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Clinical and Diagnostic Laboratory Immunology, May 2003, p. 451-458, Vol. 10, No. 3
1071-412X/03/$08.00+0     DOI: 10.1128/CDLI.10.3.451-458.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Development of a Recombinant Protein-Based Enzyme-Linked Immunosorbent Assay and Its Applications in Field Surveillance of Rodent Mice for Presence of Immunoglobulin G against Orientia tsutsugamushi

Yeau-Ching Wang,^* Ting-Yu Jian, Lih-Jeng Tarn, Yao-Wen Hung, Hai-Yuan Chao, Da-Der Ji, and Hwan-Wun Liu

Institute of Preventive Medicine, National Defense Medical Center, Taipei 100, Taiwan

Received 14 October 2002/ Returned for modification 3 December 2002/ Accepted 28 January 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
A recombinant protein containing the immunodominant conserved epitope region of the 56-kDa outer membrane protein of the Karp strain of Orientia tsutsugamushi was purified to near homogeneity using recombinant DNA techniques. The purified protein was used to immunize rabbits and produced an antibody that could recognize different strains of O. tsutsugamushi, as demonstrated both by Western blotting and immunofluorescence assay. An enzyme-linked immunosorbent assay (ELISA) based on this recombinant protein was developed to detect antibody (immunoglobulin G [IgG]) against O. tsutsugamushi in mice captured in different districts of Taiwan during 2000 to 2001. A significant difference was found in the antibody seroprevalence rates of Suncus murinus mice captured in different districts of Taiwan (²_{4, 0.95} = 26.64; P < 0.05). Furthermore, a significant difference of IgG seropositivity rates was observed among different kinds of mice (²_{5, 0.95} = 93.85; P < 0.05). Antibody seropositivity rates were higher in Bandicota indica (100%), Rattus flavipectus (96.17%), and Rattus losea (95.83%) than in Rattus norvegicus (86.05%) and Rattus mindanensis (83.67%) (²_{diff, 5, 0.95} = 12.59, P < 0.05). The lowest antibody seropositivity rate (54.4%) was observed in Suncus murinus. Antibody seropositivity rates of mice from different districts differed significantly because of the significant difference in antibody seroprevalence rates for S. murinus. The results of this study indicated that the recombinant protein ELISA developed in this study could be used to conduct large-scale surveillance of rodent mice for the presence of antibody against O. tsutsugamushi. The high seroprevalence rates in rodent mice (except S. murinus) suggest that people residing in these districts are at increased risk of developing O. tsutsugamushi infection.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Scrub typhus is an acute febrile disease endemic in Asia-Pacific regions including Korea, Japan, China, the Philippines, Thailand, and Taiwan (8, 25, 26, 28, 29, 32, 33, 35). The causative agent, Orientia (formerly Rickettsia) tsutsugamushi, is a gram-negative obligate intracellular bacterium which has been isolated from a variety of eukaryotic host cells and is transmitted via chiggers. O. tsutsugamushi causes local inflammation accompanying eschars at the site of infection, which then spreads systemically (6). Orientia isolates are antigenically diverse, resulting in numerous serotypes. Several antigenic variants, such as representative strains Gilliam, Karp, Kato, Boryong, Shimokoshi, Kawasaki, Kuroki, and other isolates have been reported (7, 8, 11, 22-24, 33, 36).

Traditionally, confirmatory diagnosis of scrub typhus is generally based on serologic procedures, such as the Weil-Felix test, the immunoperoxidase test, and the immunofluorescence (IF) test (4, 5). However, these serodiagnostic tests have shortcomings or requirements which limit their usefulness. A more practical approach to the diagnosis of scrub typhus is to detect the antibody using a specific and immunodominant protein of O. tsutsugamushi or to detect antigen using a specific antibody. The 56-kDa immunodominant protein of O. tsutsugamushi is reactive with group-specific and strain-specific monoclonal antibodies, suggesting the existence of group-specific and strain-specific epitopes in this molecule (30). The immunogenicity of this protein suggests that it is an a priori diagnostic antigen candidate. Several studies dealing with the antibody responses obtained by using this recombinant 56-kDa protein as bait in enzyme-linked immunosorbent assay (ELISA) have been reported (9, 13, 15). ELISA has been demonstrated to have, as demonstrated by Kim et al. (13) and Land et al. (15), high sensitivity and specificity for serodiagnosis of O. tsutsugamushi.

