Quantitative Real-Time PCR Assay for Detection of Ehrlichia chaffeensis
Viral and Rickettsial Zoonoses Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgiaq\$s(, 百拇医药
Received 25 February 2003/ Returned for modification 24 April 2003/ Accepted 7 May 2003q\$s(, 百拇医药
ABSTRACTq\$s(, 百拇医药
A real-time PCR assay was developed for the detection of Ehrlichia chaffeensis. The assay is species specific and provides quantitative results in the range 10 to 1010 gene copies. The assay is not inhibited by the presence of tick, human, or mouse DNA and is compatible with high sample throughput. The assay was compared with previously described assays for E. chaffeensis.q\$s(, 百拇医药
TEXTq\$s(, 百拇医药
Ehrlichia chaffeensis is the causative agent of human monocytic ehrlichiosis, a potentially fatal disease (11). The bacterium, first isolated in 1991 (4), is a tick-borne zoonotic pathogen classified within the order Rickettsiales, family Anaplasmataceae (6). The pathogen is maintained in a natural transmission cycle between the Lone Star tick (Amblyomma americanum) and mammalian hosts (1, 7, 9). Experimental studies of the acquisition and transmission of E. chaffeensis between mammals and ticks require a quantitative assay for the organism that is compatible with high sample throughput.
Existing PCR assays for E. chaffeensis include a direct PCR assay for the 16S rRNA gene (2) and a nested PCR assay that amplifies the variable-length PCR target (VLPT) (13). These PCR assays can detect the presence of pathogen DNA but do not provide quantitative data. Nested PCR is typically more sensitive than direct PCR but requires more handling of amplified PCR products, decreasing throughput capacity and increasing the risk of sample cross-contamination.l5@2h, 百拇医药
Real-time (TaqMan) PCR. The single-copy 16S rRNA gene of E. chaffeensis was selected for the development of the real-time TaqMan PCR assay (10). Primers that amplify an 81-bp region of the gene (bases 17 to 97, GenBank accession no. U86665) were selected. The assay was optimized with a Brilliant quantitative-PCR core reagent kit (Stratagene, La Jolla, Calif.), with a final reaction volume of 25 µl. The reaction mixture contained a 200 nM concentration of the forward primer ECH16S-17 (5'-GCGGCAAGCCTAACACATG-3'), an 800 nM concentration of the reverse primer ECH16S-97 (5'-CCCGTCTGCCACTAACAATTATT-3'), a 100 nM concentration of the probe ECH16S-38 (5'-6-carboxyfluorescein-AGTCGAACGGACAATTGCTTATAACCTTTTGGT-3'),and 3.0 µM magnesium chloride. Real-time PCRs and fluorescence detection were performed using an iCycler thermal cycler and iQ software (Bio-Rad Laboratories, Hercules, Calif.). The optimized thermal cycler program was 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 57°C for 1 min.
Sensitivity and specificity. Quantitative results were based on a 10-fold dilution series of a plasmid encoding the 16S rRNA gene of E. chaffeensis. The specificity of the assay was determined by testing genomic DNA from three strains of E. chaffeensis, several closely related organisms, uninfected ticks, and uninfected mammalian blood and tissues. E. chaffeensis isolates (Arkansas, St. Vincent, and Jax) were obtained from clinical samples, as described previously (4, 12). All three strains of E. chaffeensis were detected by the TaqMan PCR, and agarose gel electrophoresis revealed a single band of the appropriate size. The TaqMan assay did not amplify genomic DNA from Ehrlichia muris (AS145 strain) (15), Ehrlichia canis (Oklahoma strain) (5), Neorickettsia sennetsu (formerly Ehrlichia sennetsu, Miyayama strain) (14), or Rickettsia prowazekii (Breinl strain). As revealed by agarose gel electrophoresis, the primers used for the TaqMan PCR assay amplified the 16S rRNA genes from Ehrlichia ewingii (human clinical sample) (3) and Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila, USG3 strain) (16). However, neither A. phagocytophilum nor E. ewingii was detected during real-time amplification and fluorescence detection, indicating that the TaqMan probe is species specific for E. chaffeensis (data not shown).
