Cloning of a truncated Babesia equi gene encoding an 82-kilodalton protein and its potential use in an enzyme-linked immunosorbent assay

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.     

Identification of clone-9 antigenic protein of Theileria uilenbergi and evaluation of its application for serodiagnosis

The pathogenic protozoan parasite Theileria uilenbergi is one of the causative agents of theileriosis in small ruminants in China. The infection results in great economical losses in the northwest part of China. Efforts are underway to establish an enzyme-linked immunosorbent assay (ELISA) based on a T. uilenbergi immunodominant recombinantly expressed protein using different approaches in order to perform epidemiological studies in the area. In this study, we describe the possible use of the clone-9 protein for this purpose, which was identified as a potential immunogenic piroplasm protein by random sequencing of cDNA library clones followed by bioinformatic analyses. The clone-9 gene was partially recombinantly expressed and used for the development of an indirect ELISA for the detection of circulating antibodies in sera of T. uilenbergi-infected sheep. No cross-reactivity was observed in serum from animals infected with Theileria lestoquardi. The cut-off was calculated at 48.6% positivity using 25 serum samples from uninfected animals. A total of 101 field samples collected from an endemic area in China were used to evaluate the clone-9 ELISA for its use in the field.     

Immunochromatographic test for simultaneous serodiagnosis of Babesia caballi and B. equi infections in horses

An immunochromatographic test for the simultaneous detection of Babesia caballi- and B. equi-specific antibodies (BceICT) was developed using a recombinant B. caballi 48-kDa rhoptry protein (rBc48) and a recombinant truncated B. equi merozoite antigen 2 (rEMA-2t). An evaluation of the ability of the BceICT to detect antibodies in sera from uninfected horses and experimentally infected horses showed high sensitivities and specificities of 83.3% (10/12 sera) and 92.9% (52/56 sera), respectively, for the anti-B. caballi antibody and 94.1% (16/17 sera) and 88.2% (45/51 sera), respectively, for the anti-B. equi antibody. Results from the detection of antibodies in field-collected sera indicated that the BceICT results corresponded with those of enzyme-linked immunosorbent assays (ELISA), showing 91.8% correspondence (67/73 sera) for B. caballi and 95.9% correspondence (70/73 sera) for B. equi, and that the BceICT results also corresponded with the ICT for B. caballi and for B. equi, both of which were 98.2% (55/56 sera). The comparable results of the ICT and ELISA and the simplicity and rapidity of the performance of the ICT suggest that the BceICT would be a feasible test for the simultaneous serodiagnosis of both agents of equine babesiosis in the field.     

Expression of a gene encoding the major antigenic protein 2 homolog of ehrlichia chaffeensis and potential application for serodiagnosis

The major antigenic protein 2 (MAP2) homolog of Ehrlichia chaffeensis was cloned and expressed. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of human monocytic ehrlichiosis. The recombinant protein, which contained a C-terminal polyhistidine tag, had a molecular mass of approximately 26 kDa. The antigen was clearly identified by Western immunoblotting using antihistidine antibody. However, immune sera failed to react with the recombinant on immunoblots when the antigen was denatured by heat or reduced using beta-mercaptoethanol. The recombinant MAP2 (rMAP2) was used in an ELISA format with 60 blinded serum samples. Twenty of the serum samples were previously demonstrated to contain antibodies reactive with E. chaffeensis by indirect immunofluorescence assays (IFAs). The remaining 40 samples were seronegative. All samples negative by IFA were also found to be negative for antibodies against the rMAP2 of E. chaffeensis by using the ELISA. Only 1 of 20 IFA-positive samples tested negative in the rMAP2 ELISA. There was 100% agreement using IFA-negative samples and 95% agreement using IFA-positive samples, resulting in a 97.5% overall agreement between the two assays. These data suggest that the rMAP2 homolog of E. chaffeensis may have potential as a test antigen for the serodiagnosis of human monocytic ehrlichiosis. To our knowledge, this recombinant is unique because it is thus far the only E. chaffeensis recombinant antigen that has been shown to work in an ELISA format.     

