Immunoenzymatic assays
The screening for C. burnetii antibodies was done using an indirect C. burnetii ELISA (Q fever C. burnetii antibody test kit, IDEXX Laboratories, Switzerland), B. abortus , L. Hardjo , N. caninum , and by BVDV antigen capture ELISA according to the kit manufacturer’s instructions. After diluting test sera and positive and negative controls to 1:400 in the kit wash buffer solution, 100 µL/well of each were dispensed into 96 microtiter plate wells pre-coated with inactivated C. burnetii antigen. After incubating the plates at 37◦C for 60 min, they were washed three times with the kit wash buffer, 100 µL/ well of a peroxidase labelled anti-ruminant IgG conjugate addedm and the plates then incubated for 60 min at 37◦C. The plates were then washed three times with the kit wash buffer like before and 100 µL/well of the TMB substrate added. After stopping the reaction, the ELISA plates were read at rT in a spectrophotometer (Bio Tek Instruments. Inc. Highland Park, USA) at 450 nm and the results then expressed as a percentage of the ratio of the test sample OD450 to the positive control OD450 (S/P %). The test samples with S/P % ≥ 40% were interpreted to be positive while those with S/P% < 30% were deemed negative.
For the indirect ELISA for B. abortus antibodies, 100 µL/well of test sera, as well as the positive and negative controls, were diluted 1:10 in the kit sample diluent were placed in the 96 microtitre wells and the plates tightly sealed. After incubating at 37◦C for 60 min, the plates were washed three times using 300 µL/well of the kit wash solution and 100 µL/well of peroxidase labeled anti-ruminant IgG conjugate added. After tightly sealing the plates, they were further incubated at 37◦C for 60 min. Following the incubation step, the plates were washed three times with 300 µL/well of the kit wash buffer and 100 µL/well of 3,3’,5,5’-tetramethylbenzidine or TMB substrate then added. After stopping the reaction, the plates read at 450 nm using a spectrophotometer (Bio Tek Instruments. Inc. Highland Park, USA). Samples having S/P % < 80 % were deemed negative while those with S/P % values ≥ 80 % were determined to be positive.
The screening of test sera for L. Hardjo antibodies was done using indirect ELISA (Leptospira Hardjo Ab bovine ELISA, Demeditec Diagnostics, Germany) according to manufacturer’s instructions. After washing the plates five times with 100 µL/well of the kit wash buffer, 100 µL/well of test sera diluted at 1:100 in the kit sample diluent as well as the negative and positive controls diluted at 1:50 were separately dispensed into the microtiter plates coated withLeptospira antigen. The plates were then incubated at 37⁰C for 60 min after which they were washed five times with kit wash buffer, 100 µL/well of horse radish peroxidase labelled anti-bovine IgG conjugate added, and the plates incubated for another 60 min at 37⁰ C. After five washes, , 100 µL/well of TMB substrate were added and the plates incubated at rT in darkness for 15 min. After stopping the reaction, the plates were read at 450 nm in a spectrophotometer (Bio Tek Instruments, Inc. USA). Test samples with S/P % ≥ 34% were considered positive forL. Hardjo antibodies while samples with S/P% < 34% deemed negative.
The screening for N. caninum antibodies was done using an indirect ELISA (Neospora caninum Antibody Test Kit, IDEXX Laboratories, USA) following the kit manufacturer’s instructions. Briefly, 100 µL/well of test samples diluted at 1:100 in the kit sample diluent as well as undiluted negative and positive controls were dispensed into microtiter plates coated with N. caninum antigen. The plates were then incubated at rT for 30 min after which they were washed four times with kit wash buffer and then 100 µL/well of peroxidase labelled anti-ruminant IgG conjugate added and the plates incubated at rT for 30 min. The plates were then washed four times with kit wash buffer, 100 µL/well of TMB substrate added to each well, and the plates further incubated at rT in darkness for 15 min. After stopping the reaction, the plates were read using a spectrophotometer (Bio Tek Instruments. Inc. Highland park, USA) at 630 nm. Test samples with S/P % ≥ 50% were determined to be positive while those with S/P% <50% were deemed negative.
The detection of BVDV antigen was done using an antigen capture ELISA (Bovine Viral Diarrhoea Virus antigen test kit/serum plus, IDEXX Laboratories, Switzerland) according to the manufacturer’s kit instructions. Briefly, 50 µL of the detection antibodies were dispensed into a microtiter plate wells pre-coated with monoclonal antibodies specific for BVDV antigen. This was followed by 50 µL of the test sera alongside the negative and positive controls. The plates were then tightly sealed and incubated at 37⁰C for 2 h. The plates were then washed five times with the kit wash buffer. After incubating at rT with 100 µL/well of peroxidase labelled anti-ruminant IgG conjugate for 30 min, the plates were washed five times with the kit wash buffer after which 100 µL/well of TMB substrate were added to each well and the plates incubated at rT for 10 min. After stopping the reaction, the plates were read at 450 nm using spectrophotometer (Bio Tek Instruments. Inc. Highland park, USA). The results were expressed as corrected OD values (S-N) for each sample using negative control. Test samples with (S-N) ≤ 0.300 were considered negative, while test samples with (S-N) ≥ 0.300 were considered positive.