This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ohashi, N.
Right arrow Articles by Rikihisa, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ohashi, N.
Right arrow Articles by Rikihisa, Y.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, September 1998, p. 2671-2680, Vol. 36, No. 9
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Cloning and Characterization of Multigenes Encoding the Immunodominant 30-Kilodalton Major Outer Membrane Proteins of Ehrlichia canis and Application of the Recombinant Protein for Serodiagnosis

Norio Ohashi, Ahmet Unver, Ning Zhi, and Yasuko Rikihisa*

Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210-1093

Received 2 March 1998/Returned for modification 7 April 1998/Accepted 16 June 1998

A 30-kDa major outer membrane protein of Ehrlichia canis, the agent of canine ehrlichiosis, is the major antigen recognized by both naturally and experimentally infected dog sera. The protein cross-reacts with a serum against a recombinant 28-kDa protein (rP28), one of the outer membrane proteins of a gene (omp-1) family of Ehrlichia chaffeensis. Two DNA fragments of E. canis were amplified by PCR with two primer pairs based on the sequences of E. chaffeensis omp-1 genes, cloned, and sequenced. Each fragment contained a partial 30-kDa protein gene of E. canis. Genomic Southern blot analysis with the partial gene probes revealed the presence of multiple copies of these genes in the E. canis genome. Three copies of the entire gene (p30, p30-1, and p30a) were cloned and sequenced from the E. canis genomic DNA. The open reading frames of the two copies (p30 and p30-1) were tandemly arranged with an intergenic space. The three copies were similar but not identical and contained a semivariable region and three hypervariable regions in the protein molecules. The following genes homologous to three E. canis 30-kDa protein genes and the E. chaffeensis omp-1 family were identified in the closely related rickettsiae: wsp from Wolbachia sp., p44 from the agent of human granulocytic ehrlichiosis, msp-2 and msp-4 from Anaplasma marginale, and map-1 from Cowdria ruminantium. Phylogenetic analysis among the three E. canis 30-kDa proteins and the major surface proteins of the rickettsiae revealed that these proteins are divided into four clusters and the two E. canis 30-kDa proteins are closely related but that the third 30-kDa protein is not. The p30 gene was expressed as a fusion protein, and the antibody to the recombinant protein (rP30) was raised in a mouse. The antibody reacted with rP30 and a 30-kDa protein of purified E. canis. Twenty-nine indirect fluorescent antibody (IFA)-positive dog plasma specimens strongly recognized the rP30 of E. canis. To evaluate whether the rP30 is a suitable antigen for serodiagnosis of canine ehrlichiosis, the immunoreactions between rP30 and the whole purified E. canis antigen were compared in the dot immunoblot assay. Dot reactions of both antigens with IFA-positive dog plasma specimens were clearly distinguishable by the naked eye from those with IFA-negative plasma specimens. By densitometry with a total of 42 IFA-positive and -negative plasma specimens, both antigens produced results similar in sensitivity and specificity. These findings suggest that the rP30 antigen provides a simple, consistent, and rapid serodiagnosis for canine ehrlichiosis. Cloning of multigenes encoding the 30-kDa major outer membrane proteins of E. canis will greatly facilitate understanding pathogenesis and immunologic study of canine ehrlichosis and provide a useful tool for phylogenetic analysis.

* Corresponding author. Mailing address: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, 1925 Coffey Rd., Columbus, OH 43210-1093. Phone: (614) 292-9677. Fax: (614) 292-6473. E-mail: rikihisa.1{at}osu.edu.

