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Clinical and Diagnostic Laboratory Immunology, March 1998, p. 259-262, Vol. 5, No. 2
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Evaluation of an Enzyme-Linked Immunosorbent Assay
Using Recombinant Major Surface Protein 5 for Serological
Diagnosis of Bovine Anaplasmosis in Venezuela
Armando
Reyna-Bello,1,2
Axel
Cloeckaert,1
Nieves
Vizcaíno,1,
Mary I.
Gonzatti,3
Pedro M.
Aso,3
Gérard
Dubray,1 and
Michel S.
Zygmunt1,*
Laboratoire de Pathologie Infectieuse et
Immunologie, Institut National de la Recherche Agronomique, 37380 Nouzilly, France,1 and
Laboratorio de
Biología Experimental, Centro de Estudios Biomédicos
y Veterinarios, Universidad Simón Rodríguez, Caracas
1010,2 and
Laboratorio de
Hemoparásitos, Departamento de Biología Celular,
Universidad Simón Bolívar,
Sartenejas,3 Venezuela
Received 14 July 1997/Returned for modification 2 October
1997/Accepted 8 December 1997
 |
ABSTRACT |
An indirect enzyme-linked immunosorbent assay (ELISA) was developed
for the serological diagnosis of bovine anaplasmosis with purified
recombinant major surface protein 5 (MSP5) of Anaplasma marginale produced in Escherichia coli.
Serum antibody responses against MSP5 were detected in calves
experimentally infected with A. marginale as early as
21 days postinfection and reached maximum titers at 28 days
postinfection. The MSP5 ELISA performed with serum samples taken from
field cattle from different regions of Venezuela showed a
seroprevalence of 47%, which seems to be in accordance with the
reported epidemiological status of bovine anaplasmosis in Venezuela.
Positive results obtained in the MSP5 ELISA were further confirmed by
immunoblotting, with the recombinant MSP5 as the antigen. Thus, these
results confirmed the importance of MSP5 as a suitable antigen for the
serological diagnosis of bovine anaplasmosis.
| |
TEXT |
Anaplasma
marginale is an arthropod-borne, rickettsial hemoparasite of
ruminants. It causes a disease called bovine anaplasmosis, which is
characterized by anemia, weight loss, and death. The disease occurs
worldwide, especially in tropical and subtropical regions.
Several serological tests have been described for the diagnosis of
bovine anaplasmosis, including capillary tube agglutination, card agglutination, indirect immunofluorescence,
radioimmunoassay, and enzyme-linked immunosorbent assay (ELISA)
(5, 7-10, 16, 18, 19, 21, 23, 28, 30-32, 35). The major
drawback of these tests is that the antigens used are crude mixtures of A. marginale bodies and erythrocyte material (13,
14). Indeed, A. marginale is an obligatory parasite of
bovine erythrocytes, and efficacious methods for culturing A. marginale are not yet available. Therefore, to produce antigenic
preparations, experimental infection of calves is needed, followed by
purification of Anaplasma bodies from erythrocytes.
This implies that antigen production is costly and lacks
standardization. The current serological tests also lack acceptable
specificity and/or sensitivity, particularly in the detection of
carrier cattle (1, 5, 7-9, 16).
To improve serological diagnosis of bovine anaplasmosis, research has
focused on the identification and characterization of A. marginale antigens by gene cloning and production of recombinant proteins which would be suitable for use in developing standardized diagnostic tests. Among the antigens of interest, five major surface proteins (MSPs) have been described (1-4, 11, 12, 17, 22, 24, 25,
27, 33). These MSPs have been expressed to high levels and
purified from recombinant Escherichia coli. MSP1, -2, and -4 have potential for the development of vaccines, and MSP3 and -5 have
potential for use in improved diagnostic assays (3, 12, 20,
27).
In the present study, we evaluated MSP5 (19 kDa) as a diagnostic
antigen for bovine anaplasmosis in Venezuela, where a high prevalence
(about 50%) of the disease has been reported (10). The
choice of MSP5 was justified, since this protein has been reported to
be conserved in all recognized Anaplasma species, and at least one conserved epitope of this protein (defined by monoclonal antibody [MAb] ANAF16C1) has been found to be
immunodominant in infected cattle (3, 11, 12, 22, 33).
msp5 gene analysis.
