A. Aguado-Martínez, G. Álvarez-García, I. Arnaiz-Seco, E. Innes, L. M. Ortega-Mora 1

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1 J Vet Diagn Invest 17: (2005) Use of avidity enzyme-linked immunosorbent assay and avidity Western blot to discriminate between acute and chronic Neospora caninum infection in cattle A. Aguado-Martínez, G. Álvarez-García, I. Arnaiz-Seco, E. Innes, L. M. Ortega-Mora 1 Abstract. Avidity serological tests (avidity enzyme-linked immunosorbent assay [ELISA] and avidity Western blot) were developed and used to differentiate between acute (primary infection, reinfection, and recrudescence) and chronic Neospora caninum infection in cattle. In addition, the pattern of immunoglobulin G (IgG) avidity maturation against different specific antigens of N. caninum tachyzoites was studied. Sequential serum samples were collected from cattle naturally and experimentally infected with N. caninum. Four groups of experimentally infected cattle were included in the study and were representative of primary infection, reinfection, chronic infection, and noninfection. Serum samples were also collected from naturally infected cattle classified into nonaborting and aborting cows on the basis of clinical findings and serological profiles, and a third group composed of seronegative cows that seroconverted during the course of the experiment. All samples were tested by avidity ELISA and avidity Western blot. The IgG avidity ELISA allowed the discrimination between primary and chronic infection because all experimentally primary-infection cows showed low avidity indexes at week 4 postinfection (p.i.) compared with the high avidity values found at week 20 postinfection. However, this test did not allow the discrimination of reinfection or recrudescence from chronic infection. Regarding IgG avidity Western blot results, no antigenic markers correlating with acute (primary infection, recrudescence, and reinfection) or chronic infection were recognized. However, the 17-kD immunodominant antigen was mostly responsible for high avidity values obtained by avidity ELISA because it was intensively recognized by high-avidity antibodies in all chronically infected animals after urea treatment. Key words: Avidity ELISA; avidity Western blot; bovine abortion; Neospora caninum. Introduction Neospora caninum, a coccidian parasite closely related to Toxoplasma gondii, is one of the major causes of abortion in cattle worldwide. 4 The most frequent transmission pattern is vertical from infected dams to their offspring with resulting lifelong infection. However, cattle can be also infected postnatally through ingestion of oocysts shed by infected dogs. 12,17 Conventional serological techniques, such as indirect fluorescent antibody test and enzyme-linked immunosorbent assay (ELISA), are routinely used in adult animals and aborted fetuses for the detection of anti N. caninum antibodies. All these techniques have been validated and have proved to be quite sensitive and specific for the detection of infection in cattle. 26 However, they still have some limitations, and future chal- From the Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain (Aguado-Martínez, Álvarez-García, Ortega-Mora), the Laboratorio Regional de Sanidad y Producción Animal de Lugo, Lugo, Spain (Arnaiz-Seco), and the Department of Parasitology, Moredun Research Institute, Pentlands Science Park, EH26 OPZ Edinburgh, UK (Innes). 1 Corresponding Author: L. M. Ortega-Mora, Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Ciudad Universitaria, s/n, Madrid, Spain. lenges in diagnosis should focus on designing new serological tests to provide additional information regarding the phase of infection, the predominant route of transmission, or the abortion risk. Such information is needed to establish the most appropriate control measures in cattle herds. Serological avidity tests are known to discriminate between acute and chronic infection caused by other protozoan parasites, such as T. gondii 9,22 or Trypanosoma cruzi. 14 These tests are based on the avidity of immunoglobulin G (IgG) to the antigen that increases over time after the first contact with the pathogen and, therefore, can be used to determine the duration of infection. Three avidity ELISAs that use N. caninum proteins incorporated into immunostimulating complexes, soluble extract from tachyzoites or the affinity-purified p38 protein as antigen have been developed. 3,21,23 These ELISAs have been able to discriminate between primary and chronic N. caninum infection in experimentally infected cattle. 3,21 These tests have also been successfully used for epidemiological studies to discriminate between herds with abortion storms (usually associated with recent postnatal infection) in which most of the animals showed low IgG avidity and herds with endemic abortions (usually associated with recru- 442