In recent years, both the reported and identified cases of scrub typhus have increased in various districts of Taiwan (9a). The reasons for this increase remain to be investigated. However, epidemiologic surveillance of rates of seroprevalence against O. tsutsugamushi among different kinds of rodent mice in different districts might be an appropriate first step in exploring the reasons responsible for the increasing frequency of reports of scrub typhus cases from Taiwan. In this study, an ELISA was developed using recombinant truncated proteins which contain the epitope region of the 56-kDa outer membrane protein of the Karp strain. This test was used to survey seroprevalence rates among different kinds of rodent mice against O. tsutsugamushi in different districts of Taiwan.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and vectors. Escherichia coli DH5 or HB101 was used for cloning, and E. coli BL21(DE3) was used for overexpression of proteins under the control of the phage T7 promoter. The plasmid vector pRSET-B (Invitrogen) was used for the cloning of an expression plasmid that expresses the histidine-tagged truncated protein rP56.

Construction of plasmid expressing truncated 56-kDa outer membrane protein of O. tsutsugamushi. Genomic DNA was extracted from L929 cells infected with the Karp strain of O. tsutsugamushi using the method described by Maniatis et al. (20) with some modifications. Extracted DNA resuspended in TE buffer (10 mM Tris-Cl, pH 8.0; 1 mM EDTA, pH 8.0) was used as a template in the PCR for the amplification of amino acids (a.a.) 31 to 274 of the Karp strain p56-kDa outer membrane protein (Kp56) gene of O. tsutsugamushi. A pair of primers corresponding to nucleotides 643 to 663 and nucleotides 1357 to 1377 (STA31-KpnI, 5'-GGGGGTACCGCCGCCATGTTAGAGTGTGGTCCTTATGCT-3'; STA274-EcoRI, 5'-GGGGAATTCTTAATCACTATATATCTGAGTAAT-3', where cutting sites are underlined and start and stop codons appear in boldface) of Karp strain 56-kDa-protein-encoding sequences were designed based on the published sequences (GenBank accession number M33004). The restriction enzyme site for KpnI was incorporated at the 5' terminus of the coding sequence amplicon. An in-frame termination codon followed by an EcoRI restriction site was introduced at the 3' terminus of the coding sequence amplicon. The PCRs were carried out with an initial denaturation step of 94°C for 5 min, followed by 30 cycles of denaturation (94°C for 1 min), annealing (55°C for 1 min), and extension (72°C for 2 min), with a final prolonged extension step (72°C for 10 min). Each reaction buffer contained 5% dimethyl sulfoxide. The amplified coding sequence DNA was digested with KpnI and EcoRI enzymes, and the resulting 863-bp fragment (nucleotides 556 to 1419) of the coding sequence DNA was inserted into the KpnI and EcoRI sites of pRSETB vectors to form the plasmid pRSETB-Kp56. Recombinant plasmid DNA was sequenced and confirmed.

Preparation of bacterial competent cells. Stocked E. coli strains [DH5 or BL21(DE3)] were used to prepare competent cells according the method of Ausubel et al. (1). Competent cells were aliquoted and stored at -70°C immediately.

Transformation of bacteria. Transformation of bacteria was performed on a Luria-Bertani agar plate containing ampicillin (100 µg/ml) according to the method of Ausubel et al. (1).

Plasmid DNA preparations. Plasmid DNA used for cloning was prepared by alkaline lysis miniprep (2) or boiling miniprep methods (12) with some modifications. Briefly, the procedures of plasmid DNA preparation in this study were the same as the procedures of references listed; however, in the DNA precipitation, an equal volume of isopropanol was used and the mixture was allowed to sit for 5 min at room temperature. Plasmid DNA used to express recombinant protein or to be sequenced was prepared by large-scale alkaline lysis and a polyethylene glycol (PEG) precipitation method (17, 18, 19).