Genomic DNA was extracted from uninfected ticks, blood, and tissue samples using the IsoQuick nucleic acid extraction kit (Orca Research, Inc., Bothell, Wash.). The TaqMan PCR assay did not amplify genomic DNA from mouse blood, spleen, or liver (BALB/c and C57BL/6 strains), human or guinea pig blood, or uninfected ticks (Amblyomma americanum, Dermacentor variabilis, or Ixodes scapularis).(a!4c6, http://www.100md.com
The specificity of the TaqMan PCR assay was further tested using DNA extracted from human clinical samples submitted to the Centers for Disease Control and Prevention (Atlanta, Ga.). Three E. chaffeensis-positive samples and three E. chaffeensis-negative samples (one A. phagocytophilum-positive blood sample, one negative blood sample, and one negative lymph node sample) were tested. The TaqMan PCR was positive for all three samples containing E. chaffeensis DNA but was negative for two samples lacking E. chaffeensis DNA and for the sample containing A. phagocytophilum DNA.
Effect of background DNA. The effect of background DNA on the efficiency and sensitivity of the TaqMan PCR assay was determined. Genomic DNA was pooled from 10 adult female Amblyomma americanum ticks (35.3 µg of DNA/ml) and 10 uninfected BALB/C mice (19.9 µg of DNA/ml), and human genomic DNA (25 µg of DNA/ml) was obtained from Promega (Madison, Wis.). Reaction efficiencies were compared using a fivefold dilution series of genomic E. chaffeensis DNA in water, tick DNA, mouse DNA, or human DNA. As summarized in Table 1, the presence of background DNA did not decrease the efficiency of the TaqMan PCR. The effect of host DNA on sensitivity was determined by comparing the threshold cycles in which genomic E. chaffeensis DNA was detected in the presence of water, tick DNA, mouse DNA, or human DNA. E. chaffeensis DNA was tested at a dilution of approximately 620 bacteria per µl of blood, corresponding with a moderate degree of ehrlichemia (unpublished results). The sensitivity of the assay in the presence of background DNA was not significantly different from that in water (Table 1).
fig.ommitted0ng3%, 百拇医药
TABLE 1. Effect of host DNA on the efficiency and sensitivity of the real-time TaqMan PCR assay for E. chaffeensisa0ng3%, 百拇医药
Comparison with other assays. The analytical sensitivity and specificity of the TaqMan PCR were compared with those of three previously described PCR assays for E. chaffeensis: a direct PCR assay for the 16S rRNA gene using primers HE1 and HE3 (2), a nested PCR assay for the VLPT of E. chaffeensis (13), and a real-time PCR assay for the 16S rRNA gene, based on the SYBR Green detection system (8). All PCR assays were performed in a 25-µl final volume. For the SYBR Green assay, real-time fluorescence detection was performed by use of an ABI 7900HT sequence detection system (Applied Biosystems, Foster City, Calif.).0ng3%, 百拇医药
The sensitivities of all four PCR assays were compared by testing a fivefold dilution series of E. chaffeensis genomic DNA (Table 2). All assays were repeated to ensure reproducible results. The sensitivity of the TaqMan assay was significantly greater than that of the direct (HE1/HE3) assay and comparable to those of the real-time SYBR Green and nested VLPT assays.
fig.ommitted'#u#es, 百拇医药
TABLE 2. Relative sensitivities of four different PCR assays for E. chaffeensis DNA'#u#es, 百拇医药
The specificity of the TaqMan PCR was compared with those of the other three PCR assays for E. chaffeensis (Table 3). The HE1/HE3 direct and VLPT nested PCR assays have been evaluated by using genomic DNA from several species of Ehrlichia and Anaplasma and have been demonstrated to be species specific for E. chaffeensis (2, 13). As reported above, the TaqMan PCR is species specific for E. chaffeensis. However, the SYBR Green assay detected E. canis, E. ewingii, E. muris, and A. phagocytophilum, as well as E. chaffeensis.'#u#es, 百拇医药
fig.ommitted'#u#es, 百拇医药
TABLE 3. Specificities of four different PCR assays for E. chaffeensis DNA'#u#es, 百拇医药
Conclusions. In conclusion, the real-time PCR assay that we have developed for E. chaffeensis is very sensitive (10 gene copies), is species specific, is suitable for high-throughput applications, and is not inhibited by the presence of human, mouse, or tick DNA. The assay was found to be superior to three assays previously described for the detection of E. chaffeensis based on one or more criteria. It is more sensitive than the HE1/HE3 direct PCR assay, more specific than the SYBR Green real-time PCR assay, and has a higher throughput capacity and less sample handling than the VLPT nested PCR assay. Most importantly, the real-time TaqMan PCR assay provides quantitative data, allowing for simultaneous detection of the pathogen and determination of the infectious loads in ticks and mammals. This assay provides a powerful tool for examining the kinetics of infection with E. chaffeensis and the transmission of the pathogen between mammalian hosts and arthropod vectors.