2 Avidity tests for N. caninum diagnosis in cattle 443 descence of congenital chronic infections) in which most of the cattle showed high IgG avidity. 8,16,23 However, it is not clear whether these tests would allow the detection of a N. caninum reinfection or a recrudescence in chronically infected cattle and, thus, predict the likelihood of abortions. The aim of this study was to evaluate the utility of 2 avidity serological tests (avidity ELISA and avidity Western blot) to differentiate between acute (primary infection, reinfection, and recrudescence) and chronic infection in both experimentally and naturally infected cattle. In addition, the pattern of IgG avidity maturation against different specific antigens of N. caninum tachyzoites was studied. Materials and methods Bovine serum samples Serum samples were collected from breeding cows experimentally and naturally infected with N. caninum. All sera were tested by a conventional N. caninum tachyzoite somatic antigen ELISA a and by IgG avidity ELISA and IgG avidity Western blot. Sera from 12 breeding cows experimentally infected with N. caninum (group 1) were used. The animals had been infected with live tachyzoites (NC-1 isolate), as described previously. 7 Serum samples were collected from each animal at weeks 0, 4, and 20 after day 140 of gestation. The animals were subdivided into 4 groups as follows.1) Group 1A consisted of 3 breeding cows infected with N. caninum tachyzoites at day 140 of gestation. Specific antibodies were detected by ELISA for the first time at week 4 postinfection (p.i.), with a rise at week 20 p.i. This group was representative of primary infection. 2) Group 1B comprised 3 breeding cows infected with N. caninum tachyzoites before mating and reinfected at day 140 of gestation. Before challenge at day 140 of gestation, specific antibodies to N. caninum were detected with an increase at week 4 after day 140. This group was regarded as representative of reinfection. 3) Group 1C included 3 breeding cows infected before mating and left unchallenged throughout pregnancy. Before day 140 of gestation, specific antibodies to N. caninum were detected, and no increase in antibody levels was observed afterward. This group was considered as representative of a chronic infection. 4) Group 1D was composed of 3 uninfected breeding cows, which remained as controls. No N. caninum specific antibodies were detected. Regarding naturally infected cattle (group 2), sera from 19 naturally infected breeding cows were obtained and subdivided into 3 groups according to clinical and serological parameters. Four to eight sequential serum samples were collected from each cow of groups 2A, 2B, and 2C approximately every 3 mo. 1) Group 2A consisted of 6 N. caninum chronically infected breeding cows without Neospora-associated abortions. Cows in this group did not show any notable antibody fluctuations among different samples throughout the study period. 2) Group 2B comprised 8 N. caninum infected breeding cows with Neosporaassociated abortions and high antibody fluctuations among different sampling times. 3) Group 2C was composed of 5 breeding cows (with or without Neospora-associated abortions). All animals in this group were seronegative at the start of the experiment and then seroconverted during the experiment. Two of them seroconverted at the eighth month of pregnancy. A third cow aborted at the sixth month of pregnancy and seroconverted just 1 mo later. The remaining 2 cows of this group seroconverted at the fourth and sixth sampling, but their pregnancies were not documented. Serum samples were aliquoted and stored at 80 C before testing by both serological avidity tests. Neospora caninum IgG avidity ELISA A N. caninum tachyzoite somatic antigen ELISA a was adapted for use in an IgG avidity ELISA, as described previously. 21 Sera were tested using duplicate 4-fold dilution series starting at 1:25 up to 1:25,600. After 1 hr of incubation at 37 C, 1 dilution series was washed with washing solution and the other was washed 3 times with 6 M urea b diluted in phosphatebuffered saline (ph 7.4) containing 0.05% Tween-20 (PBST). The following steps were performed according to the manufacturer s recommendations. a The avidity ELISA cut-off was positioned in the lower linear part of the dilution curve, 21 and IgG avidity was determined as described 9 by calculating 2 end point titers (one of them after washing with urea and the other one after washing with PBST). The IgG avidity was expressed as an avidity index (AI) according to the following formula endpoint titer with urea AI 100 endpoint titer without urea Three different avidity classes were determined in a similar manner, as in previous reports 21,23 : AIs lower than 25%, between 25% and 40%, and above 40% were classified as low, intermediate, and high avidity, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and IgG avidity Western blot Neospora caninum tachyzoites were grown and maintained by serial passages in Vero cell cultures, as described. 11 To obtain the water-soluble proteins of N. caninum, tachyzoites were purified and disrupted by ultrasound treatment according to a previous proce-

