106 milk samples were obtained from cows with records of reproductive abnormalities (n=74) from various locations, including late aborted animals (n=22), cows with a record of retained placenta (n=28), repeat breeders (n=24), and seemingly healthy cows (n=32).
Milk samples were collected a non-invasive collection of milk samples by traditional hand-stripping was approved by the Egyptian Network of Research Ethics Committees (ENREC) and under the supervision of authors. Fifteen ml of the milk were sampled and subdivided into 5 ml aliquots. These were used in the serological, bacteriological, and molecular examination. Samples collected for PCR assay were kept at –80 °C until examination.
Serological examination of milk Samples
Milk Ring test (MRT)
30 μ1MRT antigen (Animal Health Veterinary Laboratories Agency (AHVLA), DEFRA, UK) was mixed with 1ml of fresh milk samples (within 2 hours after collection) mixed well, and kept at 37 °C for 3 hours. The appearance of the purple band at the top of milk was considered positive .
Indirect Enzyme-Linked Immunosorbent Assay (i-ELISA)
The anti-brucella antibodies were detected using an indirect ELISA (i-ELISA) kit (Svanova, Sweden), and the technique was conducted according to the manufacturer's instructions. Before usage, milk samples were centrifuged at 5000 rpm for 5 minutes to remove the cream layer. Using an ELISA reader, the control and test-sample wells' optical densities (OD) were adjusted at 405nm wavelength (ThermoElectron, Finland). The results were then interpreted according to the manufacturer's instructions.
Bacteriological examination of milk Samples
Milk samples were inoculated onto Brucella agar (Oxoid) plates supplemented with Brucella supplement (Oxoid) and 5% horse serum. The plates were then incubated at 37°C in 10% CO2 for 5 to 10 days and examined daily for the presence of colonies. The obtained colonies were identified and classified according to Quinn et al. .
Molecular examination of milk Samples:
DNA extraction from milk samples
DNA was extracted using NZY tissue g DNA isolation kit® according to the manual. 1400 ul of milk samples were transferred to 1.5 microcentrifuge tubes and centrifuged at 3000 rpm for 5 minutes. Then discard the supernatant (milk and cream) and pellets were transferred into new microtubes. Finally, extracted DNA was stored at -80 ºC until used.
For conventional PCR, pair of oligonucleotide primers B4: TGG CTC GGT TGC CAA TAT CAA-3, and B5: CGC GCT TGC CTT TCA GGT CTG were used and amplified a 223-bp fragment  using 2X Taq PCR mix (Tiangene Cat. no. KT201)
The optimized cycle program for PCR was as follows: at 94 ◦C for 3 min., afterthose 40 cycles of the 20s at 95◦C, 1 min at 60 ◦C, and 1 min at 72oC. The last cycle included incubation of the sample at 72 oC for 7 min. The PCR was performed in a DNA thermocycler (Perken Elmer model 9600). The negative control contained sterile water instead of the DNA template, while, the positive control was B. melitensis Rev1DNA.
Electrophoresis of PCR product:
After amplification, 5ul of the PCR product was mixed with 1ul of 6X gel loading dye and subjected to electrophoresis on 1.5% agarose gel at 100V for 30 min. The gel was stained with ethidium bromide and photographed on a UV transilluminator.
Samples were considered positive for Brucella species by using B4 and B5 primers when a single band of DNA at 223 bp was evident in the ethidium bromide-stained gels, compared with the molecular size marker (50 bp DNA ladder).
DNA amplification in Real-Time PCR:
Extracted DNA was amplified by using a real-time PCR detection kit for Brucella (Primer design, JN68G10-21593). The reaction mixture was composed of 5 ul of DNA template and 10 ul of 2x Precision TM master mix in a tube, 1 ul of primer/probe mix, and RNAse/DNAse free water till reached 20 ul. The optimized cycle program was as follows: 1 cycle 37oC for 15 min. for UNG (Uracil-N-Glycosylase) treatment: initial denaturation at 95oC for 10 min., then 50 cycles consisting of 95oC for 10 s and 60oC for 1 min. To confirm the absence of contamination and false-positive result, a negative and positive control reaction respectively were included in the reaction.
Estimation of Relative Sensitivity, specificity, and estimated false positives of different diagnostic tests:
In this study, the relative sensitivity, specificity, and estimated false positive of different serological tests employed in this study were calculated according to Parikh et al., .
Relative Sensitivity= True positive / True positive + false negative x 100
Relative Specificity= True negative / True negative + false positive x 100
Estimated false positive= False positive/ False positive + True negative x 100
Where: True positive or negative reactions are those confirmed as being positive or negative by other two or more tests. False-positive or negative reactions are those confirmed as being positive or negative by other one or non-tests.