Complement Fixation Test
The Brucella abortus strain 99-Weybridge (Pourquier CFT Brucellosis Ag, IDEXX, US) was used. This antigen was standardized to give 50% of fixation with a final dilution of 1/200 of the International Brucella abortus Standard Serum (OIEISS). The CFT was performed according to standard procedures as detailed in the OIE Terrestrial Manual (OIE, 2018, Chapter 3.1.4). Briefly, serum samples were inactivated at 59oC for 30 minutes and diluted to cover a dilution range from 1/4 to 1/512 in Veronal buffer (IDEXX, US). A total of 25 μL of diluted sera, equivalent volume of antigen diluted according to the manufacturer’s (IDEXX, US) instructions and 2U complement (IDVet, France) were added to the tubes and incubated overnight at 2-8oC. After incubation, 25 μL of 2% sheep red blood cells sensitized with rabbit anti sheep red blood cell antibodies, were added and the tubes were incubated at 37oC for 30 minutes, then centrifuged at 1000 gfor 10 minutes. Positive and negative controls were included in the test. The cutoff value for the CFT was ≥50% of hemolysis inhibition (++) at the 1/4 dilution, corresponding to 20 International Units (IU/mL). Serum was considered positive for antibodies to Brucella spp . if it was positive to both the RBT and the CFT.