Expression and purification of recombinant protein. For prokaryotic expression and purification of histidine-tagged proteins, E. coli BL21(DE3) containing plasmid pRSETB-Kp56 was used. The E. coli strain harboring this plasmid was grown to an optical density (OD) at 600 nm of 0.7 to 0.8 prior to induction with 1 mmol of IPTG (isopropyl-ß-D-thiogalactopyranoside) per ml. After 3 h of induction (see Fig. 4), cells were harvested and lysed by resuspending the bacterial pellets in sonication buffer (containing 50 mM sodium phosphate [pH 8.0], 10 µM phenylmethylsulfonyl fluoride, 0.1% Tween 20, 100 mM KCl, 500 mM NaCl, and 1 mg of lysozyme per ml) for 30 min prior to sonication. Following centrifugation at 17,000 x g (4°C for 20 min) in a Beckman J2-MI centrifuge with a JA 25.5 rotor, the pellets were resuspended in buffer B (8 M urea, 0.1 M sodium phosphate [pH 8.0], and 10 mM Tris) and stirred at room temperature for 1 h. After centrifugation, the supernatant was purified via metal chelate affinity chromatography using Ni²⁺-nitrilotriacetic acid (NTA) complexes (Qiagen). Briefly, 20 ml of supernatant was passed through a 2-ml column of Ni²⁺-NTA agarose that was prewashed with buffer B, buffer C (8 M urea, 0.1 M sodium phosphate, 10 mM Tris [pH 6.3]), and buffer F (6 M guanidine-HCl, 0.2 M acetic acid) and preequilibrated in buffer B. The column was then washed with 10 volumes of buffer B and then with 10 volumes of buffer C, and the protein was eluted with buffer D (8 M urea, 0.1 M sodium phosphate, 10 mM Tris [pH 5.9]) and buffer E (8 M urea, 0.1 M sodium phosphate, 10 mM Tris [pH 4.5]) in fractions of 0.5 ml. Protein-containing fractions were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining. To renature the protein, a stepwise dialysis was performed at 4°C against buffer B containing decreasing concentrations of urea (4, 2, 1, 0.5, 0.25, 0.125, and 0.05 M) and against buffer D (20 mM HEPES [pH 8.0], 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol [DTT], and 0.2% Nonidet P-40) alone. After dialysis and a short centrifugation at 18,000 x g in a KUBOTA 1720 centrifuge with an RA-50 JS rotor for 5 min at 4°C, the supernatant was quickly frozen in liquid nitrogen and stored at -80°C.

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FIG. 4. Recombinant protein (rP56) can induce immunized animals to produce high and long-lived antibody titers. Rabbits immunized with recombinant protein induced high and long-lived titers of antibody against the outer membrane protein of O. tsutsugamushi. Rabbits were immunized subcutaneously twice with 40 µg of recombinant truncated protein (rP56) at the 4th and 10th weeks after the basal serum collection. The sera before and after immunization were collected and mixed with 50% glycerol and stored at -20°C until use. The antibody was assayed for reactivity with rP56 by ELISA as described in Materials and Methods. Error bars indicate standard deviations.

Preparation of antisera against O. tsutsugamushi. Normal healthy rabbit, mouse, and rat sera were obtained from the Animal Center of the Institute of Preventive Medicine of National Defense Medical Center, Taipei, Taiwan. All animals were confirmed to be healthy by a licensed veterinarian. Rabbit, mouse (ICR), and rat antisera against O. tsutsugamushi were prepared by three inoculations (subcutaneous for rabbits and intramuscular for mice and rats) of recombinant truncated protein (40 µg for rabbits and 5 µg for mice and rats) mixed with Freund's adjuvant (complete adjuvant for the first inoculation and incomplete adjuvant for the second and third inoculations) at an interval of 1 month. One month after the last immunization, animals were bled and sera were centrifuged at 3,000 x g for 10 min at 4°C in a Sorvall RT7 centrifuge with an RTH-250 rotor (Dupont) after 2 h of agglutination at room temperature. Antisera were collected and mixed with 50% glycerol and stored at -20°C.

Serum samples. Sera were obtained from mice caught in different districts of Taiwan during the period from 2000 to 2001. A total of 482 serum samples were randomly selected for evaluation of immunoglobulin G (IgG) titers by recombinant ELISA. All serum samples were stored at -20°C prior to being assayed.

