INTRODUCTION

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In the Canadian cattle population, leptospirosis is predominantly caused by serovar hardjo (now generally accepted as being Leptospira borgpetersenii serovar hardjo type hardjobovis) and serovar pomona 1, 6, 7, 8, 9, 12, 13, 14, 15. Other serovars such as grippotyphosa and icterohaemorrhagiae have also been detected but at relatively lower levels 6, 7, 13. Direct detection of these organisms by microscopic examination or culture is impractical due to the low success rate and the amount of time and labor required. Instead, leptospirosis is most often diagnosed serologically with the microscopic agglutination test (MAT) 2. The MAT however, despite its widespread usage and international recognition, is encumbered with a number of limitations. These include the need to use hazardous live bacteria and the amount of time and labor required to test each serum sample against multiple serovars of this organism. In addition, the lack of standard operating procedures and source strains among laboratories and the subjective scoring of results may cause quality assurance difficulties. Due to the drawbacks of the MAT we are developing alternative diagnostic tests for the detection of Leptospira serovars which are of economic importance to Canada.

In a previous publication 20, we described two monoclonal antibodies (M897 and M898) that are suitable for incorporation into competitive enzyme-linked immunosorbent assays (ELISAs) for the specific detection of serum antibodies to serovar pomona. In this communication, we report the results of a validation study of a competitive ELISA that was developed with monoclonal antibody M898 for the detection of bovine serovar pomona antibodies.

	MATERIALS AND METHODS

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Bacterial culture and MAT. The Leptospira organisms were cultured and the MAT was performed as previously described 20.

Bovine sera. Field serum samples submitted to Canadian Food Inspection Agency laboratories across Canada were collected and tested by the MAT. Of these sera, 190 with serovar pomona MAT titers of 100 (group A) and 1,445 which were serovar pomona MAT negative at a 1:100 dilution (group B) were included in this study. Some of these sera also had MAT titers of 100 for serovars other than pomona. Two hundred and ten sera (group C) from a specific-pathogen-free (SPF) herd of cattle were also tested. These sera were negative in the MAT at a 1:100 dilution for serovars canicola, copenhageni, grippotyphosa, hardjo, pomona, and sejroe. All sera were stored at 20°C and thawed at room temperature before testing.

ELISA. The monoclonal antibody (M898) was produced as described 20. The antigen was prepared from serovar pomona cells as described 20 and then sonicated for 2 min with a 375-W cell disruptor (Heat Systems-Ultrasonics Inc., Farmingdale, N.Y.). The assay was performed as described 20 except for the following modifications. Batches of microtiter plates were coated with the antigen, incubated overnight at room temperature, and then frozen at 20°C. The plates were thawed at room temperature and washed before use. Four controls (each in quadruplicate wells) were included in every plate. In the first (uninhibited control), the bovine serum was replaced with phosphate-buffered saline-Tween (PBST). The second control consisted of a serovar pomona MAT-negative serum. Conditions of the assay were adjusted so that at 10 min of substrate-chromogen development, an optical density (OD) value of approximately 1.0 was obtained for the PBST and the negative serum controls. The third control was a medium-titer-positive serum which gave an optical density value of approximately 0.50 at 10 min, and the fourth control was a high-titer-positive serum which gave an optical density value of <0.10 at 10 min. Both of the positive control sera were obtained from cows naturally infected with serovar pomona. In the control wells all other reagents were added in the exact amounts and sequence as described.

Acceptance criteria. Results of the entire plate were rejected unless the mean OD at 414 nm (OD₄₁₄) values of the controls were within predetermined limits (established by performing the test at least 40 times) with a coefficient of variation of 10% for the medium-titer-positive serum, the negative serum, and the PBST controls. A coefficient of variation of 50% was acceptable for the high-titer-positive serum control, which gave OD₄₁₄ values of <0.10. In addition, for plates with acceptable control OD₄₁₄ values, the results for test samples were rejected unless the coefficient of variation of the duplicate OD₄₁₄ values was 10%, except for OD₄₁₄ values of <0.10, when a coefficient of variation of 50% was accepted.

