Improved enzyme-linked immunosorbent assay using C-terminal truncated recombinant antigens of Babesia bovis rhoptry-associated protein-1 for detection of specific antibodies.

Abstract

An enzyme-linked immunosorbent assay (ELISA) based on a recombinant rhoptry-associated protein-1 (RAP-1) of Babesia bovis has been previously developed, but it was imperfect because some cross-reactions were still present in Babesia bigemina-infected bovine sera. To improve its accuracy for the specific detection of the antibodies to B. bovis, we constructed three C-terminal truncated recombinant antigens of the RAP-1-rCT1 (amino acids [aa] 301 to 408), rCT2 (aa 388 to 490), and rCT3 (aa 466 to 565)-by using a baculovirus expression system and evaluated their diagnostic potentials using ELISA. rCT1 and rCT2 were better diagnostic antigens in their sensitivities and diagnostic efficiencies than rCT3, although none of the recombinant antigens showed any cross-reactivity to B. bigemina-infected bovine sera. These results confirmed that the N-terminal 300-aa region caused cross-reactivity of the entire RAP-1 antigen, and the C-terminal truncated recombinant antigens were shown to be useful reagents for species-specific serodiagnosis.

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JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2004, p. 1601–1604
0095-1137/04/$08.00?0DOI: 10.1128/JCM.42.4.1601–1604.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 42, No. 4
Improved Enzyme-Linked Immunosorbent Assay Using C-Terminal
Truncated Recombinant Antigens of Babesia bovis Rhoptry-Associated
Protein-1 for Detection of Specific Antibodies
Suthisak Boonchit,1Xuenan Xuan,1Naoaki Yokoyama,1Will L. Goff,2Suryakant D. Waghela,3
Gale Wagner,3and Ikuo Igarashi1*
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro,
Hokkaido 080-8555, Japan1; Animal Disease Research Unit, Agricultural Research Service, U.S. Department of
Agriculture, Pullman, Washington 991642; and Department of Veterinary Pathobiology, College
of Veterinary Medicine, Texas A&M University, College Station, Texas 778433
Received 14 April 2003/Returned for modification 26 May 2003/Accepted 8 August 2003
An enzyme-linked immunosorbent assay (ELISA) based on a recombinant rhoptry-associated protein-1
(RAP-1) of Babesia bovis has been previously developed, but it was imperfect because some cross-reactions were
still present in Babesia bigemina-infected bovine sera. To improve its accuracy for the specific detection of the
antibodies to B. bovis, we constructed three C-terminal truncated recombinant antigens of the RAP-1—rCT1
(amino acids [aa] 301 to 408), rCT2 (aa 388 to 490), and rCT3 (aa 466 to 565)—by using a baculovirus
expression system and evaluated their diagnostic potentials using ELISA. rCT1 and rCT2 were better diag-
nostic antigens in their sensitivities and diagnostic efficiencies than rCT3, although none of the recombinant
antigens showed any cross-reactivity to B. bigemina-infected bovine sera. These results confirmed that the
N-terminal 300-aa region caused cross-reactivity of the entire RAP-1 antigen, and the C-terminal truncated
recombinant antigens were shown to be useful reagents for species-specific serodiagnosis.
Bovine babesiosis is an economically important tick-borne
disease of cattle in tropical and subtropical regions of the
world (8). The disease is caused by the intraerythrocytic pro-
tozoan parasites Babesia bovis and Babesia bigemina. The clin-
ical signs developed as a result of B. bovis and B. bigemina
infections are similar, and they are characterized by fever,
anemia, and icterus in the infected cattle (3). Generally, the
disease caused by B. bovis is more severe and more difficult to
control than that caused by B. bigemina (9). Acute infections
are usually diagnosed by microscopic examination of blood
smears, whereas subclinical infections can be identified sero-
logically (14). Differential diagnosis between B. bovis and B.
bigemina infections will lead to a better understanding of the
epidemiology with regard to species distribution and preva-
lence and also will provide useful information for disease con-
trol strategies (3). Therefore, accurate diagnosis of B. bovis
infection is essential for the development of disease control
measures and epidemiological surveys.