ELISA. The 96-well microtiter plates (Maxisorp; Nunc) were coated with antigens (100 µl/well; 0.2 µg of purified recombinant p56-truncated protein [a.a. 31 to 274] per ml in 0.05 M carbonate buffer, pH 9.6) at 4°C overnight. The contents of the plates were dumped, and the wells were filled with phosphate-buffered saline (PBS) containing 3% skim milk (100 µl/well). Plates were then incubated for 1 h at room temperature for blocking. After dumping the contents, the wells were rinsed five times with PBST (PBS-0.05% Tween 20). This was followed by incubation with secondary antibodies (goat anti-rabbit IgG or goat anti-rat IgG [for Suncus murinus, goat anti-Peromyscus leucopus IgG-horseradish peroxidase (HRP) {catalog no. 14-33-06; Kirkegaard & Perry Laboratories} was used as secondary antibody] conjugated with HRP, 1:3,000 in PBS containing 3% skim milk [100 µl/well]) at room temperature for 1 h. After dumping the contents, the wells were rinsed five times with PBST again. The substrate solution (3,3,5,5-tetramethylbenzidine [TMB] [100 µg/ml in phosphate-citrate buffer, pH 5.0] containing 1/1,000 volume of 35% H₂O₂) was added and incubated at room temperature for 15 min. After the reaction was stopped by adding 1 M H₂SO₄ (50 µl/well), the ODs at 450 nm were measured. Sera were always assayed in duplicate. Each plate included an air blank, a negative control (preimmune serum in triplicate), as well as a row of different diluted positive controls used to establish a standard curve.

Cutoff value for ELISA. Background reactivity and possible cross-reactivity were assessed by analyzing preimmune serum specimens from healthy rabbits, mice, and rats. The cutoff values were set at ODn + 3 standard deviations, where ODn is the mean of ODs of the preimmune serum specimens. The ODs of these preimmune serum specimens varied from 0.064 to 0.131 for the rabbit anti-P56 IgG assay, from 0.05 to 0.06 for the mouse anti-P56 IgG assay, and from 0.046 to 0.053 for the rat anti-P56 IgG assay. In this way, for all of the investigations described below, sera were classified as being above or below the OD threshold.

Western immunoblotting analysis. For the identification of purified rP56-kDa truncated outer membrane protein (O. tsutsugamushi Karp strain a.a. 31 to 274), a Western immunoblot analysis (16, 17) was performed. Briefly, the purified protein was boiled in a sample buffer (125 mM Tris-HCl [pH 6.8], 100 mM DTT, 2% SDS, 20% glycerol, 0.005% bromophenol blue) for 5 min and then loaded onto an SDS-12% polyacrylamide gel. After electrophoresis, proteins were transferred to an Immobilon membrane (Millipore) by semidry methods (Hoffer). The membranes were blocked with BLOTTO-Tween blocking buffer and then incubated with mouse antihistidine antibody (1/1,000; Amersham Pharmacia Biotech). The membrane was then washed with blocking buffer and further incubated with HRP-conjugated secondary antibody. The membrane was finally washed with blocking buffer and developed with ECL Western blotting reagents (Amersham Pharmacia Biotech) or with DAB peroxidase substrate (Sigma Fast [catalog no. D-4418]; Sigma) (DAB [0.7 mg/ml] in 0.06 M Tris [pH 7.6] with H₂O₂ [0.17 mg/ml] and 0.1% NiCl₂).

For the identification of rabbit, mouse, or rat anti-rP56 antisera, sera from immunized rabbits, mice, or rats were used as primary antibodies to react with purified rP56 protein in a Western blot assay. For the identification of expressions of wild-type or truncated P56 protein, equal amounts (approximately 50 mg) of nuclear proteins from extracts of infected or transfected cells (mouse L929 or human Vero cells) were boiled in a sample buffer (125 mM Tris-HCl [pH 6.8], 100 mM DTT, 2% SDS, 20% glycerol, 0.005% bromophenol blue) for 5 min and then loaded onto an SDS-10% polyacrylamide gel. After electrophoresis, Western blotting was performed according to the methods described above.

IFA. To check the recognition capability of rabbit anti-rP56, an indirect IF assay (IFA) (14) was performed. Briefly, the mouse L929 monolayer cells infected with different strains of O. tsutsugamushi were washed three times with PBS and then fixed in 100% methanol for 5 min at -20°C. After blocking with 3% skim milk in PBS for 30 min at 37°C, monolayers were incubated for 30 min at 37°C with rat anti-rP56 antibody diluted in PBS. After washing three times with PBS, monolayers were subsequently incubated at 37°C with fluorescein isothiocyanate (FITC)-conjugated goat anti-rat IgG diluted in PBS for 30 min. Finally, the monolayers were washed three times with PBS and were mounted with glycerol-PBS (1:1). Each stained monolayer was viewed with an immunofluorescence microscope.

