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Prepared by
Claude E. Barton, DVM; Robert F. Devlin, PhD; Robert M. Studholme
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PROTOCOL FOR THE FIELD TRIAL
The serums included in this trial were tested in three separate groups as follows:
1. Routine Field Samples - There were 17,430 samples in this group. Included were samples collected at livestock markets, slaughterhouses, and on-farm herd tests. This group of blood samples was obtained from routine submissions to the Tennessee and Mississippi state-federal brucellosis laboratories.
2. Positive Herd Data - There were 1,010 samples in this group. Included were both positive and negative serums from known affected cattle herds in Tennessee and Mississippi. This group also included approximately 400 test positive samples originally tested at the Texas state-federal brucellosis laboratory that were disclosed from routine submissions.
3. Swine Data - There was a total of 635 serums in this group. These were obtained from routine submissions for pseudorabies and brucellosis surveillance at the Tennessee state-federal brucellosis laboratory. Included also were frozen serums from a B. suis affected swine herd that occurred several years ago.
The field trial itself consisted of the BAPA and the RAP tests being conducted in parallel on each serum. In the group of routine field samples, serums testing positive to the BAPA or RAP tests were further assayed with the Card test. All samples, including those negative to the BAPA and RAP tests, from the positive herd data and the one affected swine herd were tested with the Card test. Because of differences in sensitivity and subjectivity between the BAPA and RAP tests, the Card test was used as the common denominator against which the results of both tests were compared. In all cases where either the BAPA or RAP test was negative and the other positive, the tests were repeated to confirm results.
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PROTOCOL FOR THE BLIND STUDY
Serums for the double blind study were from the serum bank maintained at the NVSL, Ames, IA. This panel of 203 lyophilized samples were configured and evaluated by the Diagnostic Bacteriology Laboratory at the NVSL.
The lyophilized samples were reconstituted and stored under refrigeration until used. There were 76 distinct samples used in the study. Multiple vials of each sample were contained in the study providing 203 vials in all. The samples were chosen from three groups using information on whether organisms were isolated by culture for that animal and the serological state of that animal. These groups were:
1. Samples positive by culture and positive serology
2. Samples negative by culture and positive serology
3. Samples negative by culture and negative serology
Two sets of samples were coded and tested using the field trial protocol except that the Card test was performed on all samples. The sample panel configuration, test results and evaluator's comments are provided below.
RESULTS
Definitions:
- % positive defined as positives by the test divided by the total number of samples.
- Sensitivity is defined as positives by Card divided by positives by Card + false negatives by RAP or BAPA. Therefore sensitivity measures the prevalence of false negatives by RAP or APA.
- Specificity is defined as negatives by Card divided by negatives by Card + false positives by RAP or BAPA. Therefore specificity measures the prevalence of false positives by RAP or BAPA leading to increased testing with the Card test.
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Routine field samples, samples from positive herds, and swine samples were tested by the three methods. The number and percentage of positives found for each set of samples is shown. The RAP test yields a slight increase in the positive rate.
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Taking all samples into account the RAP & BAPA tests were 100% sensitive. The RAP test was 98.5% specific compared with 99% for the BAPA test indicating a slightly higher number of false positives; however, the total number of false positives in both tests is small with 1.43$ for the RAP test and 0.95% for the BAPA test. This difference led to only 91 more samples run with the Card test, out of 19,075 tests, than would be done with the current test protocol used in many states.
In all test groups studied, routine field samples, positive herd samples, and swine samples, the RAP and the BAPA test performed equally well as a presumptive test method when compared against the Card test.
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Double Blind Study
Both the RAP & BAPA tests were 100% sensitive compared with the Card test. The RAP test was 81.5% specific compared with 75% for the BAPA test due in large part to the subjectivity of the BAPA test. This is demonstrated by the negative culture, negative serology samples. There were 7 samples aliquoted into 9 vials for a total of 63 vials. All tests by the RAP and CARD test were negative. In 5 cases the BAPA test was scored positive on three separate samples.
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The next Figure illustrates the success achieved by the RAP method in separating the positive and negative samples. The x scale is non-linear to illustrate this separation. The RAP indicator %AGG for 600 negative samples pulled at random are compared with 625 positive results. There is a clear distinction in the valley between the positives and negatives. A typical negative sample assays with an index near 0 where typical positives distribute with indexes near 20 to 50. The +/- cutoff in the study was set at 5. If that figure is lowered more false positives are generated, however, the high separation allows it to be relatively insensitive to protocol variations.
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CONCLUSIONS
The RAP test offers a reliable, accurate and high volume method for presumptive testing of cattle and swine serums in the national brucellosis eradication program. The sensitivity and specificity of the RAP test is equal to or greater than the BAPA test, the presumptive test used widely at present. It provides added benefits of cost reduction while maintaining high throughput and objectivity. Aside from labor costs which would not differ markedly from that of tests currently being done and the initial one-time cost of the instrumentation, the cost per test is about three cents.
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