INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

Neoporosis is a newly recognized protozoan disease. The etiological agent was first isolated from a paralyzed dog and was named Neospora caninum (11). Later it was discovered that Neospora-like protozoa may cause abortion in cattle (27). The bovine and the canine parasite were shown to be identical (16, 20). At present neosporosis is recognized as a major cause of bovine abortion throughout the world (for a review, see reference 13).

The diagnosis of the infection in aborted fetuses is primarily based on the characteristic histological lesions and the immunohistochemical identification of the parasites in fetal tissues (1, 2, 32).

The complete life cycle of N. caninum is not known. At present the only described mode of infection is transplacental, from cow to calf (3, 17, 25, 31). This vertical transmission may contribute significantly to the persistence of the infection in the herd (25, 31). Congenitally infected calves sporadically show neurological signs (3, 12) but usually are in good health (25). However, several studies indicate that chronically infected cows have an increased risk of abortion (21, 26, 29, 31). The only way to identify chronically infected animals is by detection of antibodies in the blood.

The immunofluorescent antibody test (IFAT) has been widely used to detect antibodies against N. caninum (9, 23) but has subsequently been superseded by enzyme-linked immunosorbent assays (ELISAs) (5, 7, 10, 14, 18, 19, 24, 30). Different antigens have been used in the various ELISAs: extracted tachyzoite membrane proteins incorporated into immunostimulating complexes (7), whole-tachyzoite lysate antigens (10, 14, 24), recombinant antigens (18, 19), and tachyzoite surface antigens made available by chemical fixation of whole tachyzoites onto the plates (30). A monoclonal antibody-based competitive inhibition ELISA was developed by Baszler et al. (5). Recently, Dubey et al. (12) evaluated IFAT and ELISA performance in various laboratories, using sera from a herd which had experienced an outbreak of N. caninum-induced abortions. They found considerable differences between different tests and concluded that no test could be used to establish definitively that N. caninum caused the abortion in an individual cow.

In this study, we compared three ELISA methods for detection of bovine antibodies to N. caninum, using various categories of sera. Two ELISAs, referred to as test A (MAST Diagnostics, Bootel, United Kingdom) and test B (IDEXX Laboratories, Westbrook, Maine), were obtained commercially, and each was performed according to the manufacturer's instructions. The third ELISA (test C) was developed at the laboratory of the Animal Health Service (AHS). The aims of the study were (i) to determine the diagnostic relevance of the three ELISA methods in abortion cases in cattle and (ii) to evaluate their applicability for herd prevalence studies and monitoring of congenital infections.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Commercial ELISA methods (tests A and B). Test A was described by Williams et al. (30). Test B was derived from the ELISA originally described by Paré et al. (24). Both methods were performed entirely according to the manufacturer's instructions supplied with the kits. The recommended test sample dilutions (1:400 for test A and 1:100 for test B) were used. Positive and negative controls were provided with the kits (a high and a low positive for test A and a single positive for test B). Results were expressed as sample/positive control (S/P) ratios. For interpretation of the results, the cutoff values recommended by the manufacturers were used. The main characteristics of each ELISA are summarized in Table 1.

AHS ELISA (test C). (i) Antigen preparation. N. caninum (isolate NC1) tachyzoites were cultured on Vero cells. Monolayers were maintained in 75-cm² or 162-cm² tissue culture flasks at 37°C by using Hanks minimal essential medium with L-glutamine and 5% inactivated horse serum and were passaged at 7-day intervals. The flasks were seeded with 10⁷ to 10⁹ tachyzoites. Infected cultures were inspected daily with an inverted microscope. When numerous free tachyzoites were seen in the medium and >80% of the monolayer was destroyed, the remaining cells adhering to the plastic were scraped into the culture medium by means of a rubber policeman. The suspension was then homogenized by means of a Potter-Elvehjem device, followed by filtration through a 5-µm-pore-size filter. Tachyzoites were washed twice in sterile phosphate-buffered saline (PBS) by centrifugation (for 15 min at 1,000 × g). After removal of the supernatant, the resulting pellet was resuspended in PBS. Tachyzoite concentration (10⁷ to 10⁸ parasites per ml) was estimated by using a hemocytometer. Tachyzoites were then pelleted again through a 20% sucrose cushion in PBS for 1 h at 13,000 × g at 4°C. Tachyzoite pellets were suspended in PBS containing 1% (vol/vol) Triton X-100. After overnight incubation at 4°C, sodium azide was added to a final concentration of 0.025%, and the antigen preparation was aliquoted and stored at 20°C.