Previously, we demonstrated that the entire rhoptry-associ-
ated protein-1 (RAP-1) of B. bovis synthesized by a baculovirus
expression system could be used as a diagnostic enzyme-linked
immunosorbent assay (ELISA) antigen for the detection of
antibodies to B. bovis, but there were some cross-reactions with
another pathogen, B. bigemina (1). The cross-reactions may be
due to the high degree of sequence identity in the first 300
amino acids (aa) located at the N-terminal region between the
RAP-1 derivates of both parasites (11, 12). On the contrary,
the C terminus is represented by conserved sequences that
serve as B-cell epitopes that can be helpful in the development
of species-specific diagnostic assays (3a). Therefore, in order to
develop a more-specific serological test without cross-reactivity
to B. bigemina, we constructed three truncated recombinant
antigens derived from the C-terminal region of RAP-1 by the
baculovirus system and then evaluated their sensitivities and
specificities in ELISA.
MATERIALS AND METHODS
Parasites. The Texas strain of B. bovis (3b) was maintained by a microaeroph-
ilous stationary-phase culturing system (7).
Cloning of truncated RAP-1 genes. B. bovis genomic DNA was isolated from
the culture as described previously (1). Oligonucleotide primers were synthesized
with restriction enzyme-compatible ends for the subsequent cloning to find a
specific immunogenic region in C-terminal RAP-1 (Table 1). Three kinds of
DNA fragments encoding various regions of C-terminal RAP-1 were amplified
from the genomic DNA by PCR with the primer pairs RCT11-RCT12, RCT21-
RCT22, or RCT31-RCT32 (Fig. 1). Each of the amplified DNA samples was
digested with the corresponding restriction enzymes, BamHI and XhoI, and then
inserted into a pBluescript SK(?) cloning vector. The PCR amplification and
confirmative nucleotide sequencing of the target DNA fragments were per-
formed as described previously (1).
Construction of recombinant baculoviruses containing a deletion series of the
RAP-1 gene. Each of the 3?-terminal RAP-1 gene fragments was subcloned into
the BamHI and XhoI sites of a baculovirus transfer vector, pFastBac Ht (Life
Technologies, Grand Island, N.Y.), and the vectors were designated pFB/RAP-1
CT1, pFB/RAP-1 CT2, and pFB/RAP-1 CT3. By using these transfer vectors,
three kinds of recombinant viruses, AcRAP-1 CT1, AcRAP-1 CT2, and Ac-
RAP-1 CT3, were produced, respectively, according to the previously described
method (1).
Immunoblot analysis. Immunoblot analysis was carried out by using the RAP-
1-specific mouse immune serum (1).
ELISA. According to the same method described previously (1), soluble lysates
of the recombinant baculovirus-infected insect cells were prepared as ELISA
antigens. All sera were examined by using the recombinant RAP-1 (rRAP-1)
antigen (1) and three C-terminal truncated recombinant antigens, rCT1, rCT2,
* Corresponding author. Mailing address: National Research Cen-
ter for Protozoan Diseases, Obihiro University of Agriculture and
Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
Phone: 81-155-49-5641. Fax: 81-155-49-5643. E-mail: igarcpmi
@obihiro.ac.jp.
1601

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and rCT3. Ninety-six-well microtiter plates (Nunc, Roskilde, Denmark) were
coated with 50 ?l of each recombinant antigen (10 ?g/ml) at 4°C overnight. The
details of the ELISA procedure were described previously (1). The cutoff line
was defined as the mean value plus threefold standard deviations of the optical
density (OD) obtained from 30 noninfected serum samples. The following def-
initions were used to calculate the corresponding diagnostic parameters—true-
positive number (tp) of B. bovis-infected bovine sera showing a positive reading,
false-negative number (fn) of B. bovis-infected bovine sera showing a negative
reading, false-positive number (fp) of sera from healthy or B. bigemina-infected
cattle showing a positive reading, true-negative number (tn) of sera from healthy
or B. bigemina-infected cattle showing a negative reading: sensitivity ? tp ?
100/(tp ? fn); specificity ? tn ? 100/(tn ? fp); diagnostic efficiency ? (tn ? tp)
? 100/(tp ? fp ? tn ? fn) (4).
Sera. Serum samples from cattle experimentally infected with B. bovis (n ? 14)
or B. bigemina (n ? 12) and noninfected control serum samples (n ? 30) from
healthy cattle were kindly provided by Washington State University (Pullman)
and Texas A&M University (College Station).