High-level expression and purification of a truncated merozoite antigen-2 of Babesia equi in Escherichia coli and its potential for immunodiagnosis

The gene encoding a truncated merozoite antigen-2 (EMA-2t) of Babesia equi was cloned and highly expressed in Escherichia coli as a glutathione S-transferase fusion protein (G-rEMA-2t). Both G-rEMA-2t and rEMA-2t (after the removal of glutathione S-transferase) had good antigenicity. Either Western blot analysis with rEMA-2t or enzyme-linked immunosorbent assay (ELISA) with G-rEMA-2t clearly discriminated the sera of horses experimentally infected with B. equi from sera of horses infected with Babesia caballi and healthy horses, although rEMA-2t was not suitable for ELISA, probably owing to its poor absorbability to the plates. The specific antibodies in B. equi-infected horses were detectable during both acute and latent infection (6 to 244 days postinfection). Horse sera from Jilin Province, China, were examined by the two tests. The seroprevalence of B. equi was 49.2% (31 of 63 sera) by Western blot analysis with rEMA-2t and 47.6% (30 of 63 sera) by ELISA with G-rEMA-2t. The correspondence was 98.4% (62 of 63 sera) between the two tests. The results indicate that G-rEMA-2t and rEMA-2t proteins should be suitable antigens for the development of an effective immunodiagnostic assay due to their high sensitivity, specificity, and great yield.     

Cloning of a novel Babesia equi gene encoding a 158-kilodalton protein useful for serological diagnosis

In this study, we characterized a Babesia equi Be158 gene obtained by immunoscreening a B. equi cDNA expression phage library with B. equi-infected horse serum. The Be158 gene consists of an open reading frame of 3,510 nucleotides. The recombinant Be158 gene product was produced in Escherichia coli and used for the immunization of mice. In Western blot analysis, mouse immune serum against the Be158 gene product recognized 75- and 158-kDa proteins from the lysate of B. equi-infected erythrocytes. In an indirect fluorescent-antibody test with the mouse immune serum, the Be158 antigen appeared in the cytoplasm of Maltese cross-forming parasites (which consist of four merozoites) and was located mainly in the extraerythrocytic merozoite body. When the recombinant Be158 gene product was used in an enzyme-linked immunosorbent assay as a serological antigen, it was found to react to B. equi-infected horse sera, indicating that the Be158 gene product is useful as a serologically diagnostic antigen for B. equi infection.     

A monoclonal antibody defines a geographically conserved surface protein epitope of Babesia equi merozoites.

Babesiosis is a tick-borne hemoparasitic disease affecting horses worldwide. To investigate mechanisms of immunity to this parasite, the antibody response of infected horses to Babesia equi merozoite proteins was evaluated. Immunoprecipitation of B. equi merozoite antigens with sera from infected horses revealed 11 major proteins of 210, 144, 108, 88, 70, 56, 44, 36, 34, 28, and 25 kDa. Monoclonal antibody (MAb) 36/133.97, which binds to live merozoites, immunoprecipitated proteins of 44, 36, 34, and 28 kDa. When immunoprecipitations were performed with in vitro translation products of merozoite mRNA, MAb 36/133.97 immunoprecipitated proteins of 38, 28, 26, and 23 kDa which comigrated with proteins immunoprecipitated by sera from infected horses at 10(-3) to 10(-4) dilutions. In Western blot analysis, MAb 36/133.97 recognized proteins of 44, 36, 34, and 28 kDa, and a 28-kDa protein was identified by sera from infected horses at a dilution of 10(-4). MAb 36/133.97 bound to B. equi isolates from Florida and Europe. Furthermore, the binding of MAb 36/133.97 to merozoite proteins was inhibited by sera of infected horses from 19 countries. Collectively, these data indicate MAb 36/133.97 binds to a geographically conserved peptide epitope on multiple B. equi merozoite proteins, including a merozoite surface protein, and MAb 36/133.97 reacts with a B. equi protein immunodominant in infected horses.     