ACKNOWLEDGMENTS?(dum3, 百拇医药
We thank John W. Sumner (Centers for Disease Control and Prevention) for providing clinical specimens. We thank Gregory A. Dasch for his review of the manuscript. We acknowledge the Biotechnology Core Facility of the National Center for Infectious Diseases, Centers for Disease Control and Prevention, for the synthesis of oligonucleotides.?(dum3, 百拇医药
This research was supported in part by the Association of Public Health Laboratories, through an appointment of the Emerging Infectious Diseases Fellowship Program funded by the Centers for Disease Control and Prevention.?(dum3, 百拇医药
REFERENCES?(dum3, 百拇医药
Anderson, B. E., K. G. Sims, J. G. Olson, J. E. Childs, J. F. Piesman, C. M. Happ, G. O. Maupin, and B. J. Johnson. 1993. Amblyomma americanum: a potential vector of human ehrlichiosis. Am. J. Trop. Med. Hyg. 49:239-244.?(dum3, 百拇医药
Anderson, B. E., J. W. Sumner, J. E. Dawson, T. Tzianabos, C. R. Greene, J. G. Olson, D. B. Fishbein, M. Olsen-Rasmussen, B. P. Holloway, E. H. George, and A. F. Azad. 1992. Detection of the etiologic agent of human ehrlichiosis by polymerase chain reaction. J. Clin. Microbiol. 30:775-780.
Buller, R. S., M. Arens, S. P. Hmiel, C. D. Paddock, J. W. Sumner, Y. Rikhisa, A. Unver, M. Gaudreault-Keener, F. A. Manian, A. M. Liddell, N. Schmulewitz, and G. A. Storch. 1999. Ehrlichia ewingii, a newly recognized agent of human ehrlichiosis. N. Engl. J. Med. 341:148-155.&+, http://www.100md.com
Dawson, J. E., B. E. Anderson, D. B. Fishbein, J. L. Sanchez, C. S. Goldsmith, K. H. Wilson, and C. W. Duntley. 1991. Isolation and characterization of an Ehrlichia sp. from a patient diagnosed with human ehrlichiosis. J. Clin. Microbiol. 29:2741-2745.&+, http://www.100md.com
Dawson, J. E., Y. Rikihisa, S. A. Ewing, and D. B. Fishbein. 1991. Serologic diagnosis of human ehrlichiosis using two Ehrlichia canis isolates. J. Infect. Dis. 163: 564-567.&+, http://www.100md.com
Dumler, J. S., A. F. Barbet, C. P. Bekker, G. A. Dasch, G. H. Palmer, S. C. Ray, Y. Rikihisa, and F. R. Rurangirwa. 2001. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila. Int. J. Syst. Evol. Microbiol. 51:2145-2165.
Ewing, S. A., J. E. Dawson, A. A. Kocan, R. W. Barker, C. K. Warner, R. J. Panciera, J. C. Fox, K. M. Kocan, and E. F. Blouin. 1995. Experimental transmission of Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) among white-tailed deer by Amblyomma americanum (Acari: Ixodidae). J. Med. Entomol. 32:368-374..%o'm, http://www.100md.com
Li, J. S., F. Chu, A. Reilly, and G. M. Winslow. 2002. Antibodies highly effective in SCID mice during infection by the intracellular bacterium Ehrlichia chaffeensis are of picomolar affinity and exhibit preferential epitope and isotype utilization. J. Immunol. 169:1419-1425..%o'm, http://www.100md.com
Lockhart, J. M., W. R. Davidson, D. E. Stallknecht, J. E. Dawson, and S. E. Little. 1997. Natural history of Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) in the Piedmont physiographic province of Georgia. J. Parasitol. 83:887-894..%o'm, http://www.100md.com
Massung, R. F., K. Lee, M. Mauel, and A. Gusa. 2002. Characterization of the rRNA genes of Ehrlichia chaffeensis and Anaplasma phagocytophila. DNA Cell Biol. 21:587-596..%o'm, http://www.100md.com
McQuiston, J. H., C. D. Paddock, R. C. Holman, and J. E. Childs. 1999. The human ehrlichioses in the United States. Emerg. Infect. Dis. 5:635-642.
