IntroductionThe diagnosis of brucellosis largely relies on tiger red plate agglutination test (RBPT). However, it is difficult to distinguish between natural infection antibody positive and vaccination antibody positive, nevertheless, the identification of specific Brucella species natural infection.MethodsHere, we analyzed the structure of main outer membrane proteins (OMPs), OMP25 and OMP31 fromBrucella ovis(B. ovis) andBrucella melitensis(B. melitensis), which are the main pathogens of sheep brucellosis, and found the OMP25 and OMP31 could be used as the differential antigens forB. ovisandB. melitensisantibody. Then we expressed the OMP25 fromB. ovis(OMP25o) and OMP31 fromB. melitensis(OMP31m).ResultsThey have equally efficiency in antibody detection of vaccinated sheep serum, consistent with the RBPT results. However, through epidemiological investigations, we found some RBPT positive samples were negative by the OMP31m based serum antibody detection, but these samples gave positive results by the OMP25o. We verified these OMP31m negative but OMP25o positive samples byB. ovisandB. melitensisspecific primers based PCR detection, and all these samples wereB. melitensisnegative. However, four out of six samples areB. ovispositive. These results showed that we could use the OMP25o and OMP31m to diagnose sheep brucellosis antibody, especially to discriminate the infection of theB. ovis.DiscussionCurrently, China has not yet approved a vaccine based onB. ovisandB. ovispositive samples should be naturally infected. There should be some implicit transmission ofB. ovisin Jilin province. Further epidemiological investigation should be conducted to monitor theB. ovisnatural infection.