Potential Value of Major Antigenic Protein 2 for Serological Diagnosis of Heartwater and Related Ehrlichial Infections

Origins of C. ruminantium strains, E. canis, and E. chaffeensis.Three strains ofC. ruminantium from Zimbabwe (Crystal Springs, Highway, and Palm River [4]), one strain from Sudan, Um Banein (11), and the Antigua strain (3) from the Caribbean were grown in bovine aortic endothelial cells (2, 4). E. canis (Oklahoma strain) and E. chaffeensis (Arkansas isolate) were grown in the canine macrophage cell line DH82 in minimum essential medium containing 12.5% fetal bovine serum and 200 mM l-glutamine (7). Organisms were harvested from the culture following the complete lysis of host cells, and rickettsial genomic DNA was isolated as previously described (5, 20).

Amplification of the map2 genes of C. ruminantium strains of different geographic origins and of themap2-like genes of E. chaffeensis andE. canis.Primers AB249 and AB251, corresponding to flanking regions of the map2 gene (open reading frame [ORF] 1) of C. ruminantium clone pF5.2 (17), were synthesized. Primers for PCR amplification ofE. canis and E. chaffeensis were selected by using information from one of three sources: (i) sequences determined after alignment of conserved regions of the map2gene of C. ruminantium and the msp5 gene ofA. marginale, (ii) sequences obtained from newly sequenced regions of the map2-like genes of E. canisor E. chaffeensis, and (iii) primers developed to sequence the pF5.2 clone of the Crystal Springs strain of C. ruminantium (17). Primers (Table1) were synthesized by the Oligonucleotide Synthesis Laboratory (Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville) or National Biosciences, Inc. (Plymouth, Minn.). These primers were used in a PCR assay to amplify DNA of the strains of C. ruminantium,E. canis, and E. chaffeensis. Briefly, target DNA (1 ng) was amplified in a mixture of 0.4 mM deoxynucleoside triphosphates, 0.5 μM (each) primer, 20 mM MgCl₂, 100 mM KCl, 200 mM Tris-HCl (pH 8.2), 60 mM (NH₄)₂SO₄, 1% Triton X-100, 100 μg of nuclease-free bovine serum albumin/ml, and 2.5 U of nativePfu DNA polymerase. PCR assays, for the amplification ofmap2 and map2-like genes of C. ruminantium, E. canis, and E. chaffeensis, were performed at 94°C for 5 min, followed by 30 cycles of varying denaturing, annealing, and extension temperatures and times (Table 2). A final extension step at 72°C was performed for 10 min in all reactions. The amplicons were analyzed by gel electrophoresis on a 0.8% agarose gel in 1× TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM disodium EDTA). PCR products were purified by using the QIAquick Spin PCR purification kit (Qiagen, Inc., Chatsworth, Calif.) as described by the manufacturer. The DNA was eluted in 50 μl of 10 mM Tris-HCl, pH 8.3.

Cloning and restriction enzyme analysis.Amplicons were concentrated to 20 μl in a Speed Vac concentrator (Savant Instruments, Inc., Farmingdale, N.Y.) and ligated toEcoRV-digested pBluescript SK(+) (Stratagene, La Jolla, Calif.) at 16°C in the presence of T4 DNA ligase at an insert/vector molar ratio of 8:1. The recombinant plasmid was used to transform competent E. coli XL1-Blue cells, and transformants were grown on Luria-Bertani agar plates in the presence of ampicillin (50 μg/ml), 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) (50 mg/ml), and isopropyl-β-d-thiogalactopyranoside (IPTG) (0.2 mg/ml). Positive colonies were selected, inoculated into terrific broth (21) containing ampicillin (250 μg/ml), and incubated overnight at 37°C with vigorous shaking. Plasmid DNA from positive colonies was extracted by the boiling prep method (9). DNA was reconstituted in Tris-EDTA buffer (pH 8.0) containing 20 μg of DNase-free RNase/ml and was analyzed on a 0.8% agarose gel. Recombinant clones containing the map2 genes of different strains of C. ruminantium were digested with seven restriction enzymes (AccI, HincII,EcoRV, EcoRI, XbaI, BamHI, and HindIII) to compare their various sizes to those obtained for the reported Crystal Springs strain (GenBank accession no. G289922). The resulting restriction enzyme-digested DNA was analyzed on a 0.8% agarose gel.

