Bacterial strains isolation, phenotypic characterization, and
molecular typing
The phenotypic characterization of the Brucella isolates (n=41)
was done at the genus level based on colony morphology, urease activity,
oxidase, and catalase production. Then, species determination was
carried out by phage lysis using Tbilisi (Tb), Izatnagar (Iz), Weybridge
(Wb), and Rough-Canis (R/C) phages. Agglutination with monospecific A,
M, and R antisera besides, CO2 requirement, H2S production, growth on
thionin, and basic fuchsin (20 µg/ml in serum dextrose agar) were
performed to identify Brucella at the biovar level. Full typing
at these three levels was done according to Alton et al. (1988), and OIE
(2018). DNA was extracted from bacterial culture harvested in
Phosphate- buffered saline with PH 7.2 and inactivated at 100ºC
for 15 min using QIAamp® DNA Mini Kit (QIAGEN, Hilden, Germany). DNA
concentrations were measured by NanoDrop™ 2000/2000c Spectrophotometers
(Nanodrop Technologies, Bremen, Germany). Molecular typing using
AMOS-PCR described by Bricker and Halling (1994) and Bricker et al.
(2003) was conducted under the following conditions: initial
denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 30
sec, 55°C for 40 sec, and 72°C for 45 sec, with a final extension of
72°C for 10 min. The extracted genomic DNA from the B. melitensisbv3 reference strain Ether (ATCC 23458) was used for the allele
assignment control.