Paddock, C. D., J. W. Sumner, G. M. Shore, D. C. Bartley, R. C. Elie, J. G. McQuade, C. R. Martin, C. S. Goldsmith, and J. E. Childs. 1997. Isolation and characterization of Ehrlichia chaffeensis strains from patients with fatal ehrlichiosis. J. Clin. Microbiol. 35:2496-2502.:z%9, 百拇医药
Sumner, J. W., J. E. Childs, and C. D. Paddock. 1999. Molecular cloning and characterization of the Ehrlichia chaffeensis variable-length PCR target: an antigen-expressing gene that exhibits interstrain variation. J. Clin. Microbiol. 37:1447-1453.:z%9, 百拇医药
Weiss, E., G. A. Dasch, Y.-H. Kang, and H. N. Westfall. 1988. Substrate utilization by Ehrlichia sennetsu and Ehrlichia risticii separated from host constituents by renografin gradient centrifugation. J. Bacteriol. 170:5012-5017.:z%9, 百拇医药
Wen, B., Y. Rikihisa, J. Mott, P. A. Fuerst, M. Kawahara, and C. Suto. 1995. Ehrlichia muris sp. nov., identified on the basis of 16S rRNA base sequences and serological, morphological, and biological characteristics. Int. J. Syst. Bacteriol. 45:250-254.:z%9, 百拇医药
Yeh, M.-T., T. N. Mather, R. T. Coughlin, C. Gingrich-Baker, J. W. Sumner, and R. F. Massung. 1997. Serologic and molecular detection of granulocytic ehrlichiosis in Rhode Island. J. Clin. Microbiol. 35:944-947.(Amanda D. Loftis Robert F. Massung and Michael L. Levin)
Received 25 February 2003/ Returned for modification 24 April 2003/ Accepted 7 May 2003q\$s(, 百拇医药
ABSTRACTq\$s(, 百拇医药
A real-time PCR assay was developed for the detection of Ehrlichia chaffeensis. The assay is species specific and provides quantitative results in the range 10 to 1010 gene copies. The assay is not inhibited by the presence of tick, human, or mouse DNA and is compatible with high sample throughput. The assay was compared with previously described assays for E. chaffeensis.q\$s(, 百拇医药
TEXTq\$s(, 百拇医药
Ehrlichia chaffeensis is the causative agent of human monocytic ehrlichiosis, a potentially fatal disease (11). The bacterium, first isolated in 1991 (4), is a tick-borne zoonotic pathogen classified within the order Rickettsiales, family Anaplasmataceae (6). The pathogen is maintained in a natural transmission cycle between the Lone Star tick (Amblyomma americanum) and mammalian hosts (1, 7, 9). Experimental studies of the acquisition and transmission of E. chaffeensis between mammals and ticks require a quantitative assay for the organism that is compatible with high sample throughput.
Existing PCR assays for E. chaffeensis include a direct PCR assay for the 16S rRNA gene (2) and a nested PCR assay that amplifies the variable-length PCR target (VLPT) (13). These PCR assays can detect the presence of pathogen DNA but do not provide quantitative data. Nested PCR is typically more sensitive than direct PCR but requires more handling of amplified PCR products, decreasing throughput capacity and increasing the risk of sample cross-contamination.l5@2h, 百拇医药
Real-time (TaqMan) PCR. The single-copy 16S rRNA gene of E. chaffeensis was selected for the development of the real-time TaqMan PCR assay (10). Primers that amplify an 81-bp region of the gene (bases 17 to 97, GenBank accession no. U86665) were selected. The assay was optimized with a Brilliant quantitative-PCR core reagent kit (Stratagene, La Jolla, Calif.), with a final reaction volume of 25 µl. The reaction mixture contained a 200 nM concentration of the forward primer ECH16S-17 (5'-GCGGCAAGCCTAACACATG-3'), an 800 nM concentration of the reverse primer ECH16S-97 (5'-CCCGTCTGCCACTAACAATTATT-3'), a 100 nM concentration of the probe ECH16S-38 (5'-6-carboxyfluorescein-AGTCGAACGGACAATTGCTTATAACCTTTTGGT-3'),and 3.0 µM magnesium chloride. Real-time PCRs and fluorescence detection were performed using an iCycler thermal cycler and iQ software (Bio-Rad Laboratories, Hercules, Calif.). The optimized thermal cycler program was 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 57°C for 1 min.