A. marginale initial
bodies were isolated from erythrocytes from a splenectomized calf
experimentally infected with a Venezuelan A. marginale
Táchira strain. Briefly, the calf was inoculated intravenously with 4 ml of cryopreserved infected blood (15) showing about 60% rickettsemia. Sixty days later, blood samples were
taken and A. marginale bodies were isolated from infected erythrocytes by differential centrifugation as described previously (26). Genomic DNA was isolated from the A. marginale bodies by standard procedures (4). For PCR,
20-mer primers were designed to amplify the entire msp5 gene
without its signal sequence (to further produce the mature MSP5) but
with its putative transcription terminator sequence according to the
reported msp5 nucleotide sequence (33). These
primers were 19A (5'-GTGTTCCTGGGGTACTCCTA-3') and 19B
(5'-TGATCTGGTCAGCCCCAGCT-3'). PCR was performed as described previously (34). Briefly, amplification reaction mixtures
were prepared in volumes of 100 µl containing 10 mM Tris-HCl (pH
9.0), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 0.2 mg
of gelatin per ml (1× PCR buffer; Appligene, Illkirch, France), 200 µM (each) deoxynucleoside triphosphate, 1 µM (each) primer, 100 ng
of genomic DNA, and 2.5 U of Taq DNA polymerase
(Appligene). The temperature cycling for the amplification was
performed in a GeneAmp PCR system 9600 thermocycler (Perkin-Elmer) as
follows. Cycle 1 was 94°C for 5 min (denaturation); the next 30 cycles were 62°C for 30 s (annealing), 70°C for 30 s
(extension), and 94°C for 30 s (denaturation); and the last
cycle was 62°C for 30 s (annealing) and 70°C for 10 min
(extension). Identity of the PCR-amplified product (714 bp) with
msp5 was first controlled by restriction digestion with the
following restriction enzymes: AluI, EcoRI,
EcoRV, HaeIII, HindIII,
Sau3AI, SspI, StyI, and
TaqI (Appligene). The sizes of the restriction products run
on agarose gel were as expected according to the A. marginale
msp5 published nucleotide sequence (33) (data not
shown). DNA sequencing of the PCR product revealed that only 8 bp on
the entire nucleotide sequence differed from the msp5
published sequence (data not shown). These results are in accordance
with the high level of conservation of MSP5 in
Anaplasma spp. reported previously (22,
33).
Recombinant MSP5 production and purification.
The
PCR-amplified msp5 gene was first cloned in plasmid pCRII
(TA cloning kit; Invitrogen, San Diego, Calif.) according to the manufacturer's instructions, resulting in plasmid
pAR1902 (insert noncoding orientation relative to the
Plac promoter). E. coli INV
F'
(TA cloning kit; Invitrogen) was used as the host strain. Plasmid
isolation and further subcloning procedures were performed as described
by Sambrook et al. (29). Insert orientation was determined
by the sizes of fragments produced after double digestion with
SspI and XhoI. The
KpnI-XhoI fragment of plasmid pAR1902,
gel purified with the GeneClean kit (Bio 101, La Jolla, Calif.), was
further ligated into plasmid pTrcHis C (Xpress system; Invitrogen), cut
by KpnI and XhoI, and gel purified with the
GeneClean kit, resulting in plasmid pAR1903 [insert coding orientation
relative to the Ptrc (trp-lac)
promoter]. E. coli JM109 (Promega, Madison, Wis.) was used
as host strain. The Xpress system (Invitrogen) allows the production of
recombinant proteins fused to six histidine residues at the N-terminal
end of the protein, facilitating the purification of the recombinant
protein by immobilized metal affinity chromatography. For recombinant
MSP5 production and purification, an overnight culture of E. coli JM109 carrying plasmid pAR1903 grown in liquid selective
Luria-Bertani medium containing 50 µg of ampicillin per ml was
adjusted to an optical density at 600 nm of 0.1 in 50 ml of fresh
liquid selective Luria-Bertani medium. After 2 h of growth at
37°C (optical density at 600 nm reached approximately 1.0),
isopropyl-
-D-thiogalactopyranoside (IPTG) (Sigma,
St. Louis, Mo.) was added at a final concentration of 1 mM. The
cultures were allowed to grow for 6 more h, and the E. coli cells were recovered by centrifugation at 8,000 × g for 20 min. Recombinant protein production in E. coli was verified by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Coomassie
blue staining, and immunoblotting with an anti-MSP5 MAb as described
previously (6). As shown in Fig.