(ii) ELISA technique. Optimal ELISA concentrations for coating and conjugate concentration were determined by means of checkerboard titration. For coating, Nunc Polysorp microtiter plates were used. Determination of the cutoff value was based on the mean extinction plus 3 times the standard deviation for 50 bovine sera from herds with no history of N. caninum abortions and was further refined by using a frequency distribution of extinction values obtained by assaying N. caninum-infected herds and herds not suspected of having N. caninum infection. After assays at dilutions of 1:50, 1:100, 1:200 and 1:400, a dilution of 1:50 was chosen for test sera because optimal sensitivity was achieved at this dilution.

Categories of test sera. (i) Postabortion samples. Postabortion sera were collected from 59 dairy cows which aborted fetuses with histological evidence of N. caninum infection (immunohistochemically confirmed) and from 16 cows which aborted fetuses with no histological evidence of N. caninum infection and for which other agents were identified: Actinomyces pyogenes (n = 9), Listeria monocytogenes (n = 2), Salmonella dublin (n = 2), Escherichia coli (n = 1), Chlamydia sp. (n = 1), and bovine herpesvirus 1 (n = 1). All sera were obtained from dairy farms in the northern part of the Netherlands. Sera were collected within 14 days after the abortion. All fetuses had been submitted to the laboratory of the AHS and had been examined by using a standard protocol as described previously (32).

(ii) Postpartum samples. Precolostral blood samples were collected from 20 calves born to dams which had previously aborted N. caninum-infected fetuses. Samples were taken immediately after parturition and before nursing. In addition, blood and colostrum samples were taken from the dams.

(iii) Sequential calf samples. Sequential sera were taken from two calves (calves 1 and 2) which had high precolostral antibody levels, indicating congenital infection, and from two calves (calves 3 and 4) that were seronegative at birth but received colostrum from seropositive dams. Sampling was started immediately after birth and was continued at 3-week intervals for 8 to 10 months.

(iv) Sequential samples from aborting cows. Sequential sera were taken at 3- to 4-week intervals from six cows which had aborted N. caninum-infected fetuses. Two cows (cows 32 and 38) had aborted during an abortion storm. Cross-sectional samples had been taken from the herd (herd 1; see below) immediately after the onset of the abortion storm. Cows 32 and 38 aborted 3 and 10 days, respectively, after the herd sampling and were monitored for 6 months. Four cows (cows 21, 37, 45, 93) which had abortions caused by N. caninum were identified in different herds in which abortions were sporadic. These four cows were monitored until calving. Cow 93, which was not reinseminated, was monitored for another 12 months postpartum.

(v) Cross-sectional and total-herd samples. In two dairy herds (herds 1 and 2) with acute N. caninum abortion outbreaks, 20% of the animals (51 and 31 animals, respectively) were bled immediately after the onset of the outbreak. In three herds (herds 3 through 5) with histories of ongoing N. caninum abortions, suggesting endemic neosporosis, sera were collected from all animals (190, 115, and 72 animals, respectively). In one herd (herd 6) with no history of N. caninum abortions, sera were obtained from all 80 cows. The sera from this herd were used for the estimation of test specificity.

Analysis of data. The sensitivities of the tests were calculated by using sera from 59 cows which aborted N. caninum-infected fetuses (confirmed by immunohistochemistry) as the "gold standard." The specificities of the tests were assessed by using sera from 16 cows with abortions in which other (non-Neospora) abortifacients were identified and from 80 cows (from herd 6) which had no history of N. caninum abortions. Exact confidence intervals for sensitivity and specificity were calculated by using a binomial distribution. Agreement between tests was assessed by calculating the kappa statistic (28). Antibody levels were compared between etiological groups by using a chi-square test.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Postabortion samples. The proportions of positive postabortion samples for each ELISA are presented in Table 2. One of the 59 cows in the N. caninum abortion group was negative in all three tests.

