Validation and field assessment of a rapid lateral flow assay for detection of bovine antibody to Anaplasma marginale.

The lateral flow assay (LFA) is a rapid diagnostic test which may be performed under most conditions and is especially useful for field applications. This type of assay was applied to the detection of antibody to bovine Anaplasma marginale using sera from endemic areas and from areas which have been free from infection for more than 25 years. Briefly, the test uses recombinant A. marginale major surface protein 5 peptide (Msp5), immobilized on a cellulose acetate membrane. A serum sample is added to a pad containing a monoclonal antibody specific for bovine IgG(1), conjugated with colloidal gold, located at one end of the strip. The sample and gold conjugate are wicked along the membrane and if antibody is present in the serum, a visible line will form between the Msp5-antibody-conjugate immune complex in minutes. An additional band of recombinant protein A/G was added to the membrane as a positive control reaction of the monoclonal antibody conjugate. For comparison, direct examination of blood smears and a nested polymerase chain reaction (PCR) were performed on some of the samples. Using samples from herds in one endemic area, the PCR gave a sensitivity value of 9.2% while a commercial competitive enzyme immunoassay (CELISA) gave a sensitivity value of 17.2% and the LFA values of 20.5%. In a second endemic area, selected samples, all positive by direct examination gave a 71.7% sensitivity values with the PCR, 94.5% with the CELISA and 95.5% with the LFA. Using sera from a disease-free area, the specificity values were 100% for the PCR (testing a proportion of randomly selected samples), 99.5% for the CELISA and 98.0% for the LFA. It is envisaged that the validated LFA will be a useful tool for screening cattle moving from an area with infection to a disease-free area.

Development of a lateral flow assay for rapid detection of bovine antibody to Anaplasma marginale.

A rapid lateral flow assay for detection of bovine antibody to Anaplasma marginale was developed. The assay used a recombinant peptide of major surface protein 5 as the antigen and a monoclonal antibody specific for bovine IgG(1) conjugated with colloidal gold beads for detection. Serum and anticoagulated blood samples were obtained from cattle in an area where anaplasmosis was endemic. The samples were selected based on positive identification of the organism in blood smears. The unclotted blood samples were used for PCR determination of the presence of A. marginale while the sera were tested by a commercial competitive enzyme immunoassay (CELISA) and by the lateral flow assay (LFA). Similar samples, collected at a Canadian sales barn, were tested by the CELISA and LFA and 10% were tested by PCR for the presence of A. marginale nucleic acid. In addition, stored serum samples from a second endemic area were tested by CELISA and LFA. Of the 114 smear positive samples, all were positive by CELISA and LFA. All samples were also positive by PCR. Samples from Canadian sources (n=524) were negative in the CELISA but 11 sera gave false positive reactions in the LFA. All samples tested were PCR negative. Of 113 samples from herds with anaplasmosis, 53 were positive in the CELISA and 50 were LFA positive.

Comparison of serological tests for the detection of ovine and caprine antibody to Brucella melitensis.

The indirect enzyme-linked immunosorbent assay (IELISA), the competitive enzyme-linked immunosorbent assay (CELISA) and the fluorescence polarisation assay (FPA) were evaluated with sera from sheep experimentally infected with Brucella melitensis and negative Canadian sheep. The sensitivity and specificity of the assays were as follows: IELISA: 91.7% and 97.6%, CELISA: 75.0% and 99.8% and FPA: 91.7% and 89.5%. Sera from the same experimental population were divided according to serological reaction in the rose bengal agglutination test (RBT) and the complement fixation test (CFT). Reactivity relative to the RBT positive and CFT positive sera were as follows: IELISA: 99.7%, CELISA: 93.2% and FPA: 99.1%. Since sera from goats with proven B. melitensis infection were not available, 699 sera from goats judged positive in the buffered antigen plate agglutination test (BPAT) and CFT and 982 BPAT/CFT negative Canadian goats were used. The sensitivity and specificity of the assays relative to the BPAT and CFT positive sera were: IELISA: 99.4% and 98.0%, CELISA: 95.4% and 97.1% and FPA: 92.7% and 99.8%.

Validation of the fluorescence polarization assay for detection of milk antibody to Brucella abortus.

A fluorescence polarization assay (FPA) for detection of antibody to Brucella abortus in individual milk samples was developed and validated. Samples from 190 cattle from which B. abortus was isolated; milk samples from cattle in herds infected with B. abortus (n = 1,086) and positive in the milk ring test (MRT), as well as milk samples from Canadian cattle (with no evidence of brucellosis, n = 2,974) were tested by the indirect enzyme immunoassay (IELISA) and the FPA. The sensitivity (based on samples from culture positive cattle) and specificity (based on Canadian milk samples) of the IELISA and the FPA were 100%. The relative sensitivity value obtained with milk from cattle of infected herds and the specificity values of the IELISA were 98.5 and 99.9%, respectively. The relative sensitivity and specificity of the FPA with the same samples were 82.2 and 99.4% using a cutoff value of 90 millipolarization units (mP). The low relative sensitivity value of the FPA was shown, by competitive enzyme immunoassay (CELISA), to be due to vaccinal antibody (assumed as vaccinal antibody against B. abortus Sl19 is excluded by the FPA and CELISA but not by the MRT and the IELISA), present in some of the milk samples. The FPA is a homogeneous assay which, unlike the MRT and the IELISA, may be used for testing in the field.

Fluorescence polarization assay for the diagnosis of bovine brucellosis: adaptation to field use.