DNA sequencing and sequence data analysis.The DNA insert in pBluescript SK(+) was sequenced as double-stranded DNA by using Sequenase (United States Biochemical) as recommended by the manufacturer. T3 and T7 plasmid-specific promoter primers were used in initial reactions, and then new oligonucleotides were synthesized based on the sequences obtained (“primer walking”). Insert DNA was completely sequenced on both strands. DNA sequences were analyzed with the PCGENE (Intelligenetics) and Genetics Computer Group (University of Wisconsin) programs. Database searches were performed with the BLAST program of the National Center for Biotechnology Information (Bethesda, Md.).

C. ruminantium recombinant MAP2 production and purification.The 627-bp ORF encoding MAP2 of C. ruminantium was subcloned into the expression vector pFLAG (International Biotechnologies, Inc., New Haven, Conn.) and transformed into E. coli for expression of recombinant protein (17). An E. coli colony expressing MAP2 was selected and expanded in L broth (1% tryptone–0.5% yeast extract–1% NaCl), induced with 0.1 mM IPTG, and purified by monoclonal antibody affinity chromatography, as described by the manufacturer of the pFLAG vector system. The protein concentration was determined, and the size and purity were analyzed on a 12% acrylamide gel. The antigenicity of the protein was analyzed by using an indirect ELISA and hyperimmune sheep serum.

Antisera.Positive sera were collected from sheep born and reared under Amblyomma-free conditions in Zimbabwe and experimentally infected with the Welgevonden strain of C. ruminantium (17). False-positive sera were obtained from sheep (numbers 056, 060, 062, and 066) born and raised in regions of Zimbabwe where neither Amblyomma ticks nor the disease has ever been observed. Animals were confirmed free of infection by tick pickup, PCR, and challenge (18). Negative sera were obtained from sheep born and reared under Amblyomma-free conditions in Zimbabwe. Normal and anti-E. canis dog sera (obtained from the Veterinary Medical Teaching Hospital at the University of Florida) were confirmed positive or negative by an indirect fluorescent antibody test.

Indirect ELISA.ELISA plates were coated by using isolated recombinant MAP2 (4 μg/ml) in 0.14 M NaCl–20 mM Na₂HPO₄–3 mM KH₂PO₄(pH 7.2) containing 0.02% (wt/vol) sodium azide (phosphate-buffered saline [PBS]-azide) at 50 μl per well and incubated overnight at 4°C. After five washes with buffer containing PBS-azide and 0.05% (vol/vol) Tween-20, the wells were blocked for 60 min at room temperature with 1.0% (wt/vol) bovine serum albumin in PBS-azide. The plates were washed five times and incubated for 60 min with sera or supernatant diluted in PBS-azide (50 μl per well). The plates were washed (five times) and incubated at room temperature for a further 60 min in the presence of rabbit anti-species-specific immunoglobulin G conjugated to alkaline phosphatase (1:1,000; 50 μl per well). After another five washes, the substrate, p -nitrophenylphosphate (1 mg/ml; Sigma), in 0.16 M NaHCO₃–0.14 M Na₂CO₃–0.02 M MgCl₂ (pH 9.6) was added at 50 μl per well to each well and incubated for 30 min at room temperature. Absorbance was read at 405 nm.

Immunoblot of C. ruminantium and E. canis antigens. C. ruminantium (Fig. 5a, lanes A) or E. canis (Fig. 5a, lanes B) lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Membranes were blocked with Tris-buffered saline (TBS) (0.1 M Tris HCl–0.9% NaCl [pH 8.0])–0.25% gelatin, then reacted with an anti-C. ruminantium antiserum (from sheep 483; Crystal Springs strain), anti-recombinant MAP2 antiserum (from sheep 177), or anti-E. canis antiserum (dog Mukanya; E. canis Oklahoma). Membranes were washed three times in TBS containing 0.25% Tween 20, reacted with peroxidase-labeled protein G (Zymed) for 2 h, washed three times as described above, and developed by incubation with 4CN peroxidase substrate (Kirkegaard and Perry, Gaithersburg, Md.).

Nucleotide sequence accession numbers. The nucleotide sequences of the map2 genes have been assigned the following GenBank accession numbers: Antigua map2 gene, AF117726 ; Highway map2 gene, AF117727 ; Palm River map2gene, AF117728 ; Um Banein map2 gene, AF117729 . The nucleotide sequences of the map2-like genes have been assigned the following GenBank accession numbers: E. canis map2-like gene, AF117730 ; E. chaffeensis map2-like gene, AF117731 .