Data expression and analysis. The results of the assay were expressed as percent inhibition of the binding of the monoclonal antibody to the antigen (%I), which was calculated as described 11. Receiver operating characteristic (ROC) curve analysis (MedCalc Software, Mariakerke, Belgium) was performed on the ELISA results to determine the optimal cutoff point (at which the sum of the sensitivity and specificity values is highest) for distinguishing between positive and negative results. The area under the ROC curve (AUC), which can be used as a measure of the accuracy of the test, was also calculated. Separate analyses were performed on the ELISA results of (i) serum groups A and B; (ii) serum groups A and C; and (iii) serum groups A, B, and C.

	RESULTS

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In this competitive ELISA, the majority of the negative sera yielded %Is of 10 and the majority of the positive sera yielded %Is of 20. There was also an area of overlap involving relatively few numbers of both positive and negative sera (Fig. 1). This indicated that the cutoff point for differentiating between positive and negative samples would be %I between 10 and 20. ROC curve analysis was then used to optimize the cutoff point.

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Frequency distribution of the competitive ELISA results of 1,655 serovar pomona MAT-negative (NEG) and 190 serovar pomona MAT-positive (POS) bovine sera. %I of the binding of the monoclonal antibody to the antigen is shown in 10% increments (x axis) against the number of observations (y axis).

The ROC curve analysis of the combined ELISA results of serum groups A and B yielded an AUC of 0.975 and recommended a cutoff point of 13% inhibition of the binding of the monoclonal antibody to the antigen. At this cutoff point, the sensitivity estimate was 93.7% (±3.5% with 95% confidence limits) and the specificity estimate was 96.3% (±0.97% with 95% confidence limits). The analysis of the combined results of serum groups A and C yielded an AUC of 0.987 and recommended a cutoff point of 12% inhibition, with sensitivity and specificity estimates of 94.7% (± 3.2% with 95% confidence limits) and 96.2% (±2.6% with 95% confidence limits), respectively. The analysis of the combined results of serum groups A, B, and C yielded an AUC of 0.977 and recommended a cutoff value of 13%, with a sensitivity estimate of 93.7% (±3.5% with 95% confidence limits) and a specificity estimate of 96.3% (±0.91% with 95% confidence limits).

The ELISA sensitivity and specificity estimates that were obtained from the three analyses and the respective AUC values are summarized in Table 1. The ROC curve obtained from the analysis of the combined ELISA results of serum groups A, B, and C is shown in Fig. 2.

View larger version (26K): [in this window] [in a new window]   FIG. 2.   ROC curve obtained from analysis of the ELISA results of all of the bovine sera tested (groups A, B, and C). The true-positive rate (sensitivity [y axis]) is plotted against the false-positive rate (100  specificity [x axis]) for each %I cutoff point applied. An optimal %I cutoff point of 13 is indicated.

	DISCUSSION

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Many countries stipulate that animals be tested for specific Leptospira serovars as part of their requirements for the international trade of livestock and their products. In addition, some countries also require that cattle which are resident in artificial insemination centers be similarly tested on a periodic basis. In Canada, cattle in artificial insemination centers are tested for a number of serovars, including canicola, copenhageni (represents icterohaemorrhagiae), grippotyphosa, hardjo, pomona, and sejroe. Due to the problems associated with the MAT, we have undertaken the development of sensitive and specific ELISAs to detect antibodies to each of the six serovars listed above. The availability of such a package of ELISAs would then present a practical alternative to the MAT for the diagnosis of these important Leptospira serovars. As part of this endeavor, we have already reported an indirect ELISA 18 and a competitive ELISA 19 for detection of antibodies to serovar hardjo type hardjobovis and an indirect ELISA 17 for detection of antibodies to serovar pomona. In this communication, we report the development and evaluation of a monoclonal antibody-based competitive ELISA for detection of bovine antibody to serovar pomona. The monoclonal antibody (M898) that has been incorporated into this ELISA is very specific for serovar pomona. This monoclonal antibody does not cross-react with any of 13 other pathogenic serovars, including those known to occur in North America 1, 3, 6, 7, 8, 9, 12, 13, 14, 15, 16, 21, 22 and thus the most likely cause of cross-reaction in the local cattle population, nor does it cross-react with L. biflexa serovar patoc, an ubiquitous nonpathogen 20.