Postpartum samples. The proportions of positive postpartum samples for each ELISA are presented in Table 3.

View larger version (50K): [in this window] [in a new window]   FIG. 1.   Precolostral N. caninum antibodies (ELISA test A) in 20 calves compared with serum and colostrum antibodies of their dams, which had histories of N. caninum abortions (colostrum samples 4 and 14 are missing).

Sequential calf samples. Results obtained in each ELISA for sequential samples from two congenitally infected calves and two calves with colostrum-derived antibodies are presented in Fig. 2.

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Profiles of antibodies to N. caninum, as determined with three different ELISAs, in two congenitally infected calves (calves 1 and 2) and two calves (calves 3 and 4) which received colostrum from N. caninum-seropositive dams.

Sequential samples from cows which aborted N. caninum-infected fetuses. Results obtained in each ELISA for sequential samples from aborting cows are presented in Fig. 3 (data from two cows monitored for 6 months) and Fig. 4 (data from four cows monitored until calving or longer). Cows 21, 45, and 93 gave birth to calves which had high levels of precolostral antibodies to N. caninum, indicating congenital infection (data not shown), whereas cow 37 gave birth to a seronegative calf.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Profiles of immune response to N. caninum, as determined with three different ELISAs, in two cows which aborted during an abortion storm.

View larger version (23K): [in this window] [in a new window]   FIG. 4.   Profiles of immune response to N. caninum in four cows that aborted, as determined with three different ELISAs during 1 to 2 years postabortion. These cows were from herds in which abortions were sporadic.

Cross-sectional and total-herd samples. Considerable differences were found between the various ELISAs when the cross-sectional and total-herd samples were tested (Table 4). In particular, the assessed seroprevalences of the three herds (herds 3 through 5) with endemic neosporosis showed great variations depending on which ELISA was used (Table 4). For all six herds, there was a high level of agreement between tests B and C ( = 0.83). Agreement of test A with tests B and C was low ( = 0.39 for test B and  = 0.34 for test C; these values increased to 0.47 and 0.42, respectively, if the samples that were suspect with test A were included in the calculation).

Sensitivity and specificity. Sensitivity and specificity estimates for the three ELISAs, obtained by using 75 postabortion sera and 80 sera from a herd not suspected of having N. caninum infection, are presented in Table 5.

Labor intensiveness. There were no significant differences between the three ELISAs in labor intensiveness with respect to personnel required, the length of the assay, and numbers of samples run at a given time.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Good agreement between the three ELISAs was found with postabortion sera, as shown in Table 2. Antibodies were detected in a high proportion of cows which had recently aborted N. caninum-infected fetuses. These results were similar to those obtained by IFAT by other researchers testing acute-phase abortion sera (22). Sensitivity and specificity estimates with the postabortion sera were satisfactory for all tests. Sensitivity was slightly lower in test A, but this was not statistically significant. Likewise, precolostral calf sera gave similar results in all three ELISAs (Table 3).

However, considerable differences among the tests were found in the antibody profiles of serial samples (Fig. 2 through 4) and in the seroprevalences based on cross-sectional and total-herd sampling (Table 4). Apparently, the sensitivity of test A was inadequate to detect low antibody levels in some animals during chronic stages of the infection.