Sensitivity and specificity. Quantitative results were based on a 10-fold dilution series of a plasmid encoding the 16S rRNA gene of E. chaffeensis. The specificity of the assay was determined by testing genomic DNA from three strains of E. chaffeensis, several closely related organisms, uninfected ticks, and uninfected mammalian blood and tissues. E. chaffeensis isolates (Arkansas, St. Vincent, and Jax) were obtained from clinical samples, as described previously (4, 12). All three strains of E. chaffeensis were detected by the TaqMan PCR, and agarose gel electrophoresis revealed a single band of the appropriate size. The TaqMan assay did not amplify genomic DNA from Ehrlichia muris (AS145 strain) (15), Ehrlichia canis (Oklahoma strain) (5), Neorickettsia sennetsu (formerly Ehrlichia sennetsu, Miyayama strain) (14), or Rickettsia prowazekii (Breinl strain). As revealed by agarose gel electrophoresis, the primers used for the TaqMan PCR assay amplified the 16S rRNA genes from Ehrlichia ewingii (human clinical sample) (3) and Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila, USG3 strain) (16). However, neither A. phagocytophilum nor E. ewingii was detected during real-time amplification and fluorescence detection, indicating that the TaqMan probe is species specific for E. chaffeensis (data not shown).
Genomic DNA was extracted from uninfected ticks, blood, and tissue samples using the IsoQuick nucleic acid extraction kit (Orca Research, Inc., Bothell, Wash.). The TaqMan PCR assay did not amplify genomic DNA from mouse blood, spleen, or liver (BALB/c and C57BL/6 strains), human or guinea pig blood, or uninfected ticks (Amblyomma americanum, Dermacentor variabilis, or Ixodes scapularis).(a!4c6, http://www.100md.com
The specificity of the TaqMan PCR assay was further tested using DNA extracted from human clinical samples submitted to the Centers for Disease Control and Prevention (Atlanta, Ga.). Three E. chaffeensis-positive samples and three E. chaffeensis-negative samples (one A. phagocytophilum-positive blood sample, one negative blood sample, and one negative lymph node sample) were tested. The TaqMan PCR was positive for all three samples containing E. chaffeensis DNA but was negative for two samples lacking E. chaffeensis DNA and for the sample containing A. phagocytophilum DNA.
Effect of background DNA. The effect of background DNA on the efficiency and sensitivity of the TaqMan PCR assay was determined. Genomic DNA was pooled from 10 adult female Amblyomma americanum ticks (35.3 µg of DNA/ml) and 10 uninfected BALB/C mice (19.9 µg of DNA/ml), and human genomic DNA (25 µg of DNA/ml) was obtained from Promega (Madison, Wis.). Reaction efficiencies were compared using a fivefold dilution series of genomic E. chaffeensis DNA in water, tick DNA, mouse DNA, or human DNA. As summarized in Table 1, the presence of background DNA did not decrease the efficiency of the TaqMan PCR. The effect of host DNA on sensitivity was determined by comparing the threshold cycles in which genomic E. chaffeensis DNA was detected in the presence of water, tick DNA, mouse DNA, or human DNA. E. chaffeensis DNA was tested at a dilution of approximately 620 bacteria per µl of blood, corresponding with a moderate degree of ehrlichemia (unpublished results). The sensitivity of the assay in the presence of background DNA was not significantly different from that in water (Table 1).