Micro-IFA. O. tsutsugamushi strains Karp, Kato, and Gilliam were obtained (from the Institute of Preventive Medicine, Nan Kong) and used to infect mouse L929 cells. The cells infected with O. tsutsugamushi were spread on the glass slide and were fixed with acetone for 15 min at -20°C. After blocking with 3% bovine serum albumin (catalog no. B 4287; Sigma) in phosphate buffer, pH 7.4, at room temperature for 30 min, mouse serum (1:40), normal mouse serum (negative control, 1:40), and positive control serum (ascites or serum [with an antibody titer of >1:40], of ICR mice inoculated with the Karp strain of O. tsutsugamushi, 1:40) were added, and the reaction mixture was incubated at room temperature for 1 h. After washing three times (10 min per washing) with phosphate buffer, pH 7.4, both secondary antibodies (mixture of goat anti-mouse IgG-FITC conjugate [catalog no. 115-015-008; Jackson Immuno Research] and goat anti-rat IgG-FITC conjugate [catalog no. 112-095-008; Jackson Immuno Research], 1:100 in PBS, pH 7.4) and DAPI (4',6-diamidino-2-phenylindole) (1:1,000 in PBS, pH 7.4; catalog no. D 9542; Sigma) were added and incubated at room temperature for 30 min and 5 min, respectively. After being washed three times (10 min per washing) with phosphate buffer, pH 7.4, glass slides were mounted and examined by immunofluorescence microscopy.

Statistical analysis. The ² test was used to examine the differences in the seroprevalence rates among different kinds of mice or IgG seropositivity rates of mice caught in different districts of Taiwan. The kappa statistic test was used to examine the reproducibility of results obtained from recombinant ELISA and immunofluorescence assay. A P of <0.05 was considered statistically significant.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Purification of recombinant truncated outer membrane protein of O. tsutsugamushi. A conserved sequence, which contains the immunodominant epitope region (that encompassed ADI-ADII and part of ADIII) of the antigen (28), encoding the outer membrane proteins of different strains of O. tsutsugamushi was obtained from GenBank using the CLUSTAL W (version 1.8) multiple sequence alignment program (Fig. 1a). This coding sequence, corresponding to amino acids 31 to 274 of the P56 outer membrane protein of Karp strain, was amplified by PCR and cloned into the prokaryotic expressional vector containing a six-histidine tag coding sequence, pRSET-B, to produce pRSETB-Kp56 plasmid. The pRSETB-Kp56 plasmid allows overexpression of the six-histidine-tagged truncated outer membrane protein in E. coli BL21(DE3), and the recombinant protein could be purified by nickel chromatography since the histidine region of the fusion protein will bind nickel ions. After sequencing, the expression plasmid was transformed into E. coli [strain BL21(DE3)], and the recombinant protein was induced by IPTG to confirm the correctness of expression. Recombinant protein was purified to near homogeneity using Ni²⁺-NTA agarose (Qiagen) affinity chromatography (Fig. 1b, lane 8) and was confirmed by Western blotting with mouse antihistidine monoclonal antibody (data not shown). Results from this study demonstrated that the purified protein was a recombinant fusion protein containing the truncated outer membrane protein of O. tsutsugamushi.

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FIG. 1. Cloning and purification of recombinant truncated outer membrane protein of Karp strain O. tsutsugamushi. (A) Alignment of amino acids corresponding to the conserved region of outer membrane protein among different strains of O. tsutsugamushi. A conserved sequence which contains the epitope region of the antigen and encodes the outer membrane proteins of different strains of O. tsutsugamushi was found in GenBank using the CLUSTAL W (version 1.8) multiple sequence alignment program. Arrows indicate the initiation and stop points of amino acids amplified by PCR. (B) Purification of recombinant p56 outer membrane protein (O. tsutsugamushi Karp strain a.a. 31 to 274). Histidine-tagged truncated rP56 expressed in E. coli BL21(DE3) was purified by nickel chelate chromatography as described in Materials and Methods. The protein was resolved by SDS-12% PAGE and stained with Coomassie blue. Lane 1, marker; lanes 2 and 3, total cell lysates from E. coli transformed with pRSETB vector (lane 2) and pRSETB-karp56 (a.a. 31 to 274) plasmid DNA (lane 3), respectively; lane 4, fractions washed with buffer B; lane 5, fractions washed with buffer C; lanes 6 and 7, fractions eluted with buffer D; lanes 8 and 9, fractions eluted with buffer E. The arrow indicates the predicted histidine-tagged p56 truncated protein. *, protein induction for 3 h by IPTG.

