Competitive Enzyme-Linked Immunosorbent Assay Based on a Rhoptry-Associated Protein 1 Epitope Specifically Identifies Babesia bovis -Infected Cattle

Abstract

The competitive enzyme-linked immunosorbent assay (cELISA) format has proven to be an accurate, reliable, easily standardized, and high-throughput method for detecting hemoparasite infections. In the present study, a species-specific, broadly conserved, and tandemly repeated B-cell epitope within the C terminus of the rhoptry-associated protein 1 of the hemoparasite Babesia bovis was cloned and expressed as a histidine-tagged thioredoxin fusion peptide and used as antigen in a cELISA. The assay was optimized with defined negative and positive bovine sera, where positive sera inhibited the binding of the epitope-specific monoclonal antibody BABB75A4. The cELISA accurately differentiated animals with B. bovis-specific antibodies from uninfected animals and from animals with antibodies against other tick-borne hemoparasites (98.7% specificity). In addition, B. bovis-specific sera from Australia, Argentina, Bolivia, Puerto Rico, and Morocco inhibited the binding of BABB75A4, confirming conservation of the epitope. The assay first detected experimentally infected animals between 13 and 17 days postinfection, and with sera from naturally infected carrier cattle, was comparable to indirect immunofluorescence (98.3% concordance). The assay appears to have the characteristics necessary for an epidemiologic and disease surveillance tool.

Validation and Field Evaluation of a Competitive Enzyme-Linked Immunosorbent Assay for Diagnosis of Babesia bovis Infections in Argentina

“…A cELISA format based on an epitope located in the C-terminal region of the B. bovis rhoptry-associated protein 1 (CT-RAP-1) has been shown to have high specificity and sensitivity (20,21). However, the first version of this test has been applied to only 130 field serum samples from Morocco, Bolivia, and Puerto Rico, while its validity for Argentina or any other geographic region with large cattle herds has not been assessed (20).…”

“…However, the first version of this test has been applied to only 130 field serum samples from Morocco, Bolivia, and Puerto Rico, while its validity for Argentina or any other geographic region with large cattle herds has not been assessed (20). In its second version, this cELISA features a different cutoff than the first version (21% versus 40%) and was exclusively applied to a fixed set of known-positive and -negative samples in different laboratories in order to validate the reproducibility of its results (21).…”

“…Serological assays are currently most likely to meet these requirements, as B. bovis infection finally leads to an asymptomatic carrier animal state in which the parasite commonly escapes direct detection by PCR, reverse line blot hybridization (RLB), or Giemsa-stained blood smears (10,23). A number of serological methods have been established for the detection of B. bovis-specific antibodies, of which ELISA is considered the most advantageous for epidemiological investigations, as it offers greater sensitivity and objectivity and can be easily adapted to test large numbers of serum samples (3,4,6,7,8,11,20,21,27,31,35,37). Furthermore, this format is amenable to the use of recombinant antigens, which makes it independent of the necessity to produce large amounts of parasites in vivo or in vitro, facilitates standardization, and has the potential to overcome limitations caused by cross-reactivity (22,32).…”

Validation of a Competitive Enzyme-Linked Immunosorbent Assay for Detection ofBabesia bigeminaAntibodies in Cattle

“…We previously described the development of a competitive ELISA for B. bovis that used a similar species-specific epitope within the C terminus of RAP-1 from B. bovis (7) and included an international validation (8). The usefulness of this detection format is evidenced by the recent recognition by the U.S. Department of Agriculture of similar cELISA formats as the official detection tests for the detection of both B. caballi and B. equi (11,12) and by the recognition by the Office International des Epizooties (OIE) of the cELISA as the international standard assay for the detection of these babesial species.…”

“…In a previous study, we demonstrated the high specificity afforded by use of a purified recombinant B. bovis RAP-1 C-terminal construct as the antigen and the inhibition of binding of MAb to a RAP-1-specific epitope by serum antibodies. In addition, we established the ability of the assay to detect both relatively early and long-term carrier infections (7). Finally, we validated the use of the assay for international application (8).…”