Expression of Babesia equi Merozoite Antigen 1 in Insect Cells by Recombinant Baculovirus and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay

Abstract

The gene encoding the entire Babesia equi merozoite antigen 1 (EMA-1) was inserted into a baculovirus transfer vector, and a recombinant virus expressing EMA-1 was isolated. The expressed EMA-1 was transported to the surface of infected insect cells, as judged by an indirect fluorescent-antibody test (IFAT). The expressed EMA-1 was also secreted into the supernatant of a cell culture infected with recombinant baculovirus. Both intracellular and extracellular EMA-1 reacted with a specific antibody in Western blots. The expressed EMA-1 had an apparent molecular mass of 34 kDa that was identical to that of native EMA-1. The secreted EMA-1 was used as an antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA differentiated B. equi-infected horse sera from Babesia caballi-infected horse sera or normal horse sera. The ELISA was more sensitive than the complement fixation test and IFAT. These results demonstrated that the recombinant EMA-1 expressed in insect cells might be a useful diagnostic reagent for detection of antibodies to B. equi.Babesia equi is a tick-borne hemoprotozoan parasite that causes piroplasmosis in horses. Equine piroplasmosis is an economically important disease that is characterized by fever, anemia, and icterus and that is mostly prevalent in tropical and subtropical areas as well as in temperate climatic zones (15). Areas of endemicity include many parts of Europe, Africa, Arabia, and Asia (15). Due to the almost worldwide distribution of various tick vectors, the introduction of a carrier into areas of nonendemicity should be prevented.The complement fixation test (CFT) and indirect fluorescent-antibody test (IFAT) have commonly been used to detect B. equi infection. However, these serologic tests are generally restricted by the antibody detection limits and cross-reactivity (4, 5). Besides CFT and IFAT, the enzyme-linked immunosorbent assay (ELISA) with B. equi lysate antigen has been used for detection of antibodies to B. equi (19). However, the ELISA is hindered by a limited antigen supply and poor specificity (4,5,19).Merozoite antigen 1 (EMA-1) is the major surface protein of B. equi (8). It is considered an important candidate with which to develop a diagnostic reagent for detection of antibodies to B. equi (9, 10). A competitive inhibition ELISA (CI-ELISA) that can detect antibodies to B. equi based on a monoclonal antibody to EMA-1 has been developed by Knowles et al. (11), who demonstrated that it can be more sensitive than CFT in detecting antibodies to B. equi. The CI-ELISA offers the advantage of a high degree of specificity but the disadvantage of the requirement of a complicated operating procedure. Therefore, there is a need to develop a simple ELISA method.Here, we established a highly specific, sensitive, and simple ELISA method using recombinant EMA-1 expressed in insect cells by baculovirus. Our data indicated that the recombinant baculovirus-expressed EMA-1 should be a useful diagnostic reagent for detection of antibodies to B. equi in horses. MATERIALS AND MET...

Seroepidemiological Evidence for the Possible Presence of Babesia (Theileria) equi and Babesia caballi Infections in Donkeys in Western Xinjiang, China

“…Several reports on the prevalence of equine babesiosis in Mongolia, which shares a border with the Xinjiang, have been published [1,3,14]. These studies demonstrated that both B. equi and B. caballi infections in horses are widespread in Mongolia.…”

Diagnosis of Equine Piroplasmosis in Brazil by Serodiagnostic Methods with Recombinant Antigens.

“…However, these serological tests are generally restricted by antibody detection limits and cross reactivity [1,8]. Recently, we developed a latex agglutination test (LAT) and enzyme-linked immunosorbent assay (ELISA) with recombinant antigens to diagnose B. equi infection and B. caballi infection, respectively, and demonstrated that both methods are highly specific and sensitive [3,4,11,12]. In the present study, we investigated the prevalence of equine piroplasmosis in Brazil by the LAT and ELISA.…”

“…LAT with recombinant equi merozoite antigen-1 (EMA-1) expressed in insect cells by baculovirus for diagnosis of B. equi infection in horses was performed as described previously [11,12]. ELISA with recombinant P48 protein expressed in Escherichia coli by pGEX vector for diagnosis of B. caballi infection in horses was carried out as described elsewhere [3,4].…”

“…LAT was considered positive when agglutination was observed at dilutions of 1:4 and above. d) Antibodies to B. caballi were detected by ELISA with recombinant P48 antigen expressed in E. coli (11,12). ELISA was considered positive when an optical density at 415 nm equal to or greater than 0.2 was observed at dilutions of 1:80 and above.…”

Seroepidemiologic Studies on Babesia equi and Babesia caballi Infections in Horses in Jilin Province of China

“…However, these serological tests are generally restricted by antibody detection limits and cross reactivity [1,4]. Previously, we have developed enzyme-linked immunosorbent assays (ELISAs) using recombinant antigens, and demonstrated that the ELISAs can be used as an alternative to CFT and IFAT for diagnosis of B. equi and B. caballi infections, respectively [2,3,5,6]. In the present study, we investigated the prevalence of equine piroplasmosis in Jilin province of the People's Republic of China by the ELISAs using recombinant antigens.…”