fig.ommitted0ng3%, 百拇医药
TABLE 1. Effect of host DNA on the efficiency and sensitivity of the real-time TaqMan PCR assay for E. chaffeensisa0ng3%, 百拇医药
Comparison with other assays. The analytical sensitivity and specificity of the TaqMan PCR were compared with those of three previously described PCR assays for E. chaffeensis: a direct PCR assay for the 16S rRNA gene using primers HE1 and HE3 (2), a nested PCR assay for the VLPT of E. chaffeensis (13), and a real-time PCR assay for the 16S rRNA gene, based on the SYBR Green detection system (8). All PCR assays were performed in a 25-µl final volume. For the SYBR Green assay, real-time fluorescence detection was performed by use of an ABI 7900HT sequence detection system (Applied Biosystems, Foster City, Calif.).0ng3%, 百拇医药
The sensitivities of all four PCR assays were compared by testing a fivefold dilution series of E. chaffeensis genomic DNA (Table 2). All assays were repeated to ensure reproducible results. The sensitivity of the TaqMan assay was significantly greater than that of the direct (HE1/HE3) assay and comparable to those of the real-time SYBR Green and nested VLPT assays.
fig.ommitted'#u#es, 百拇医药
TABLE 2. Relative sensitivities of four different PCR assays for E. chaffeensis DNA'#u#es, 百拇医药
The specificity of the TaqMan PCR was compared with those of the other three PCR assays for E. chaffeensis (Table 3). The HE1/HE3 direct and VLPT nested PCR assays have been evaluated by using genomic DNA from several species of Ehrlichia and Anaplasma and have been demonstrated to be species specific for E. chaffeensis (2, 13). As reported above, the TaqMan PCR is species specific for E. chaffeensis. However, the SYBR Green assay detected E. canis, E. ewingii, E. muris, and A. phagocytophilum, as well as E. chaffeensis.'#u#es, 百拇医药
fig.ommitted'#u#es, 百拇医药
TABLE 3. Specificities of four different PCR assays for E. chaffeensis DNA'#u#es, 百拇医药
Conclusions. In conclusion, the real-time PCR assay that we have developed for E. chaffeensis is very sensitive (10 gene copies), is species specific, is suitable for high-throughput applications, and is not inhibited by the presence of human, mouse, or tick DNA. The assay was found to be superior to three assays previously described for the detection of E. chaffeensis based on one or more criteria. It is more sensitive than the HE1/HE3 direct PCR assay, more specific than the SYBR Green real-time PCR assay, and has a higher throughput capacity and less sample handling than the VLPT nested PCR assay. Most importantly, the real-time TaqMan PCR assay provides quantitative data, allowing for simultaneous detection of the pathogen and determination of the infectious loads in ticks and mammals. This assay provides a powerful tool for examining the kinetics of infection with E. chaffeensis and the transmission of the pathogen between mammalian hosts and arthropod vectors.
ACKNOWLEDGMENTS?(dum3, 百拇医药
We thank John W. Sumner (Centers for Disease Control and Prevention) for providing clinical specimens. We thank Gregory A. Dasch for his review of the manuscript. We acknowledge the Biotechnology Core Facility of the National Center for Infectious Diseases, Centers for Disease Control and Prevention, for the synthesis of oligonucleotides.?(dum3, 百拇医药
This research was supported in part by the Association of Public Health Laboratories, through an appointment of the Emerging Infectious Diseases Fellowship Program funded by the Centers for Disease Control and Prevention.?(dum3, 百拇医药
REFERENCES?(dum3, 百拇医药
Anderson, B. E., K. G. Sims, J. G. Olson, J. E. Childs, J. F. Piesman, C. M. Happ, G. O. Maupin, and B. J. Johnson. 1993. Amblyomma americanum: a potential vector of human ehrlichiosis. Am. J. Trop. Med. Hyg. 49:239-244.?(dum3, 百拇医药
Anderson, B. E., J. W. Sumner, J. E. Dawson, T. Tzianabos, C. R. Greene, J. G. Olson, D. B. Fishbein, M. Olsen-Rasmussen, B. P. Holloway, E. H. George, and A. F. Azad. 1992. Detection of the etiologic agent of human ehrlichiosis by polymerase chain reaction. J. Clin. Microbiol. 30:775-780.
