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.