Antibody against recombinant outer membrane protein (rP56) can recognize different strains of O. tsutsugamushi. After immunization of the rabbit, the polyclonal antibody was obtained and confirmed to react with both recombinant rP56 protein and outer membrane protein of O. tsutsugamushi by Western blotting (Fig. 2). This antibody was further confirmed to recognize different strains of O. tsutsugamushi by IFA (Fig. 3). Antibody against O. tsutsugamushi was measured and confirmed to be long-lived with a titer higher than 204,800 even at 6 months after the last immunization (Fig. 4). Furthermore, it was demonstrated that mice and rats immunized with this purified recombinant protein could also produce high titers of antibody (IgG) against the outer membrane protein (P56) of O. tsutsugamushi (data not shown). These anti-rP56 mouse and rat antibodies were used as positive controls and used to create a standard curve by which IgG titers of mouse and rat sera measured at different days by ELISA could be calculated and adjusted (data not shown).

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FIG. 2. Rabbit anti-rP56 antiserum specifically recognizes outer membrane protein of O. tsutsugamushi by Western blotting. Histidine-tagged truncated rP56 expressed in E. coli BL21(DE3) was purified by nickel chelate chromatography. The protein was resolved by SDS-PAGE (12% gel) and was assayed by Western blotting as described in Materials and Methods. Lane 1, marker; lanes 2 and 3, total cell lysates from E. coli transformed with pRSETB vector (lane 2) and pRSETB-karp56 (a.a. 31 to 274) plasmid DNA (lane 3), respectively; lane 4, lysates (1/10 volume) from L929 cells infected with Karp strain of O. tsutsugamushi; lane 5, purified protein. Arrow 1 indicates the 56-kDa outer membrane protein of O. tsutsugamushi. Arrow 2 indicates the predicted rP56 (32-kDa) truncated protein.

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FIG. 3. Antibody against recombinant outer membrane protein (rP56) can recognize different strains of O. tsutsugamushi by IFA. (A) IFA using rat anti-rP56 antibody recognized different strains of O. tsutsugamushi. Mouse L929 monolayer cells infected with different strains of O. tsutsugamushi were assayed by IFA as described in Materials and Methods. Arrows indicate different strains of O. tsutsugamushi recognized by the antibody. I, II, and IV represent cells infected with the standard strains Karp, Gilliam, and Kato, respectively; III and V represent cells infected with two strains of O. tsutsugamushi isolated from mice captured in Kinmen County; VI represents mouse L929 cells without any infection. (B) Detection of P56 outer membrane protein of O. tsutsugamushi with Western blotting. Lanes: 1 to 4, L929 cells infected with Karp (lane 1), Gilliam (lane 2), Kiman (lane 3), and Kato (lane 4) strains of O. tsutsugamushi, respectively; lane 5, purified truncated protein (Karp strain, 31 to 274 a.a.). Arrow 1 indicates the predicted 56-kDa outer membrane protein of O. tsutsugamushi. Arrow 2 indicates the predicted 32-kDa truncated protein.

Antibody assays with recombinant protein ELISA obtain good reproducibility with traditional micro-IFA test using native O. tsutsugamushi antigen. Results of mouse IgG against O. tsutsugamushi obtained by ELISA were comparable with the results obtained by traditional micro-IFA. Of a total of 144 random serum samples assayed for their IgG antibody reactions against O. tsutsugamushi, findings were consistent for 134 samples. The coefficient was 0.735 with a kappa statistic equal to 8.96 (P < 0.05) (Table 1). These results showed that the recombinant ELISA used in this study had good reproducibility based on comparison with the traditional micro-IFA test.

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TABLE 1. Comparison of mice IgG reaction obtained by recombinant protein ELISA with traditional IFA testa

Seroprevalence rates of IgG against O. tsutsugamushi differ significantly among different species of mice. ELISA was used to evaluate the presence of antibody against O. tsutsugamushi in mice captured in different districts during 2000 and 2001. No significant differences were found in the antibody seroprevalence rates for the same strains of mice captured in different districts. However, different seroprevalence rates of IgG against O. tsutsugamushi among different strains of mice were observed (²_{5, 0.95} = 93.85; P < 0.05). Antibody seropositivity rates were higher in Bandicota indica (100%; 3 of 3), Rattus flavipectus (96.17%; 176 of 183), and Rattus losea (95.83%; 23 of 24) than in Rattus norvegicus (86.05%; 74 of 86) and Rattus mindanensis (83.67%; 41 of 49) (²_{diff, 5, 0.95} = 12.59; P < 0.05). The lowest antibody seropositivity rate (54.4%; 68 of 125) was observed in S. murinus (Table 2). Significant differences in seroprevalence rates among different strains of mice suggested potential for involvement with the reported cases of scrub typhus in different districts. However, this implication requires further detailed study.