The ELISA data were subjected to ROC curve analysis. This type of analysis has been used to evaluate the ability of a test to discriminate between infected and healthy subjects 10, 23 and to compare the diagnostic performance of two or more tests 5. With ROC curve analysis, the sensitivity and specificity values are estimated for every possible cutoff point that is selected to distinguish between a positive and a negative result. The cutoff point at which the sum of the sensitivity and specificity estimates is highest is usually recommended. However, depending on the intended application of the test, a higher sensitivity or specificity than that recommended may be desired, and this can be achieved by an appropriate adjustment of the cutoff value. The ROC curve is obtained by plotting the true-positive rate (sensitivity) as a function of the false-positive rate (100  specificity) that is associated with each cutoff point. The AUC can be used as a measure of the accuracy of the test. If the test cannot distinguish between infected and normal populations the AUC will be equal to 0.5 and the ROC curve will coincide with the diagonal. On the other hand, if the test is 100% sensitive and specific, then the AUC will be equal to 1 and the curve will reach the upper left corner.

The AUC values obtained from the three ROC curve analyses conducted on the ELISA data were all relatively high, indicating that this assay is very accurate. Each analysis also recommended approximately the same cutoff point, with similar sensitivity and specificity estimates. Two of the three analyses only differed by the source of the pomona-negative serum population included in each (sera from an SPF herd and negative field sera), and the third contained both of these negative serum populations. These results indicate that in regards to their reactivity in this ELISA, there was little difference between the SPF sera and the pomona-negative field sera that were used in this study. At either of the two recommended cutoff points, more than 96% of these pomona MAT-negative sera were also negative in the ELISA. The approximately 4% of these sera that were positive in the ELISA may have contained low levels of antibodies that were not detectable in the MAT at a 1:100 dilution. It is also possible that some of these sera may have contained nonagglutinating anti-pomona antibodies which cannot be detected by a test such as the MAT, but which can be detected by an ELISA which measures the primary binding function of antibodies. At either of the two recommended cutoff points, this ELISA detected anti-pomona antibodies in approximately 94% of the sera that had pomona MAT titers of 100. This indicated that the epitope recognized by monoclonal antibody M898 is highly immunodominant in cattle. The sera that were not detected by the ELISA could have been falsely positive in the MAT or perhaps contained antibodies to epitopes on the pomona antigen which are different from the one recognized by the M898 monoclonal antibody.

At this point it is not known whether this ELISA can distinguish between the antibodies produced in response to infection and those stimulated by vaccination. The MAT cannot make this distinction 4, and it would be useful to develop assays that can, so that vaccinated animals are not unnecessarily treated with antibiotics.

The repeatability of this ELISA is 100% when performed under the conditions described. The protocol for production of the antigen used in this ELISA is relatively simple and highly reproducible. The use of frozen antigen-coated plates allows for testing during every working day.

In conclusion, we report the development and validation of a highly sensitive and specific competitive ELISA for the detection of bovine antibodies to L. interrogans serovar pomona. This test uses nonhazardous reagents, is repeatable, is scored objectively, is semiautomated, and can be subjected to stringent quality assurance protocols. This competitive ELISA has the potential to become a useful tool for the diagnosis of serovar pomona infection in cattle.