Buller, R. S., M. Arens, S. P. Hmiel, C. D. Paddock, J. W. Sumner, Y. Rikhisa, A. Unver, M. Gaudreault-Keener, F. A. Manian, A. M. Liddell, N. Schmulewitz, and G. A. Storch. 1999. Ehrlichia ewingii, a newly recognized agent of human ehrlichiosis. N. Engl. J. Med. 341:148-155.&+, http://www.100md.com
Dawson, J. E., B. E. Anderson, D. B. Fishbein, J. L. Sanchez, C. S. Goldsmith, K. H. Wilson, and C. W. Duntley. 1991. Isolation and characterization of an Ehrlichia sp. from a patient diagnosed with human ehrlichiosis. J. Clin. Microbiol. 29:2741-2745.&+, http://www.100md.com
Dawson, J. E., Y. Rikihisa, S. A. Ewing, and D. B. Fishbein. 1991. Serologic diagnosis of human ehrlichiosis using two Ehrlichia canis isolates. J. Infect. Dis. 163: 564-567.&+, http://www.100md.com
Dumler, J. S., A. F. Barbet, C. P. Bekker, G. A. Dasch, G. H. Palmer, S. C. Ray, Y. Rikihisa, and F. R. Rurangirwa. 2001. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila. Int. J. Syst. Evol. Microbiol. 51:2145-2165.
Ewing, S. A., J. E. Dawson, A. A. Kocan, R. W. Barker, C. K. Warner, R. J. Panciera, J. C. Fox, K. M. Kocan, and E. F. Blouin. 1995. Experimental transmission of Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) among white-tailed deer by Amblyomma americanum (Acari: Ixodidae). J. Med. Entomol. 32:368-374..%o'm, http://www.100md.com
Li, J. S., F. Chu, A. Reilly, and G. M. Winslow. 2002. Antibodies highly effective in SCID mice during infection by the intracellular bacterium Ehrlichia chaffeensis are of picomolar affinity and exhibit preferential epitope and isotype utilization. J. Immunol. 169:1419-1425..%o'm, http://www.100md.com
Lockhart, J. M., W. R. Davidson, D. E. Stallknecht, J. E. Dawson, and S. E. Little. 1997. Natural history of Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) in the Piedmont physiographic province of Georgia. J. Parasitol. 83:887-894..%o'm, http://www.100md.com
Massung, R. F., K. Lee, M. Mauel, and A. Gusa. 2002. Characterization of the rRNA genes of Ehrlichia chaffeensis and Anaplasma phagocytophila. DNA Cell Biol. 21:587-596..%o'm, http://www.100md.com
McQuiston, J. H., C. D. Paddock, R. C. Holman, and J. E. Childs. 1999. The human ehrlichioses in the United States. Emerg. Infect. Dis. 5:635-642.
Paddock, C. D., J. W. Sumner, G. M. Shore, D. C. Bartley, R. C. Elie, J. G. McQuade, C. R. Martin, C. S. Goldsmith, and J. E. Childs. 1997. Isolation and characterization of Ehrlichia chaffeensis strains from patients with fatal ehrlichiosis. J. Clin. Microbiol. 35:2496-2502.:z%9, 百拇医药
Sumner, J. W., J. E. Childs, and C. D. Paddock. 1999. Molecular cloning and characterization of the Ehrlichia chaffeensis variable-length PCR target: an antigen-expressing gene that exhibits interstrain variation. J. Clin. Microbiol. 37:1447-1453.:z%9, 百拇医药
Weiss, E., G. A. Dasch, Y.-H. Kang, and H. N. Westfall. 1988. Substrate utilization by Ehrlichia sennetsu and Ehrlichia risticii separated from host constituents by renografin gradient centrifugation. J. Bacteriol. 170:5012-5017.:z%9, 百拇医药
Wen, B., Y. Rikihisa, J. Mott, P. A. Fuerst, M. Kawahara, and C. Suto. 1995. Ehrlichia muris sp. nov., identified on the basis of 16S rRNA base sequences and serological, morphological, and biological characteristics. Int. J. Syst. Bacteriol. 45:250-254.:z%9, 百拇医药
Yeh, M.-T., T. N. Mather, R. T. Coughlin, C. Gingrich-Baker, J. W. Sumner, and R. F. Massung. 1997. Serologic and molecular detection of granulocytic ehrlichiosis in Rhode Island. J. Clin. Microbiol. 35:944-947.(Amanda D. Loftis Robert F. Massung and Michael L. Levin)