3 444 Aguado-Martínez et al. dure. 2 After this process, the protein content of the supernatant was quantified by the Micro BCA protein assay method, c,24 aliquoted, and stored at 80 C until further use. The N. caninum soluble proteins were separated by electrophoresis under reducing conditions in 12.5% sodium dodecyl sulfate polyacrylamide gel. 10 Separated proteins were transferred to a nitrocellulose membrane, d and the Western blot analysis was performed, as described. 2 The sera were diluted 1:25. To estimate the IgG avidity for each N. caninum antigenic fraction, each serum sample was tested on 2 identical N. caninum nitrocellulose strips. After incubating with serum samples, one of these strips was washed with Tris-buffered saline containing 0.05% Tween-20 (TBST) and the other one with 6 M urea in TBST 3 times for 5 min. 15 The apparent molecular weights of the proteins recognized by the sera were estimated using the molecular weight standard and commercial software. e Four categories were established for the intensity of recognition (no recognition, -0-; low, -1-; medium, -2-; and high -3-). The IgG avidity against N. caninum antigenic fractions was estimated, as previously described, 19 by comparing the intensity of each band before and after the treatment with urea. Thus, a strong decrease of intensity of recognition (3/0, 2/0, 1/0) was assessed as low avidity ( ), and a similar pattern (3/ 3, 2/2, 1/1, 3/2, 2/1) or a minimal decrease of intensity (3/1) was assessed as high avidity ( ) and intermediate avidity ( ), respectively. Because of the high individual avidity variations against the minor antigens, the study focused on the avidity maturation against immunodominant antigens (IDAs) (17, 34 35, 37, and kd antigenic bands). 2 Data analysis Efficacy of the serological avidity tests was evaluated by studying the avidity evolution over time in each group and comparing the avidity results obtained among the different groups. For these purposes, nonparametric Mann Whitney U-test and Kruskal Wallis H-test were used to study intragroup variations of AIs obtained by ELISA test over time in experimentally infected cattle and intergroup variations in experimentally and naturally infected cattle. A linear regression and the Student s t-test were used to study the variations of AIs over time for each of the naturally infected cattle. To analyze the capability of the avidity tests for detecting cows at risk of abortion, 2 different approaches were used. First, intragroup variations in aborting cattle were studied by comparing results obtained at 3 different times during the study period (2 4 mo before abortion, 1 mo after abortion, and 3 5 mo after abortion) by Kruskal Wallis H-test. Second, an intergroup comparison of results obtained in aborting and nonaborting cattle was also performed by Mann Whitney U-test. All statistical analyses were performed with commercial software packages. f,g Results Experimentally infected cattle Avidity ELISA. Group 1A showed AI below 20% at week 4 p.i., with a significant increase (U 9; P 0.05) above 40% at week 20 p.i. Groups 1B and 1C consistently showed AI above 40% throughout the period of the study (Fig. 1) without statistically significant differences between sampling times (H 0.09; P 0.05 and H 2.08; P 0.05, respectively). When group 1A was compared with groups 1B and 1C, significant differences were only detected at week 4 p.i. (U 9; P 0.05 and U 9; P 0.05, respectively). Avidity Western blot. The IgG avidities against N. caninum IDAs recognized by avidity Western blot are summarized in Fig. 2. Low avidity against all IDAs was detected in 1 cow of group 1A at week 4 p.i., with an increase to high avidity at week 20 p.i. The remaining 2 cows of this group showed high or intermediate avidity values at week 4 p.i. and high avidity at week 20 p.i. However, a clear increase in avidity against 17-kD IDA between both samplings was observed. Regarding groups 1B and 1C, high avidity against 17- and 37-kD IDAs and against 17-kD and 34- to 35-kD IDAs was consistently detected, respectively (Fig. 2). Naturally infected cattle Avidity ELISA. High avidity values (above 40%) were consistently obtained in nonaborting (group 2A) and aborting breeding cattle (group 2B) during the period of the study (Fig. 3). No abortion-associated AI fluctuations were detected in aborting cattle (group 2B), either when comparing the different samplings (2 4 months before abortion, 1 month after abortion, and 3 5 months after abortion) (H 1.75; P 0.05) or when comparing AI between aborting and nonaborting cattle (U 20; U 28; U 33, P 0.05). Two different patterns of avidity fluctuations were observed in seroconverted breeding cattle (group 2C). One cow initially showed low AI coinciding with the seroconversion followed by a significant increase of AI to high avidity over months (t 4.46; P 0.05). The remaining 4 cows showed high AI in all seropositive sera collected during the course of the study. Avidity Western blot. The Western