Characterization of the major antigenic protein 2 of Ehrlichia canis and Ehrlichia chaffeensis and its application for serodiagnosis of ehrlichiosis

Canine monocytic ehrlichiosis, caused by Ehrlichia canis or Ehrlichia chaffeensis, can result in clinical disease in naturally infected animals. Coinfections with these agents may be common in certain areas of endemicity. Currently, a species-specific method for serological diagnosis of monocytic ehrlichiosis is not available. Previously, we developed two indirect enzyme-linked immunosorbent assays (ELISAs) using the major antigenic protein 2 (MAP2) of E. chaffeensis and E. canis. In this study, we further characterized the conservation of MAP2 among various geographic isolates of each organism and determined if the recombinant MAP2 (rMAP2) of E. chaffeensis would cross-react with E. canis-infected dog sera. Genomic Southern blot analysis using digoxigenin-labeled species-specific probes suggested that map2 is a single-copy gene in both Ehrlichia species. Sequences of the single map2 genes of seven geographically different isolates of E. chaffeensis and five isolates of E. canis are highly conserved among the various isolates of each respective ehrlichial species. ELISA and Western blot analysis confirmed that the E. chaffeensis rMAP2 failed to serologically differentiate between E. canis and E. chaffeensis infections.     

The cloned C-terminal region of a Trypanosoma cruzi P ribosomal protein harbors two antigenic determinants.

A DNA strategy was designed to characterize the antigenic site(s) within a lambda gt11 cloned 35-amino-acid antigenic peptide, identified with antibodies from patients with chronic Chagas' heart disease (cChHD) and systemic lupus erythematosus (SLE) as the C-terminal portion of a Trypanosoma cruzi P ribosomal protein. The 198-bp cDNA insert was digested with AluI, resulting in two DNA segments that were recloned in lambda gt11. To identify specific antigenic determinants, the recombinant phage and the purified recombinant antigens were probed with sera from clinically characterized subjects. Chronic ChHD and SLE sera defined a linear epitope common to both diseases within the 15 C-terminal residues of the parasite P ribosomal protein. It is also shown that the cloned 35-amino-acid peptide contained an antigenic site unique to cChHD.     

Identification of antigenic epitopes in an alanine-rich repeating region of a surface protein antigen of Streptococcus mutants.

A surface protein antigen (PAc) of Streptococcus mutans with a molecular mass of 190 kDa is considered to play an important role in the initial attachment of this streptococcus to the tooth surface. Two internal repeating amino acid sequences are present in the PAc molecule. One repeating region located in the N-terminal region is rich in alanine (A-region), and the other, located in the central region, is rich in proline (P-region). To identify antigenic epitopes on the A-region of the PAc protein, 82 sequential overlapping synthetic decapeptides covering one of the repetitive units of the A-region were synthesized. In the epitope scanning analyses using murine antisera raised against recombinant PAc (rPAc), multiple antigenic epitopes were found in the repetitive unit of the A-region, and some of them reacted with antisera to rPAc from BALB/c, B10, B10.D2, and B10.BR mice. In particular, a peptide YEAALKQY (residues 366 to 373) was recognized by anti-rPAc sera from all four strains of mice. The reactivities of anti-rPAc sera in the epitope scanning were confirmed by using a purified synthetic peptide, NAKATYEAALKQYEADLAA (corresponding to residues 361 to 379). Furthermore, antisera against a surface protein antigen PAg (SpaA) of Streptococcus sobrinus from BALB/c mice reacted strongly to residues 330 to 337, 362 to 369, and 366 to 373 of the PAc protein by the epitope scanning analysis. An AKATYEAALKQY (residues 362 to 373 of the PAc protein)-like sequence, AKANYEAKLAQY, was found within the A-region of S. sobrinus PAg, suggesting that the amino acid sequences AKA-YEA and YEA-L-QY may be major cross-reactive epitopes of the S. mutans PAc protein and the S. sobrinus PAg protein.     

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

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.     