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TABLE 2. Seroprevalence rates of mouse IgG against O. tsutsugamushi among different districts in Taiwan, 2000 to 2001

Different compositions of mouse strains contributed to the differences in seroprevalence rates of mice against O. tsutsugamushi among different districts. Significant differences were seen in antibody (IgG) seroprevalence rates of mice in different districts of Taiwan. Antibody seroprevalence rates of mice were higher in Penghu County (91.58%; 87 of 95), Kinmen County (84.82%; 218 of 257), and Orchid Island (83.67%; 41 of 49) than in Hwalien County (56.86%; 29 of 51) and Lienkiang County (55.56%; 10 of 18) (²_{diff, 4, 0.95} = 35.24; P < 0.05) (Table 2). However, significant differences in the antibody seroprevalence rates of mice among different districts were due to differences of proportional rates of S. murinus in the mouse populations (²_{4, 0.95} = 26.64, P < 0.05) (Table 2). Excluding S. murinus from the analysis, no significant differences in antibody seroprevalence rates were found for mice captured from different districts (²_{4, 0.95} = 6.082; P > 0.05) (Table 2). Results obtained from this study indicated that the seroprevalence rates of mice against O. tsutsugamushi were generally greater than 60% (except S. murinus).

Top Abstract Introduction Materials and Methods Results Discussion References

 
In recent years, both reported and identified cases of scrub typhus have increased in various districts in Taiwan. Meanwhile, the identification rate of scrub typhus cases is still low (9a). Rodent mice are one of the major natural hosts of O. tsutsugamushi. The data of this study suggest that the increasing rate of reported cases of scrub typhus is associated with an increasing infection rate in rodent mice and that differences of mouse strains in different districts may be associated with differences in scrub typhus prevalence. Data from epidemiological surveillance and seroprevalence of antibody against O. tsutsugamushi among the different strains of rodent mice in different areas of Taiwan are important to programs to prevent the occurrence of scrub typhus. For reasons of safety and specificity and to meet the need for evaluation of large-scale samples in a short period of time, synthetic antigens, such as recombinant proteins, have been proposed as suitable alternatives except for antigen preparations. In this study, the recombinant truncated 56-kDa immunodominant protein from O. tsutsugamushi was used to develop a serological ELISA for the surveillance of IgG of rodent mice caught in districts in different geographical regions of Taiwan.

The recombinant truncated P56 protein, corresponding to the conserved region of the outer membrane protein among different strains of O. tsutsugamushi, has been purified to near homogeneity (Fig. 1 and 2) and has been demonstrated to induce high and long-lived antibody levels in different animals, including rabbits, mice, and rats (Fig. 4 and data not shown). The difference in reactivity between the native and the recombinant protein might be due to the different interaction affinity with the rabbit anti-rP56 antibody; however, the amount of native protein in the 56-kDa protein has not been measured. To resolve this problem, the infection ratio of bacteria in L929 should be calculated and cells infected with bacteria should be sorted (27). In this study, however, the purified recombinant protein was confirmed by Western blotting using mouse antihistidine antibody (data not shown) and rabbit anti-rP56 antibody.

The induced rabbit antibody was also demonstrated to recognize different strains of O. tsutsugamushi in both IFA and Western blotting assays (Fig. 3). Possible explanations for the different intensities of Western blot seen between various bacterial strains might be due to the different infection rates of L929 cells by various strains of O. tsutsugamushi. We could not rule out, however, the possibility that these differences in infection rate may have been due to different interaction affinity between various bacteria strains with rabbit anti-rP56antibody. Data obtained from L929 cells transfected with truncated 56-kDa protein expression plasmids revealed that the recognition ability of the rP56-induced rabbit antibody was almost equal for different strains of bacteria (data not shown). These findings indicate that the purified recombinant protein and its induced antibody could be used to detect antibody as well as various strains of O. tsutsugamushi.

A standard curve of ELISA results was established using rat antibody against rP56 (data not shown), and IgG titers of rodent mouse sera were measured and adjusted by comparison to the standard curve. The recombinant ELISA used in this study was shown to have good reproducibility in comparison with IFA ( = 0.735; kappa statistic = 8.96; P < 0.05). Since ELISA is easy to perform, can assay large samples in a short time, and provides semiquantitative absorbance values, it is a more suitable method for use in epidemiological surveillance of serum antibodies for rodent mice than IFA.

