Cloning of a Truncated Babesia equi Gene Encoding an 82-Kilodalton Protein and Its Potential Use in an Enzyme-Linked Immunosorbent Assay

Abstract

To isolate Babesia equi genes encoding immunodominant proteins, a cDNA expression library prepared from B. equi mRNA was immunoscreened with B. equi-infected horse serum. Eighteen positive cDNA clones were obtained, and the clone that showed the strongest immunoreactivity, designated Be82, was further characterized. The Be82 gene consisted of 1,953 bp and contained a partial open reading frame lacking the 5-terminal sequence. As shown by Western blot analyses, immune sera from mice intraperitoneally injected with the Be82 gene product recognized the 82-and 52-kDa proteins of B. equi but not those of Babesia caballi. The glutathione S-transferase fusion protein expressed in Escherichia coli that was purified and used as the antigen in the enzyme-linked immunosorbent assay reacted specifically with B. equi-infected horse sera. These results suggest that the Be82 gene product is a potential diagnostic antigen candidate in the detection of B. equi infection in horses that will be useful both in the performance of epidemiological studies and in the granting of quarantine passes.Equine piroplasmosis is an economically important tickborne protozoan disease of horses reported worldwide. This disease is caused by Babesia equi and Babesia caballi (18). Babesia parasites destroy erythrocytes and induce fever, anemia, and icterus in infected horses (12). These parasites are usually detected in blood smears only during the acute stage of the infection, and animals that recover from the disease remain parasite carriers. These carriers, as well as those previously exposed or infected, can be identified serologically (6).Standard serological tests for babesiosis are the complement fixation test (CFT) and the indirect fluorescent antibody test (IFAT) (5), both of which require large amounts of parasite antigens, particularly in large-scale seroepidemiological surveys. Because of its low sensitivity and specificity, the CFT is unable to detect latent infection and fails to accurately discriminate between negative and carrier animals (24). With the IFAT, on the other hand, standardization is difficult, considering the subjectivity of the reader in assessing the results (3, 4). Weiland (25) has demonstrated the strong cross-reactivity of anti-B. caballi horse serum with the lysate of B. equi-infected erythrocytes using an enzyme-linked immunosorbent assay (ELISA). However, with Western blot analysis, Ikadai et al. (8) have noted an erratic or inconsistent cross-reactivity between the anti-B. equi serum and the lysate of B. caballi-infected erythrocytes. In view of the absence of equine piroplasmosis in Japan and the increasing number of horses imported to the country from places where the infection is endemic, the development of a highly specific and sensitive diagnostic system for B. equi is of urgent necessity.In this study, we isolated and determined the nucleotide sequence of a cDNA clone from a cDNA expression library prepared from B. equi mRNA and we attempted to develop and assess the efficacy of the ELISA system using the g...

Immunokinetics of Theileria equi specific antibodies: a comparison in serial and single dilution ELISA antibody end titres

“…Application of conventional serial dilution for detection of antibodies against T. equi parasite is now well established (6,10,12,20). The diagnostic specificity and sensitivity of single dilution ELISA for detection of T. equi antibodies in equine serum has previously been reported (6).…”

Production of recombinant EMA-1 protein and its application for the diagnosis of Theileria equi using an enzyme immuno assay in horses from São Paulo State, Brazil

“…The arrow indicates the full-length fusion protein with 34.2 kDa, based on the theoretical molecular weight. baculovirus insect cell system have been developed (JAFFER et al, 2010;SALIM et al, 2008;HUANG et al, 2003;CUNHA et al, 2002;HIRATA et al, 2002;TANAKA et al, 1999;XUAN et al, 2001a, b). However, to our best knowledge this is the fi rst report of the development of a recombinant EMA-1 expressed by the gene from T. equi Jaboticabal strain.…”

“…Although good crude antigenic preparations from infected erythrocytes can be used in sensitive and specific ELISA, the test is hindered by a limited antigen supply (BALDANI et al, 2004). More recently, several ELISA tests have been developed with the use of recombinant antigens, demonstrating that it can be a useful test for the identification of chronically infected T. equi horses (JAFFER et al, 2010;SALIM et al, 2008;HUANG et al, 2003;CUNHA et al, 2002;HIRATA et al, 2002;XUAN et al, 2001a, b;TANAKA et al, 1999).…”

Identification of a Specific Antigenic Region of the P82 Protein of Babesia equi and Its Potential Use in Serodiagnosis

“…The ELISA with the Be82 gene antigen was shown to be highly specific only for B. equi antibodies when a cutoff value was set at a relatively high level (4). Although the number of serum samples tested was insufficient to evaluate whether the cross-reactivity of the GST/Be82 antigen with B. caballi-infected sera was authentic, there was a possibility that the B. equi Be82 protein might have an antigen in common with B. caballi or have antigenicity similar to that of B. caballi.…”

“…We recently reported on the efficacy of the Be82 gene product with glutathione S-transferase (GST/Be82) in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of B. equi infection (4). The ELISA with the Be82 gene antigen was shown to be highly specific only for B. equi antibodies when a cutoff value was set at a relatively high level (4).…”