Development of specific immunoglobulin Ga (IgGa) and IgGb antibodies correlates with control of parasitemia in Babesia equi Infection

In this study, the kinetics of specific immunoglobulin G (IgG) isotypes were characterized in Babesia equi (Theileria equi)-infected horses. IgGa and IgGb developed during acute infection, whereas IgG(T) was detected only after resolution of acute parasitemia. The same IgG isotype profile induced during acute infection was obtained by equi merozoite antigen 1/saponin immunization.     

Expression of P32 protein of goatpox virus in Pichia pastoris and its potential use as a diagnostic antigen in ELISA

The present study was undertaken to express goatpox virus (GTPV) P32 protein in Pichia pastoris and evaluate its potential use as a diagnostic antigen in ELISA. The amplified P32 gene of GTPV was cloned into pPICZαA vector and characterized by PCR, restriction enzyme digestion and sequencing. The characterized linear recombinant plasmids were transformed in Pichia host GSII5 strain by electroporation and the zeocin resistant Pichia transformant containing P32 gene was selected and confirmed by PCR. The expression of P32 protein in Pichia was induced with 0.5% methanol at 30 °C. The optimum expression was observed at 72 h post-induction and the yield was 100 mg/L of culture. The expressed protein was precipitated with polyethylene glycol and analyzed by SDS-PAGE and Western blot using GTPV specific serum and GTPV-P32 protein specific monoclonal antibody. Further, the protein precipitated with acetone was evaluated as diagnostic antigen in indirect ELISA in order to replace the whole GTPV. The standardized P32 protein based indirect ELISA had relative specificity and sensitivity of 84.2% and 94.2–100%, respectively when compared with serum neutralization test and whole virus based indirect ELISA. This study showed a potential of the yeast expressed GTPV-P32 protein as safe antigen in ELISA for seroepidemiological study of the capripox infection in sheep and goats, in India as well as capripox enzootic countries.     

Molecular cloning of a Babesia caballi gene encoding the 134-kilodalton protein and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay

A Babesia caballi gene encoding the 134-kDa (BC134) protein was immunoscreened with B. caballi-infected horse serum. An enzyme-linked immunosorbent assay (ELISA) using recombinant BC134 protein could effectively differentiate B. caballi-infected horse sera from Babesia equi-infected or noninfected control horse sera. These results suggest that the recombinant BC134 protein is a potential diagnostic antigen in the detection of B. caballi infection.     

Recombinant Helicobacter bilis protein P167 for mouse serodiagnosis in a multiplex microbead assay

Infection of mice with Helicobacter bilis is widespread in research and commercial mouse colonies. Therefore, sensitive, specific, and high-throughput assays are needed for rapid and accurate testing of mice in large numbers. This report describes a novel multiplex assay, based on fluorescent microbeads, for serodetection of H. bilis infection. The assay requires only a few microliters of serum to perform and is amenable to a high-throughput format. Individual microbead sets were conjugated to purified, H. bilis-specific, recombinant proteins P167C and P167D and bacterial membrane extracts from H. bilis and Helicobacter hepaticus. For detecting H. bilis infection in the microbead multiplex assay, P167C and P167D provided significantly higher sensitivities (94 and 100%, respectively) and specificities (100 and 95%, respectively) than membrane extract (78% sensitivity and 65% specificity). Microbead multiplex assay results were validated by enzyme-linked immunosorbent assay. Purified recombinant proteins showed low batch-to-batch variation; this feature allows for ease of quality control, assay robustness, and affordability. Thus, recombinant antigens are highly suitable in the multiplex microbead assay format for serodetection of H. bilis infection.     