This study found that seroprevalence rates were significantly different among different strains of mice. Even after the exclusion of S. murinus from calculations, this difference remained significant (²_{4, 0.95} = 13.62; P < 0.05) (Table 2). Results also indicated that seroprevalence rates of mice against O. tsutsugamushi (except S. murinus) were at least 86%. In fact, scrub typhus is often underreported and may go undiagnosed (10, 21). Secondary infection is also often present subclinically in patients with scrub typhus (3). Therefore, it is very likely that many more of the people residing in the districts included in this study may be infected with O. tsutsugamushi or become infected in the future if proper protective measures are not instituted. Thus, our findings suggest the need for public health education in Taiwan about personal protection practices against scrub typhus and the pathogen O. tsutsugamushi.

Significant differences were seen in the antibody (IgG) seroprevalence rates of mice captured in different districts (²_{4, 0.95} = 83.41; P < 0.05). Differences in the proportion of different mouse strains might contribute to the differences in antibody seroprevalence rates of mice against O. tsutsugamushi among different geographic areas. For example, in this study the antibody seropositivity rates were higher in B. indica (100%), R. flavipectus (96.17%), and R. losea (95.83%) than in R. norvegicus (86.05%) and R. mindanensis (83.67%) (²_{diff, 5} = 12.59, P < 0.05) (Table 2). However, the major contribution to the differences in IgG seropositivity rates came from the lower seropositivity rate of S. murinus (54.4%). After excluding S. murinus from the calculation, no significant differences were found in antibody seroprevalence rates of mice from different districts (² = 6.082; P > 0.05) (Table 2).

To determine whether different affinities of the secondary HRP conjugate antibody could explain the different levels of reactivity among the different species of mice, goat anti-rat or goat anti-mouse IgG-HRP secondary antibodies were used to detect rat and mouse IgG, and goat anti-P. leucopus IgG-HRP secondary antibody was used to detect IgG of S. murinus. Results showed a 77% (50 of 65) consistency between the two detection assays using different secondary antibodies (goat anti-rat IgG-HRP and goat anti-mouse IgG-HRP conjugates). This finding showed a good reproducibility ( = 0.497, z = 4.107, P < 0.05; data not shown) between tests using these two secondary antibodies. The differences of reactivity for goat anti-rat IgG-HRP with different species of rodent mice were not significant. Thus, it seems unlikely that different levels of reactivity contributed to the different affinities of the secondary HRP conjugates. However, in the surveillance of the seroprevalence of antibodies, use of pooled secondary antibody mixture for different kinds of rodent mice might be helpful.

In this study, S. murinus had lower antibody (IgG) response rates, implying either that this strain of mice might not be easily infected with O. tsutsugamushi or that it could not efficiently produce antibody against the pathogen after infection. Our data obtained by nested PCR showed that S. murinus could be infected by O. tsutsugamushi (data not shown). The reasons why this strain of mice infected with O. tsutsugamushi could not induce an antibody (IgG) response efficiently remain to be determined; however, cell surface receptors or microbe-binding proteins of lymphocyte or defects of unknown components of the immune system might be involved (35). Further exploration of the mechanism of invasiveness and potential proteins that might be associated with outer membrane protein of O. tsutsugamushi is needed. Determination of the mechanism causing the inability of S. murinus to efficiently respond to the infection of O. tsutsugamushi will contribute to our understanding of host cell immunity against this pathogen and facilitate the development of methods to prevent scrub typhus.

 
This work was supported by a grant from the Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan, and a grant (NSC 91-2320-B-016-050) from the National Science Council (NSC), Taipei, Taiwan, awarded to Y.-C.W.

We are very grateful to Lu Pai, from the Institute of Public Health, National Defense Medical Center, for advice on statistical analysis.

 
* Corresponding author. Mailing address: Institute of Preventive Medicine, National Defense Medical Center, P.O. Box 90048-700, San-Hsia, Taiwan. Phone: 886-2-26711082, ext. 19870. Fax: 886-2-26736994. E-mail: yeauching{at}ndmctsgh.edu.tw.

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Clinical and Diagnostic Laboratory Immunology, May 2003, p. 451-458, Vol. 10, No. 3
1071-412X/03/$08.00+0     DOI: 10.1128/CDLI.10.3.451-458.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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