Several factors may have contributed to these discrepant results. In the first place, the sample dilution may have been important. In test A a dilution of 1:400 was used, whereas in tests B and C dilutions of 1:100 and 1:50, respectively, were used. Secondly, differences in antigen preparation may have played a role. In test A intact tachyzoites were used, and therefore mainly external-membrane antigen determinants and possibly secretory antigens adhering to the surface were exposed. In the other two tests whole-tachyzoite lysates were prepared either by sonication (test B) or by detergent solubilization (test C). Lysates contain soluble cytoplasmatic antigens as well as membrane antigens. It has been suggested that antibodies to soluble cytoplasmic antigens are be formed later in the course of infection, whereas membrane antigens are likely to be recognized first (15). This could account for the longer duration of reactivity with a lysate-based ELISA, compared to a membrane antigen-based ELISA. In addition, the recognized antigens of N. caninum may vary among cows (4, 5). This could explain the fact that one of the four aborting cows from herds with sporadic abortions was mainly negative in test A, which is in strong contrast with the data from tests B and C (Fig. 4). In the third place, the use of different conjugates may have contributed to the variation in test results. In the two commercial tests an anti-immunoglobulin G (IgG) conjugate was used, whereas in test C an anti-Ig conjugate was used. However, differences were particularly evident during the chronic stage of infection, when probably only IgG was detectable. Other researchers have also found discrepant results with different ELISAs, particularly for low antibody titers, but they did not determine the sensitivities and specificities of the different tests (12).

In some cows a sharp rise in antibodies was evident during gestation, most clearly with test A. Such a rise was seen in cows 38 (Fig. 3) and in cows 21, 45, and 93 (Fig. 4). This increasing immune response suggests a recrudescence of the infection (26). This may have caused the intrauterine infection of these cows' calves, as evidenced by the presence of precolostral antibodies (3, 17, 25, 26, 31). The birth of a seronegative calf to cow 37, which showed no increase in antibodies during gestation in any test, corroborates this hypothesis.

Specificity is not easy to assess, because of the lack of a negative "gold standard." All the 16 control sera from cows which aborted due to identified causes other than N. caninum were negative for N. caninum in tests A and C, but 2 samples were positive in test B. This might suggest a lower specificity of test B. However, it is conceivable that a cow which aborts due to a cause other than N. caninum may still be infected with N. caninum. The additional use of sera from a herd with no abortion history for assessment of specificity in this study is debatable, as some of the positive samples had high optical density values, suggesting that these were true positives.

Antibodies to the closely related parasites T. gondii (5, 8) and Sarcocystis spp. (5) have been observed to cross-react with multiple N. caninum antigens by immunoblot assay. Therefore, Baszler et al. (5) state that any serological assay with whole Neospora organisms as an antigen source has the potential for reduced specificity. However, other researchers observed negligible binding of immunodominant Neospora proteins with anti-T. gondii sera in immunoblot analyses (6). Also, Paré et al. (24) did not find cross-reactions between anti-Toxoplasma sera in their Neospora ELISA, from which a commercial ELISA (test B) was derived. Only Björkman et al. (7) mentioned poor specificity due to cross-reacting Toxoplasma antibodies with a crude-antigen ELISA, compared to an ELISA utilizing extracted tachyzoite proteins incorporated into immunostimulating complexes. Dubey et al. (14) observed a weak response to N. caninum in the sera of calves experimentally infected with S. cruzi, using a tachyzoite lysate ELISA. In the present study we did not find detectable cross-reactivity in the ELISA developed at the AHS (test C) with antisera to T. gondii and S. cruzi or with sera to C. parvum, B. bigemina, B. divergens, or E. alabamensis. Only in a B. bovis-infected cow was a transient reaction seen, exceeding the cutoff point in 1 sample in a series of 11. Since we used an anti-Ig conjugate, this transient reaction may be explained by aless specificIgM response. Therefore, we consider insufficient specificity not a practical problem in herd screening for neosporosis with this test. The chemically fixed intact tachyzoite ELISA (test A) was developed to circumvent the problem of cross-reactivity of lysate antigen-based tests (30), but it appears from this study that its use may underestimate the seroprevalence of N. caninum in herds.

We suggest that the use of the detergent in the antigen preparation of test C resulted in the solubilization of additional antigens, which produced a high sensitivity. Similar results were obtained with solubilization of T. gondii antigens (15). Apparently, the released antigens did not cross-react with antibodies to related parasites. Based on the data obtained, the ELISA developed in our laboratory appears to be a highly sensitive and sufficiently specific test for detection of N. caninum antibodies in cattle.