat better agreement between the laboratories. In our diagnostic laboratories, avidity samples are often analysed in singular when we receive several sera
from a herd and can identify outlier results and repeat the analysis of samples with such unexpected results. Since the samples in this study were tested blind no outlier results could be detected, probably resulting in a somewhat lower precision than in the diagnostic set up. Sera from experimentally infected animals are useful reference samples for validation of diagnostic tests. Here we used sera collected from cows inoculated with either tachyzoites or oocysts. Most laboratories showed an increase in avidity with time after inoculation in the tachyzoite-infected cows, thus, confirming results from previous evaluations of the individual tests (Bjo¨rkman et al., 1999; Schares et al., 2002). The odd low results found at different time points by different tests reflect the variability in the test performance. In the oocyst-infected group the results were less consistent. Overall the avidities were lower than in the tachyzoite-infected animals, and one laboratory detected in these animals no avidity maturation at all (UCM Madrid). This could suggest that avidity maturation of specific antibodies for particular antigens might occur – if at all – only late after infection and might be influenced by the infection dose. In contrast to the tachyzoite-infected dams that were infected by 5 108 parasites, the oocyst-infected animals were inoculated with an equivalent of only 4800 sporozoites. However, we cannot rule out that the inconsistencies are caused by the above-mentioned variabilities in the test performance. If an ELISA method is to be useful for avidity assays, it is necessary that it can detect the antibody response elicited during early infection; otherwise no avidity values can be estimated for acutely infected animals. Here we were able to measure 2–4 weeks after injection with tachyzoites, whereas it took 4–8 weeks until results were obtained in cows that had been given oocysts. However, one has to be careful when interpreting low avidity results from animals that also have low N. caninum antibody levels. They can indeed reflect that the animal is acutely infected with Neospora but might also be an effect of nonspecific reactions between the antigen and other agents than N. caninum specific antibodies. SLU/SVA Uppsala usually does not report such results without having access to complementary information about the animal or the herd, which is why several of the
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animals that were found seropositive do not have avidity results. The objective of avidity testing is mostly to identify whether an animal or herd has been recently infected or is suffering from a chronic Neospora infection. For this purpose the categorical results are the most useful. Each of our laboratories uses its own cut-off value to discriminate between low and high avidity, i.e. recent and chronic infection. Thus, IPB Berne, which had consistently lower test results than the other laboratories, applies a lower cut-off. This resulted in an overall substantial agreement between the laboratories, especially when only two categories were used, e.g. when the intermediate class results reported by three of the four laboratories were moved to the low class. This overall substantial agreement between the laboratories is also reflected in the results we obtained in herds that had experienced a recent N. caninumassociated abortion storm. All laboratories detected low to intermediate antibody responses in the aborting dams. In addition, the laboratories agreed that a high proportion of the sera from non-aborting dams in these herds had low avidity antibodies suggesting a recent infection with N. caninum in these herds. A discrepancy between Berne and Uppsala has previously been reported (Sager et al., 2003). When serial samples collected from cows that aborted were analysed by the two tests, chronically infected cows had a stable high avidity by the SLU/SVA Uppsala test, whereas the IPB Berne found an increase in avidity after abortion. In the present study we were not able to confirm this previous observation because no serial samples of aborting cows were available. Moreover, no abortion-associated fluctuations were detected by Aguado-Martinez et al. (2005). Taken together, this first ring test for N. caninum IgG avidity assays showed a modest agreement between the assays used by the different laboratories to estimate the IgG avidity. Our experience suggests that avidity tests are sometimes less robust than conventional ELISAs. Therefore, it is essential that they are carefully standardised and their performance continuously evaluated. Recurrent ring tests including a larger panel of reference sera from experimentally infected animals might lead to a better agreement between the laboratories. However, an important obstacle is our lack of a detailed understanding of which types of epitopes are subject for affinity
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maturation (Beghetto et al., 2003). As long as we lack that knowledge we will not be able to adjust for differences in cut-offs unless identical test systems are used. Nevertheless, this generation of avidity ELISAs will continue to be useful tools for investigations of N. caninum infections.
Acknowledgements This study was funded by the European Union (Research Project QLK2-CT-2001-01050 ‘‘Diagnosis, epidemiology and prevention of N. caninum associated bovine abortions’’). The work was also a part of COST-Action 854 ‘‘Protozoal reproduction losses in farm ruminants’’. We thank Andrea Ba¨rwald, Ursula Broennimann, Katarina Na¨slund and Ruth Rauho¨ft for technical assistance. We also acknowledge Thomas Dijkstra for providing sera from naturally infected cattle, and Alexander Trees and Stephen Maley for sera from experimentally infected cows.
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