Brucellosis is a chronic zoonotic disease of great public health significance caused by Gram-negative, facultative, intracellular bacteria of the genus Brucella. There are 6 classic types in the genus Brucella, namely, B. abortus, B. melltensis, B. ovis, B. Neotormae, B. suis,and B. canis. The disease causes animals to miscarry and become infertile, it can also be transmitted to humans by touching infected animals or eating infected food, such as dairy products. B.melitensis, B.bovis, and B.suis are three major types of Brucella that are prevalent in China, of these, brucellosis in sheep is most common, followed by Brucella in cattle, all of these are the most common sources of brucellosis in humans. Also, infected animals also develop orchitis, arthritis, and other symptoms. Animals have varying degrees of susceptibility to the disease. In recent years, the number of human and animal infections caused by Brucella has been increasing, which seriously threatens people's health and the development of animal husbandry. Over 0.5 million new human brucellosis cases are reported every year worldwide[4, 5]. In China, the number of sheep and goat populations infected with Brucella is on the rise and is highly endemic in some areas. Because brucellosis is mostly a chronic disease ,and there are still some problems with diagnostic methods, it is still an important public health problem in many countries, especially in backward rural areas. Following Brucella is infected, treatment is difficult, so the emphasis on brucellosis is prevention and control. Conventional live, attenuated strains B. melitensis and B. abortion B19 are currently the most commonly used vaccines. However, these vaccines also have some disadvantages, such as the possibility of miscarriage in pregnant animals. Also, vaccination often complicates the distinction between naturally infected and vaccinated animals, which is not conducive to the diagnosis and control of brucellosis. So, a timely and accurate diagnosis is important for the prevention and control of the disease.
There are many methods available for the diagnosis of brucellosis in animals, such as serological diagnosis, etiological diagnosis, molecular biology diagnosis, etc.. Although the bacteriological diagnosis is the gold standard, it is not only time consuming and laborious, but also easily causes operator infection. Molecular biology pathogen detection technology has the characteristics of safety, reliability, high sensitivity, strong specificity, and easy operation. It has begun to be applied in the detection of Brucella. The most widely used method is the polymerase chain reaction (PCR) method. Since PCR is a method for detecting nucleic acids, it requires very precise equipment. Generally, the expensive equipment prevents this method from being universally popularised. Therefore, it is necessary to design a convenient, rapid and economical diagnosis method. Enzyme-linked immunosorbent assay (ELISA), the rose bengal test (RBT) and standard tube agglutination test (SAT) can discover the existence of Brucella infection by detecting antibodies in the serum. However, methods have some disadvantages, like false positive and false negative results. Compared with other diagnostic methods, the outstanding advantages of ELISA are higher sensitivity, specificity, and simple operation. Therefore, it has become one of the most widely used detection methods.
When performing ELISA tests, the choice of antigen is important if you want to achieve higher accuracy. Compared with other antigenic molecules of Brucella, lipopolysaccharide (LPS) of smooth Brucella genus is the major virulence determinant currently known, and infected animals are prone to produce antibodies against it. Therefore, LPS is the most commonly used antigen in the serological diagnosis of brucellosis. However, serum diagnostic tests for smooth Brucella LPS as a diagnostic antigen cannot distinguish infections caused by those cross-reactive species, examples are Yersinia enterocolitica O9, Vibrio cholerae, Escherichia, and Salmonella, those bacteria often lead to false positive. Many studies have proved that the Brucella outer membrane proteins (OMPs) have strong immunoreactive, and are suitable for the diagnosis of brucellosis as a substitute for LPS, mainly including OMP16, periplasmic protein 26 (BP26), OMP2b, and OMP31[7, 14–16]. False positive results from cross-reactive antibodies can be reduced by using OMPs to replace previous diagnostic antigens.
Although some previous studies have used Brucella OMPs to diagnose brucellosis and achieved good experimental results, there are still many deficiencies that need to be improved. In this research, we used some bioinformatics tools to predict the B and T cell epitopes of the four Brucella OMPs mentioned above. Immediately afterwards, we designed a recombinant multiepitope protein (rMEP) and evaluated this protein as a serum diagnostic antigen for the detection of goat and bovine brucellosis.