A fluorescence polarization assay (FPA) was used to test whole blood samples prepared by mixing blood cells from cattle without exposure to Brucella abortus (B. abortus) with sera from animals with confirmed (bacteriologically) infection. A cut-off value between negative and positive values was initially established to be 87.2mP. This value was changed to 95mP to increase assay specificity without loss of sensitivity when testing blood samples from negative animals. The FPA technology was applied to whole blood samples in the field and to stored whole blood samples using two diluent buffers. Relative sensitivity and specificity values for the FPA performed in the field, based on buffered antigen plate agglutination test and competitive enzyme immunoassay results were 95.3 and 97.3%, respectively. However, to obtain maximum sensitivity and specificity, a cut-off value of 105mP was determined for fresh whole blood samples. The relative sensitivity and specificity values of the FPA when testing stored whole blood samples were 100% each using a 95mP cut-off.The usefulness of the FPA for testing whole blood samples in the field was demonstrated.

Validation of the fluorescence polarization assay as a serological test for the presumptive diagnosis of porcine brucellosis.

Sera from Canadian pigs (brucellosis free, n = 14037) and sera from pigs infected with Brucella suis (n = 401) were tested by the buffered antigen plate agglutination test, the complement fixation test, an indirect and a competitive enzyme immunoassay and a fluorescence polarization assay. The results were analysed and assay sensitivity and specificity estimates were calculated. The sensitivity and specificity of the tests were as follows: the buffered antigen plate agglutination test, 77.1 and 96.9%; the complement fixation test (considering anticomplementary sera as negative), 93.3 and 95.5%; the complement fixation test (considering anticomplementary sera as positive), 58.1 and 99.9%; the indirect enzyme immunoassay, 94.0 and 97.9%; the competitive enzyme immunoassay, 90.8 and 96.6%; and the fluorescence polarization assay, 93.5 and 97.2%; respectively. It was concluded that the fluorescence polarization assay was a valuable asset to the diagnosis of porcine brucellosis because of its accuracy, ease of performance and relative cost.

Evaluation of a fluorescence-polarization assay for the diagnosis of bovine brucellosis in México.

A homogeneous fluorescence-polarization assay (FPA) was used for the serological diagnosis of bovine brucellosis in México. The assay uses O-polysaccharide prepared from Brucella abortus lipoplysaccharide (20-30 kDa) conjugated with fluorescein isothiocyanate as a tracer. To measure the fluorescence polarization, a FPM-1 fluorescence-polarization analyzer was used with the procedure described by Nielsen et al. (1996b). A cut-off value of 90 millipolarization (mP) units was used for testing 560 bovine sera from different areas of México. (305 positive sera and 255 negative sera according to the complement fixation test; CFT.) Some were tested with the Rose Bengal plate (RB) test (n = 490) and some with the rivanol-agglutination (RIV) test (n = 190). Sensitivities were 98.3%, 99.3% and 99.0%, and specificities were 68.8%, 55.4% and 96.9%, respectively, for RB, RIV and FPA. The FPA gave a kappa coefficient of agreement with respect to CFT of 0.96, while RB and RIV (relative to the CFT) gave coefficients of 0.70 and 0.61, respectively. Finally, ROC analysis suggested a cut-off value which agreed with the one recommended in the test procedure. We concluded that FPA is a suitable test to be used instead of the CFT in Mexican conditions.

Diagnosis of bovine brucellosis using a homogeneous fluorescence polarization assay.

To evaluate the fluorescence polarization assay (FPA) for the serological diagnosis of bovine brucellosis, 118 sera from cattle which were culture positive for Brucella abortus, 1751 sera from cattle from premises containing cattle infected with B. abortus, 1222 sera from cattle vaccinated with B. abortus strain 19 and 1199 sera from cattle with no evidence of brucellosis were tested in Argentina, Chile, Mexico and in the American states of Iowa, Missouri and Texas. Initial determination of serological positivity and negativity was based upon reactivity in currently used serological tests, consisting of a rapid screening test, the rose-bengal or the buffered plate antigen tests, followed by a second serological test, the complement fixation test. Sensitivity of the FPA (sera from culture positive animals) ranged from 87.5% to 100%. Serological positivity of cattle from infected premises ranged from 65.5% to 99.0% while the % negative cattle in herds without evidence of brucellosis was between 94.9 and 100%. Of B. abortus strain 19 vaccinated cattle which were positive in at least one in-use serological tests, 88.2% were negative in the FPA. In contrast, previous Canadian studies, sensitivity values were 99.0% and 100% and the specificity in both cases was 100%. This discrepancy was probably due to the use of less well characterized sera in the current study.

Development and validation of an indirect enzyme immunoassay for detection of antibody to Anaplasma marginale in bovine sera.

An indirect enzyme immunoassay (IELISA) for detection of bovine antibody activity to Anaplasma marginale was developed. This assay used a crude antigen prepared from erythrocytes of infected calves, immobilized in a polystyrene matrix and a mouse monoclonal antibody to bovine IgG1, conjugated with horseradish peroxidase. Negative sera (n = 1842) were tested and the diagnostic specificity was 98.4 +/- 0.6% before retesting 29 positive samples. After retesting, eight samples remained positive and the specificity was calculated to be 99.6 +/- 0.3%. The diagnostic sensitivity, using 831 serum samples collected from naturally or experimentally infected cattle in Argentina, 370 from Mexico and 525 sera from experimentally vaccinated or infected cattle from Texas, was 87.3 +/- 1.6%.