blot patterns of the IgG avidity maturation against the N. caninum antigen fractions corresponding to 1 cow of each group are summarized in Fig. 4. High or intermediate IgG avidity against 17-kD IDA was detected in nonaborting breeding cattle (group 2A) during the course of the experiment (Fig. 4). In contrast to this finding, high individual variations in IgG avidity against the remaining IDAs and minor antigens were found. In aborting breeding cattle (group 2B), some abortion-associated avidity fluctuations were detected against IDAs and especially against 17-kD IDA (Fig. 4). However, these fluctuations were not statistically significant (H 3.92, P 0.05; H 2.65, P 0.05; and H 1.62, P 0.05) and significant avidity differences compared with group 2A were not found

4 Avidity tests for N. caninum diagnosis in cattle 445 either. Two patterns of avidity fluctuations similar to those observed by avidity ELISA were found in group 2C. Although the IgG avidity detected against the IDAs was generally low, one of the cows showed an increase in IgG avidity against 34- to 35-kD IDA and against a minor antigen of 28 kd in a serum sample collected at the end of the study, 1 month after abortion (Fig. 4). This increase in avidity was detected by Western blot at the same time that an increase in the IgG avidity by ELISA was found. The remaining 4 seroconverted cows showed low avidity values against almost all proteins despite the high AI obtained by avidity ELISA. Figure 1. Avidity indexes obtained by avidity ELISA in experimentally infected breeding cows (group 1). 1A: cows experimentally infected at day 140 of gestation; 1B: cows experimentally infected before pregnancy and rechallenged at day 140 of gestation; and 1C: cows experimentally infected before pregnancy and left unchallenged throughout pregnancy. Discussion An IgG avidity ELISA and, for the first time, an IgG avidity Western blot have been developed, and their ability to discriminate between primary infection, reinfection, recrudescence, and chronic infection in cattle infected with N. caninum are discussed. In the group of experimentally infected animals, recent infection in group 1A was confirmed by the low AI found in this group at week 4 p.i. by avidity ELISA and the significant avidity increase detected between weeks 4 and 20 p.i. These results confirm the ability of the IgG avidity ELISA to discriminate between recent and past infection in experimentally infected cattle and agree with results obtained by other authors who found that in experimentally infected calves, low AIs initially found at week 3 p.i. were followed by a rapid increase above 35% detected at week 10 or 11 p.i. 3,21 In contrast, groups 1B and 1C showed consistently high AIs (above 40%) during the course of the study. These results suggest that the IgG avidity ELISA does not accurately allow the discrimination between a chronic infection and a reinfection as has been described in human rubella reinfection. 1 According to these observations, in a reinfection, the newly synthesized antibodies would bind the antigen with high avidity because of the adaptative immune response. Similar results were obtained using avidity Western blot in experimentally infected cattle. An increase of the IgG avidity at least against one IDA between weeks 4 and 20 p.i. in group 1A might be suggestive of a recently acquired infection, whereas chronic highavidity antibodies developed against 17-kD IDA and other antigens were found in both groups 1B and 1C, characteristic of a mature immunological response and suggestive of a chronic infection. In naturally infected cattle, both nonaborting and aborting cattle (groups 2A and 2B, respectively) consistently showed high AI during the study period, suggesting a past infection. Similarly, avidity fluctuations associated with N. caninum abortion were not detected in group 2B, suggesting that the IgG avidity ELISA does not allow discrimination between chronic infection and a recrudescence associated with abortion. In other reported studies, 23 high IgG avidity values were found in aborting cattle belonging to herds with an endemic abortion pattern, and other authors 5 did not detect any differences in avidity values when trying to detect a recrudescence associated with vertical transmission. This is similar to the situation in human immunodeficiency virus seropositive humans in whom a reactivation of a T. gondii does not result in any de-

5 446 Aguado-Martínez et al. Figure 2. Pattern of avidity recognition of the Neospora caninum tachyzoite antigens by Western blot in experimentally infected breeding cows over time (group 1). 1A: a cow experimentally infected at day 140 of gestation; 1B: a cow experimentally infected before pregnancy and rechallenged at day 140 of gestation; and 1C: a cow experimentally infected before pregnancy and left unchallenged throughout pregnancy. *: weeks 0, 4, and 20 p.i.; u : without urea, u : with urea; ND: not determined; : low avidity; : intermediate avidity; : high avidity.