RESULTS
Cloning and expression of RAP-1 deletion clones. Three
3?-terminal RAP-1 gene fragments, RAP-1 CT1 (encoding aa
301 to 408), RAP-1 CT2 (encoding aa 388 to 490), and RAP-1
CT3 (encoding aa 466 to 565), were cloned by PCR (Fig. 1)
and inserted into the recombinant baculovirus vectors. The
extracts of recombinant baculovirus-infected insect cells with
the corresponding AcRAP-1 CT1, AcRAP-1 CT2, or Ac-
RAP-1 CT3 were subjected to immunoblot analysis with the
anti-RAP-1-specific mouse serum. The serum recognized the
expected recombinant proteins with a molecular mass of ap-
proximately 16 kDa in the recombinant baculovirus-infected
cell extracts, but it did not react with the noninfected cell
extract (Fig. 2). All recombinant proteins reacted with B. bovis-
infected bovine serum but not with B. bigemina-infected bovine
serum (data not shown). The results indicated the successful
expression of three of the recombinant proteins derived from
the C-terminal B. bovis RAP-1 in the insect cells.
Evaluation of ELISAs with rRAP-1 deletion antigens. In
order to improve the specificity of the rRAP-1 antigen, three
C-terminal truncated antigens were constructed and subjected
to ELISA, and their sensitivity and specificity were evaluated.
From the control results using 30 noninfected sera, cutoffs for
the OD at 415 nm were determined as 0.141, 0.140, 0.136, and
0.133 for rRAP-1, rCT1, rCT2, and rCT3, respectively (Table
2). The actual reactivities of the recombinant antigens against
each group of the serum samples are shown in Fig. 3. Maxi-
mum OD values were observed in the rRAP-1 ELISA (Fig.
3A), whereas the absorbance decreased in the rCT1, rCT2, and
rCT3 ELISAs (Fig. 3B to D). All of the B. bovis-infected serum
samples showed a certain positive value in the rRAP-1, rCT1,
and rCT2 ELISAs (Fig. 3A to C), but one of these samples had
a lower OD value than the defined cutoff line in the rCT3
ELISA (Fig. 3D), resulting in sensitivities of 100% for the
rRAP-1, rCT1, and rCT2 ELISAs and 92.8% for the rCT3
ELISA (Table 2). No OD value from the B. bigemina-infected
bovine serum samples was over the cutoff in the rCT1, rCT2,
and rCT3 ELISAs (Fig. 3B to D), whereas 10 B. bigemina-
infected bovine serum samples had OD values above the cutoff
in the rRAP-1 ELISA (Fig. 3A), resulting in specificities of
100% for the rCT1, rCT2, and rCT3 ELISAs and 76.2% for the
rRAP-1 ELISA (Table 2). The diagnostic efficiency of the
rCT1 and rCT2 antigens showed a perfect percentage, 100%
(Table 2). The results indicated that the ELISA with the rCT1
or rCT2 antigen provided a highly specific and sensitive system
for the serological diagnosis of B. bovis infection.
DISCUSSION
The effective serodiagnosis of bovine babesiosis largely de-
pends on the availability and quality of prepared antigens.
Recombinant protein-based serological tests may achieve high
sensitivity and specificity because of the high concentration of
TABLE 1. Oligonucleotides used for DNA amplification
OligonucleotideSequence (5? to 3?)
RCT11........................ACGGATCCAGATGGATAAAGAAA
RCT12........................ACCTCGAGTTGACCGATGTTTTC
RCT21........................ACGGATCCGAGTTTTTCAGGGAA
RCT22........................ACCTCGAGAAACTCATGTATGAT
RCT31........................ACGGATCCGCTACCATGAAAGTCTTG
RCT32........................ACCTCGAGAAACGCATCTCATCAG
FIG. 1. Schematic representation of C-terminal fragments of the
RAP-1 gene. Nucleotide (nt) and amino acid positions are indicated by
numbers based on the sequence.
FIG. 2. Immunoblot analysis of C-terminal recombinant antigens
of B. bovis RAP-1 expressed in insect cells. Noninfected (lane 1) and
infected insect cells with AcRAP-1 CT1 (lane 2), AcRAP-1 CT2 (lane
3), and AcRAP-1 CT3 (lane 4) were reacted with mouse anti-B. bovis
RAP-1 polyclonal antibody.