1, recombinant MSP5 (detected in
immunoblotting by anti-MSP5 MAb ANAF16C1) was the most abundant protein
in the E. coli (pAR1903) whole-cell lysate. Recombinant
protein purification was performed under denaturing conditions by
immobilized metal affinity chromatography with the Probond resin of the
Xpress system according to the manufacturer's instructions. Purity was
assessed by SDS-PAGE and Coomassie blue staining. Although the eluate
from the affinity column still contained some minor E. coli
protein bands (Fig. 1), the degree of purity of recombinant MSP5
appeared to be satisfactory to develop the MSP5 ELISA.
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FIG. 1.
SDS-PAGE and Coomassie blue staining of whole-cell
lysate of E. coli(pAR1903) (expression of
msp5 was induced with IPTG) (lane 1) and MSP5 affinity
purified from E. coli(pAR1903) (lane 2).
|
|
Recombinant MSP5 ELISA.
Conditions for optimal specificity and
sensitivity were determined by testing negative control sera, sera from
calves experimentally infected with the Venezuelan A. marginale Táchira strain, and anti-MSP5 MAb ANAF16C1 on
microtiter plates coated with different concentrations of purified
recombinant MSP5. The optimal serum dilution was determined as well.
Consequently, the recombinant MSP5 ELISA was performed as follows.
Ninety-six-well polystyrene plates (Nunc, Roskilde, Denmark) were
coated by passive adsorption of recombinant MSP5, used at a
concentration of 1 µg/ml (100 µl per well) and diluted in
phosphate-buffered saline (pH 7.2), overnight at room temperature.
The wells were emptied and washed five times with washing
solution (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France), and then nonspecific binding sites of wells were blocked by incubation for 1 h at 37°C with phosphate-buffered saline
containing 5% skim milk (100 µl per well). After five additional
washings, the serum samples diluted 1/200 in sample dilution buffer
(Sanofi Diagnostics Pasteur) were applied (100 µl per well).
Following incubation for 1 h at 37°C, the wells were again
washed and filled with 100 µl of horseradish peroxidase anti-bovine
immunoglobulin conjugate (Sanofi Diagnostics Pasteur) diluted 1/50,000
in washing solution containing 10% horse serum. After incubation for
1 h at 37°C, the conjugate solution was discarded and the plates
were washed with washing solution. The wells were filled with 100 µl of substrate solution containing TMB (3,3',5,5'-tetramethylbenzidine) (Sanofi Diagnostics Pasteur), and the plates were left at room temperature in darkness for 30 min. Color development was stopped by
adding 50 µl of stopping solution (H2SO4 [2
M]) (Sanofi Diagnostics Pasteur) per well.
A450-630 values were then recorded with an
EL312 plate reader (Bio-Tek Instruments, Highland Park, Vt.) which was
interfaced with a computer.
The cutoff of the MSP5 ELISA was determined by testing 100 negative
control serum samples (taken from cattle in regions of France where
cases of bovine anaplasmosis have not yet been reported) (data not
shown). The mean absorbance value was 0.212, with a standard deviation
of 0.116. Animals which presented serum antibody reactivities with
absorbance values above 0.560 (i.e., mean absorbance value for negative
sera + 3 standard deviations) were scored as positive.
The three calves experimentally infected with the Venezuelan
A. marginale Táchira strain developed serum antibodies from
the
21st day after experimental infection (Fig.
2). The antibody
reactivities
reached maximum absorbance values from the 28th day
until the last day
of experimental infection (day 60). The antibody
kinetics were quite
similar among the three experimentally infected
calves. Thus, the
recombinant MSP5 ELISA appeared to be convenient
for the early
serological diagnosis of bovine anaplasmosis.
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FIG. 2.