6 Avidity tests for N. caninum diagnosis in cattle 447 Figure 3. Avidity indexes evolution by avidity ELISA in naturally infected breeding cows (group 2). 2A: nonaborting infected cows; 2B: aborting infected cows; and 2C: an aborting seroconverted cow. *: abortion date. crease in the IgG avidity attributed to a secondary immune response to recall antigens. 6,18 In group 2C, the 2 different patterns identified by avidity ELISA confirm that this test could discriminate between a seroconversion associated with a recently acquired horizontal transmission (low avidity) and a seroconversion associated with antibody fluctuations during chronic infection (high avidity). Similar results have been described for experimentally infected calves where IgG avidity levels remained consistently elevated after

7 448 Aguado-Martínez et al. Figure 4. Pattern of avidity recognition of the Neospora caninum tachyzoite antigens by Western blot in naturally infected breeding cows over time (group 2). 2A: a nonaborting infected cow, 2B: an aborting infected cow 2C: an aborting seroconverted cow. *: sequential serum samples collected every 3 months; u : without urea, u : with urea; : serum sample collected 1 month after abortion; : not recognition; : low avidity; : intermediate avidity; : high avidity.

8 Avidity tests for N. caninum diagnosis in cattle 449 week 12 p.i., whereas antibody levels measured by IgG ELISA fluctuated, falling below the cut-off in some cases. 13 The avidity Western blot analysis of naturally infected cattle provided complementary information. The predominantly high avidity values against 17-kD IDA in nonaborting cows (group 2A) confirmed the detection of a chronic infection. However, the abortion-associated avidity fluctuations against the IDAs (and predominantly against 17-kD IDA) detected in some aborting cows of group 2B may be because of a recrudescence of a N. caninum chronic infection. However, differences were not statistically significant, and further work is required to examine a larger number of serum samples. Finally, in group 2C, 1 seroconverted cow initially recognized Neospora antigenic bands with low avidity showing an avidity increase against 2 antigens in the last sample. Similar avidity increase was observed by avidity ELISA. These concordant results between both tests suggest a recent postnatal infection by N. caninum in this animal. However, conflicting results between the 2 avidity tests were obtained in the remaining 4 seroconverted cows in this group, which had low antibody titers throughout the sampling period. In these animals, persistent low avidity antibodies against almost all recognized proteins were detected by avidity Western blot during the course of the study, whereas high AIs were observed by avidity ELISA. These results can be explained by the different principles of each technique. Avidity Western blot is a qualitative technique, and the avidity estimation may be imprecise in sera with low antibody titers despite these animals being chronically infected, as confirmed by the high AIs obtained with the avidity ELISA. 13 Moreover, 3 of these cows became seronegative again during the course of the experiment, indicating that the seroconversion may not have been because of a first contact with the parasite and was possibly related to antibody fluctuations known to occur in chronically infected cattle. 20,25 In agreement with other authors, the authors of this study have found that the avidity ELISA using whole tachyzoite antigen is a good tool for the detection of a primary infection and may also be capable of confirming the route of infection in seroconverted animals. This information may be important in designing effective control measures against this important infection in herds. However, this technique does not allow discrimination between a recrudescent infection or a reinfection and a chronic infection. Although the method of avidity estimation by Western blot used in this study tries as much as possible to minimize the effect of antibody titer, 14,19 results of Western blot avidity should be cautiously interpreted because of the qualitative nature of the test. Despite this limitation, the avidity Western blot has shown to be a complementary tool for identifying the main antigens responsible for the avidity maturation. Among the Neospora tachyzoite antigens, no markers of acute and chronic infection were found, but the 17-kD IDA seems to be a good candidate for designing an avidity ELISA because it is the antigen most involved in the avidity maturation and it could reduce individual avidity variations related to minor antigens. 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