TABLE 2. Diagnostic performance of recombinant RAP-1 and C-
terminal truncated antigens by ELISA
Antigen
OD415cutoff
valuea
Sensitivity
(%)
Specificity
(%)
Diagnostic efficiency
(%)
rRAP-1
rCT1
rCT2
rCT3
0.141
0.140
0.136
0.133
100
100
100
92.8
76.2
100
100
100
82.1
100
100
98.2
aOD415, optical density at 415 nm.
1602BOONCHIT ET AL. J. CLIN. MICROBIOL.

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the immunoreactive antigen and the lack of host protein com-
ponents from the crude antigen preparations (2). We previ-
ously introduced a serodiagnostic ELISA using an entire
RAP-1 gene product of B. bovis for the detection of antibodies
to B. bovis in cattle (1). However, some cross-reactions re-
mained in the B. bigemina-infected bovine sera, and further
improvement was required. The B. bovis RAP-1 shows a high
identity to B. bigemina RAP-1 (p58) in the N-terminal 300-aa
region (11). It has been demonstrated that the C terminus
contains conserved, repeated amino acid sequences (13). Fur-
thermore, a competitive ELISA using a monoclonal antibody
specific for the repeat region of the C terminus has recently
FIG. 3. Reactivities of several panels of bovine sera in ELISA with the C-terminal recombinant antigens of RAP-1. Maximum and minimum
values (bars) and median values (?) from the ELISA are shown. The bar represents the OD at 415 nm of B. bovis-infected bovine sera (n ? 14)
(column 1), B. bigemina-infected bovine sera (n ? 12) (column 2), and noninfected bovine sera (n ? 30) (column 3).
VOL. 42, 2004IMPROVED ELISA FOR DETECTION OF B. BOVIS INFECTION1603

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been shown to possess diagnostic potential with exquisite spec-
ificity (3a). In the present study, we synthesized three C-ter-
minal recombinant antigens of B. bovis RAP-1 and then eval-
uated whether the recombinants were available for species-
specific serodiagnosis without any cross-reactions to B.
bigemina infection.
The present results showed that the entire recombinant an-
tigen derived from the C-terminal region of RAP-1 had no
cross-reactivity to B. bigemina-infected bovine sera, confirming
that the N-terminal region of RAP-1 caused the cross-reactiv-
ity in ELISA (12). The rCT1 and rCT2 ELISAs gave satisfac-
tory results, and their sensitivities, specificities, and diagnostic
efficiencies were 100%. However, the number of bovine sera
was small, and further evaluation with a large number of bo-
vine serum samples will be necessary. The CT1 and CT2 frag-
ments contain a large region of repeated 23-aa sequences (10).
The periodicity of a tandem repeated sequence is 7 from aa
317 to 477 of RAP-1, and making a secondary structure with a
predicted high antigenicity is considered necessary (10). When
the same B. bovis-infected bovine sera were used, the OD
values of B. bovis-infected bovine sera in the rCT1 ELISA were
higher than those in the rCT2 and rCT3 ELISAs but lower
than those in the rRAP-1 ELISA, indicating that rCT1 con-
tains a larger immunodominant region capable of inducing a
stronger humoral immune response to B. bovis infection than
rCT2 and rCT3 but which induces a weaker response than
rRAP-1. Various genes have been identified in Babesia para-
sites. They have been used to produce their corresponding
recombinant antigens and evaluated for their diagnostic po-
tentials in ELISA for Babesia infections (5, 6, 13, 15). The
selection of target recombinants that are highly specific to the
parasites is important for the development of novel serodiag-
nosis (2).
In conclusion, we described the construction of three trun-
cated C-terminal recombinant antigens of B. bovis RAP-1,
rCT1, rCT2, and rCT3 and evaluated their diagnostic potential
in ELISA. The results of ELISA indicated that rCT1 and rCT2
are novel diagnostic antigens. These antigens are available for
the serodiagnostic ELISA of B. bovis infection. This serodiag-
nosis will allow accurate epidemiological surveys as well.
ACKNOWLEDGMENTS
This work was supported by Grants-in-Aid for Scientific Research
from the Japan Society for the Promotion of Science and by a grant
from The 21st Century COE Program (A-1), Ministry of Education,
Culture, Sport, Science, and Technology, Japan.
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