Identification of Babesia bovis L-lactate dehydrogenase as a potential chemotherapeutical target against bovine babesiosis

In this study, we characterized a novel Babesia bovis cDNA clone obtained by immunoscreening the cDNA expression phage library with B. bovis-infected bovine serum. The genetic analyses showed that it contained an open reading frame of 993 bp, which was considered to encode B. bovis L-lactate dehydrogenase (BbLDH: E.C. 1.1.1.27) because of the strikingly high amino acid identities of its gene product to the LDHs of Plasmodium falciparum and Toxoplasma gondii. Immunological analyses with the anti-recombinant BbLDH mouse serum showed that 36 kDa of the native BbLDH was expressed not only in the cytoplasm of intra- and extraerythrocytic parasites but also along the membrane of infected erythrocytes. The kinetic properties of recombinant BbLDH proved a certain enzymatic activity of LDH, and the activity was significantly inhibited by the addition of gossypol, a competitive inhibitor of protozoan LDHs. Moreover, 100 microM of the gossypol irretrievably arrested the in vitro growth of B. bovis. The results demonstrated that BbLDH provides a suitable drug target for the design of novel babesial chemotherapeutics.     

Characterization of the Babesia gibsoni P18 as a homologue of thrombospondin related adhesive protein

Thrombospondin related adhesive proteins (TRAPs) are well conserved among several apicomplexans. In this study, we reported the identification of the Babesia gibsoni P18, designated by our group previously, as a homologue of TRAP and renamed the P18 as the B. gibsoni TRAP (BgTRAP). The amino acid sequence of BgTRAP consists of several typical regions, including a signal peptide, a vonWillebrand factor A domain, a thrombospondin type 1 domain, a transmembrane region, and a cytoplasmic C-terminus. The B. gibsoni infected dog serum recognized recombinant BgTRAP expressed in E. coli by Western blotting. The antiserum against recombinant BgTRAP recognized an 80kDa protein in the lysate of infected erythrocytes (RBCs), which was detectable in the micronemal area of the parasites by confocal microscopic observation. The BgTRAP showed a bivalent cation-independent binding to canine RBC, and the specific antiserum was found to inhibit the growth of B. gibsoni in the infected severe combined immune deficiency mice given canine RBC. These results suggest that the BgTRAP is a new member of TRAP family identified from the merozoites of B. gibsoni and functionally important in merozoite invasion; this protein may be useful as a vaccine candidate against canine B. gibsoni infection.     

Antiphagocytic function of an IgG glycosyl hydrolase from Streptococcus equi subsp. equi and its use as a vaccine component

EndoSe from Streptococcus equi subsp. equi is an enzyme hydrolyzing glycosyl groups on IgG, analogous to EndoS from Streptococcus pyogenes. We here show that the activity of EndoSe leads to an antiphagocytic function and may thus be a contributory factor to immune evasion of S. equi. Despite the damaging effect that EndoSe has on IgG, antibodies against EndoSe can neutralize its function. Antibodies against EndoSe restored the opsonic activity of specific opsonizing antibodies. Mice infected with either S. equi subsp. equi or subsp. zooepidemicus or S. pyogenes could be protected by vaccination with EndoSe. It is speculated that EndoSe could be a suitable vaccine candidate against streptococcal infections.     

Inhibitory effect of allicin on the growth of Babesia and Theileria equi parasites

Allicin is an active ingredient of garlic that has antibacterial, antifungal, antiviral, and antiprotozoal activity. However, the inhibitory effects of allicin on Babesia parasites have not yet been examined. In the present study, allicin was tested as a potent inhibitor against the in vitro growth of bovine and equine Babesia parasites and the in vivo growth of Babesia microti in a mouse model. The in vitro growth of Babesia bovis, Babesia bigemina, Babesia caballi, or Theileria equi was inhibited by allicin in a dose-dependent manner and had IC₅₀ values of 818, 675, 470, and 742 μM, respectively. Moreover, allicin significantly inhibited (P?<?0.001) invasion of B. bovis, B. bigemina, B. caballi, and T. equi into the host erythrocyte. Furthermore, mice treated with 30 mg/kg of allicin for 5 days significantly (P?<?0.05) reduced the parasitemia of B. microti over the period of the study. To further examine the potential synergism of allicin with diminazene aceturate, growth inhibitory assays were performed in vitro and in vivo. Interestingly, combinations of diminazene aceturate with allicin synergistically potentiated its inhibitory effects in vitro and in vivo. These results indicate that allicin might be beneficial for the treatment of babesiosis, particularly when used in combination with diminazene aceturate.