A semi-automated test system for brucellosis antibody presumptive testing in cattle and swine has been developed. The system utilizes brucella antigen that has been in use for many years, uses automated reading of the agglutination pattern to minimize subjectivity and enhance lab-to-lab uniformity and is designed for high volume applications.

BACKGROUND

The Cooperative National Brucellosis Program currently utilizes three different presumptive tests in the serologic diagnosis of brucellosis in cattle and swine. They are the Buffered Acidified Plate Antigen (BAPA) test, The Rapid Screening Test (RST), and the Particle Concentration Fluorescence Immunoassay (PCFIA). The BAPA and RST are very economical tests but are prone to subjectivity and variation between individual technicians and laboratories. The PCFIA, while less subjective, is comparatively much more expensive.

The RAP test was developed as a procedure that could standardize presumptive testing of cattle and swine serums for brucellosis, reduce the subjectivity and variation of the manual tests, and reduce overall costs of presumptive testing in the brucellosis eradication program.

While incorporating sophisticated instrumentation, a major objective throughout its development has been to maintain low per-test cost within this high volume environment. The technology of the RAP test is adapted from the Autolex® Latex Agglutination test for pseudorabies that was developed by Viral Antigens, Inc., Memphis, TN.

Three versions of this test have been evaluated over the past 18 months. The first utilized diluted BAPA antigen. The second utilized diluted Card test antigen. While the instrumentation performed consistently well, in both cases the antigen-antibody ratio was such that there was a tendency toward prozoning in high tittered serums. The third, the RAP test reported here, utilizes undiluted Card test antigen in a ratio to serum that optimizes the agglutination process while minimizing the prozone phenomena. During development, a total of approximately 60,000 serum samples have been tested with this system.

THE INSTRUMENTATION

The instrument used in this test is the BIO-TEK® Ceres 900c Scanning Autoreader which uses a tungsten lamp filtered to 690 nm and fiber optics to illuminate 12 wells across the microtiter plate as it is drawn into the reader. Twelve detectors are used to read the total light transmitted at 26 locations within each well.

Each of the 26 initial reads are subtracted from the final reads generating transmission change profiles representing the degree of agglutination of the Card test antigen. With no antibody present in the sample there is no change in the scan profile from the initial scan to the final scan. When antibody is present the Card antigen agglutinates and forms a pellet in the bottom of the well reducing the transmission in the center and increasing the transmission at the edge. The degree of agglutination is represented as %AGG.

Percent agglutination (%AGG) is a parameter defined by subtracting the change in the transmission in the center of the microtiter well from the change in transmission at the edge.

A hard copy report is printed indicating the positives, negatives, and potential problem samples. The plate ID, read timing and other information are provided at the top of each printout. Samples can be identified, using a plate setup feature of the software program, to provide the case and tube numbers.

A positive and negative control are run in column 12 of each microtiter plate. These results are summarized at the top of the results printout and stored on the reader for comparisons from plate-to-plate.

Readings are controlled by parameters stored in the reader. These parameters setup assay timing, scan wavelength and error checking. Each reading is compared with these parameters to alert the operator if the reading is out of these preset limits. Also, the reference value to designate a sample as positive or negative is stored in this file.

The use of the brucellosis RAP software enables close tracking of samples and results through case and tube numbers as well as bar codes. All timing is preset with beeps to assist the operator. Stored quality assurance parameters help identify problem samples, equipment and materials.

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.

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 was 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 BAPA.

- 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.

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.

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.

In all three culture and serology groups studied the RAP and the BAPA test performed equally well as a presumptive test method when compared against the Card test.

Figure 5 illustrates the success achieved by the RAP method in separating the positive and negative samples. The x scale is nonlinear 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.

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 onetime cost of the instrumentation, the cost per test is about three cents.