Evolution of antibody responses to recombinant MSP5 in
three calves experimentally infected with A. marginale by
ELISA.
|
|
Among serum samples taken from 137 bovines from different regions
representative of the bovine population of Venezuela, 64
were found to
be positive in the recombinant MSP5 ELISA. The range
of absorbance
values, from 0.563 to 2.469, showed a certain degree
of heterogeneity
in the antibody responses against MSP5 (Table
1). Nevertheless, most of these positive
animals showed serum
antibody reactivities with absorbance values above
1.0. The high
number of positive animals detected (46.7%) seems to be
in accordance
with previously reported seroprevalence (57.7%) in
Venezuela by
an indirect immunofluorescence assay (
10).
Fifty-eight percent
of the serum samples tested in the present study
were positive
by indirect immunofluorescence.
Recombinant MSP5 immunoblotting.
SDS-PAGE and
immunoblotting with purified recombinant MSP5 were performed as
described previously (6). The amount of MSP5 loaded per
lane on the gel was 1 µg. The dilution used for bovine sera was 1/50.
The conjugate used to reveal bound bovine serum antibodies in
immunoblotting was peroxidase-conjugated anti-bovine immunoglobulin G (Jackson ImmunoResearch Laboratories, Baltimore, Md.). Anti-MSP5 MAb ANAF16C1 was used as a positive control. Binding of
this MAb was revealed with rabbit anti-mouse immunoglobulin antiserum
(Nordic Immunology, Tilburg, The Netherlands) and
peroxidase-conjugated protein A (Sigma) as described previously
(6).
The sera of the three calves experimentally infected with
A. marginale showed antibody reactivities against
recombinant MSP5
in immunoblotting as early as 21 days postinfection,
thus confirming
the results of the recombinant MSP5 ELISA (Fig.
2 and
3 and data
not shown). Of the 64 serum
samples from Venezuelan cattle that
were found to be positive in the
MSP5 ELISA, 57 reacted positively
with the MSP5 band in immunoblotting
(data not shown).
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FIG. 3.
Immunoblotting after SDS-PAGE of affinity-purified
recombinant MSP5 with sera from a calf experimentally infected with
A. marginale at days  7 (lane 1), 0 (lane 2), 7 (lane 3),
14 (lane 4), 21 (lane 5), 28 (lane 6), 35 (lane 7), 42 (lane 8), and 63 (lane 9) of infection; lane 10, immunoblotting with anti-MSP5 MAb
ANAF16C1.
|
|
In conclusion, both the recombinant MSP5 ELISA and the immunoblotting
results confirmed the importance of MSP5 as a suitable
antigen for the
serological diagnosis of bovine anaplasmosis.
However, to obtain a
sensitivity close to that obtained in indirect
immunofluorescence,
further improvement, which might be achieved
by the inclusion of MSP3
as a diagnostic antigen, is needed (
1,
17). MSP3 has been
found to be particularly useful for detecting
cattle that are long-term
carriers of
A. marginale (
17). However,
as MSP3
has recently been reported to be encoded by a polymorphic,
multigene
family (
2), it remains to be determined which
msp3 alleles could be useful for producing recombinant
protein which
should contain immunodominant conserved epitopes useful
for diagnostic
purposes.
| |
ACKNOWLEDGMENTS |
We thank G. H. Palmer of Washington State University for
providing the MAb ANAF16C1, H. Caballero of Universidad Simón
Bolívar for the sera from the three experimentally infected
calves, and C. Rey of Universidad Simón Bolívar and
Universidad Nacional Experimental Francisco de Miranda for sera from
field cattle.
A. Reyna was supported by the Programme de Coopération
Post-Gradué of the Ministère des Affaires Etrangères
of France.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Pathologie Infectieuse et Immunologie, INRA, 37380 Nouzilly,
France. Phone: 33 2 47 42 78 67. Fax: 33 2 47 42 77 79. E-mail: zygmunt{at}tours.inra.fr.
Present address: Dpto. Microbiología y Genética,
Edificio Departamental, Universidad de Salamanca, Avda. Campo Charro
s/n, 37007 Salamanca, Spain.
| |
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Clinical and Diagnostic Laboratory Immunology, March 1998, p. 259-262, Vol. 5, No. 2
1071-412X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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