The Study On Key Proteins For The Identification Of Brucella Species

BackgroundBrucella is a facultative intracellular pathogen responsible of a zoonotic disease called brucellosis. Brucellosis is one of the B category infectious diseases according to the People’s Republic of the Communicable Disease Prewention Act and it is one of the most common zoonotic diseases worldwide. In China, the incidence of human brucellosis has increased gradually since 2000, which causes significant economic and human health losses.The gold standard in the diagnosis of brucellosis is the isolation of Brucella bacteria. However, culture sampling sensitivity is often low, depending on the disease stage, Brucella species, culture medium, quantity of circulating bacteria and blood culture technique employed. A variety of serologic tests, such as rose-bengal plate agglutination test(RBPT), standard serum agglutination test(SAT) and complement fixation test(CFT), have been applied to brucellosis, of which the SAT is the most widely used. Additionally, ELISA coated by Brucella lipopolysaccharide is also gradually used in the diagnosis of brucellosis. However, all these classical serological methods can’t distinguish vaccinated from infected animals. With the development of genomics and proteomics, a lot reaserches try to solve this problem in protein area, but the results are not satisfactory.Human brucellosis prevention and control is inseparable with animal brucellosis control. It is reported that the trend of human brucellosis is consistent with the trends of animal brucellosis. In China, infected cattles and sheeps are the main infection source of brucellosis, therefore, the control and prevention of brucellosis in cattle and sheep contributes to the reduction of prevalence of human brucellosis and is of great significance to the reduction of the economic losses of people.As infected cattles are one of the main infection sources of brucellosis, the prevention of cattle brucellosis is very important. The live attenuated strain is considered to be the best vaccine of brucellosis. However, traditional diagnostic methods can’t distinguish between natural infection and immunization, so it is hard to decide whether the seropositive cattles should be killed and whether vaccinated cattles have been killed by mistake. If infected cattle been mistaken for vaccinated one, it would damage people’s health. Oppositely, It would cause great loses of economics. So, we urgently need a method to distinguish vaccinated or infected.At present, the most universal cattle vaccines are M5 and S2, which are brucella melitensis and brucella suis attenuated strain, respectively. But, the mainly Brucella abortus is the naturally infected specie. Some researchers have found that the gene of Omp31 is existed in all brucella species except Brucella abortus. So we can distinguish Brucella abortus infected serum from others by detecting the antibody of Omp31, and then differentiate between vaccination and natural infection.ObjectiveThe objective of the study is to differentiate between vaccinated and natural infected cattle, accord to distinguish burucella abortus from others by detecting the antibody of identifying protein in seropositive serums and then differentiate between vaccination and natural infection. Therefore to provide laboratory evidence to decide if seropositive cattle should be killed.MethodsClone and express these two proteins of brucella with the prokaryotic expression system, and detected the antigenicity of recombinant proteins by Western blot methods. Verify the ability of differentiating burucella abortus from others by western blot method, using serum of immune by different brucella strains as primary antibodies. Then summary all the results and get the conclusion.Results1. Two recombinant plasmids were successfully constructed.2. Two recombinant proteins were successfully expressed and purified.3. Western blot results:Of the seven M5 vaccine immune sera, six were positive for the Bp26 and Omp31. While five of the six S2 vaccine immune sera were positive for the Bp26 and Omp31, and nine of the ten natural infected sera were positive for Bp26 but negative for Omp31.ConclusionOmp31 and Bp26 were expressed successfully. It was verified that these two proteins can distinguish brucella abortus from others. Vaccinated and natural infected sera of cattles can be differentiated. And we can also use these proteins to identify the non-specific agglutination between some microbes and brucella antigens of SAT.