Journal of Clinical Microbiology, September 1998, p. 2671-2680, Vol. 36, No. 9
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Luo, T., Zhang, X., Wakeel, A., Popov, V. L., McBride, J. W. (2008). A Variable-Length PCR Target Protein of Ehrlichia chaffeensis Contains Major Species-Specific Antibody Epitopes in Acidic Serine-Rich Tandem Repeats. Infect. Immun. 76: 1572-1580 [Abstract] [Full Text]  
  • Zhang, C., Xiong, Q., Kikuchi, T., Rikihisa, Y. (2008). Identification of 19 Polymorphic Major Outer Membrane Protein Genes and Their Immunogenic Peptides in Ehrlichia ewingii for Use in a Serodiagnostic Assay. CVI 15: 402-411 [Abstract] [Full Text]  
  • Lopez, L., Venteo, A., Aguirre, E., Garcia, M., Rodriguez, M., Amusategui, I., Tesouro, M. A., Vela, C., Sainz, A., Rueda, P. (2007). Development of a sensitive and specific indirect enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen for detection of specific antibodies against Ehrlichia canis. jvdi 19: 635-642 [Abstract] [Full Text]  
  • Nethery, K. A., Doyle, C. K., Zhang, X., McBride, J. W. (2007). Ehrlichia canis gp200 Contains Dominant Species-Specific Antibody Epitopes in Terminal Acidic Domains. Infect. Immun. 75: 4900-4908 [Abstract] [Full Text]  
  • Miura, K., Rikihisa, Y. (2007). Virulence Potential of Ehrlichia chaffeensis Strains of Distinct Genome Sequences. Infect. Immun. 75: 3604-3613 [Abstract] [Full Text]  
  • Cardenas, A. M., Doyle, C. K., Zhang, X., Nethery, K., Corstvet, R. E., Walker, D. H., McBride, J. W. (2007). Enzyme-Linked Immunosorbent Assay with Conserved Immunoreactive Glycoproteins gp36 and gp19 Has Enhanced Sensitivity and Provides Species-Specific Immunodiagnosis of Ehrlichia canis Infection. CVI 14: 123-128 [Abstract] [Full Text]  
  • Doyle, C. K., Nethery, K. A., Popov, V. L., McBride, J. W. (2006). Differentially Expressed and Secreted Major Immunoreactive Protein Orthologs of Ehrlichia canis and E. chaffeensis Elicit Early Antibody Responses to Epitopes on Glycosylated Tandem Repeats. Infect. Immun. 74: 711-720 [Abstract] [Full Text]  
  • Collins, N. E., Liebenberg, J., de Villiers, E. P., Brayton, K. A., Louw, E., Pretorius, A., Faber, F. E., van Heerden, H., Josemans, A., van Kleef, M., Steyn, H. C., van Strijp, M. F., Zweygarth, E., Jongejan, F., Maillard, J. C., Berthier, D., Botha, M., Joubert, F., Corton, C. H., Thomson, N. R., Allsopp, M. T., Allsopp, B. A. (2005). The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy number. Proc. Natl. Acad. Sci. USA 102: 838-843 [Abstract] [Full Text]  
  • Brayton, K. A., Kappmeyer, L. S., Herndon, D. R., Dark, M. J., Tibbals, D. L., Palmer, G. H., McGuire, T. C., Knowles, D. P. Jr. (2005). Complete genome sequencing of Anaplasma marginale reveals that the surface is skewed to two superfamilies of outer membrane proteins. Proc. Natl. Acad. Sci. USA 102: 844-849 [Abstract] [Full Text]  
  • Fenollar, F., La Scola, B., Inokuma, H., Dumler, J. S., Taylor, M. J., Raoult, D. (2003). Culture and Phenotypic Characterization of a Wolbachia pipientis Isolate. J. Clin. Microbiol. 41: 5434-5441 [Abstract] [Full Text]  
  • Lin, Q., Rikihisa, Y., Ohashi, N., Zhi, N. (2003). Mechanisms of Variable p44 Expression by Anaplasma phagocytophilum. Infect. Immun. 71: 5650-5661 [Abstract] [Full Text]  
  • Knowles, T. T., Alleman, A. R., Sorenson, H. L., Marciano, D. C., Breitschwerdt, E. B., Harrus, S., Barbet, A. F., Belanger, M. (2003). Characterization of the Major Antigenic Protein 2 of Ehrlichia canis and Ehrlichia chaffeensis and Its Application for Serodiagnosis of Ehrlichiosis. CVI 10: 520-524 [Abstract] [Full Text]  
  • McBride, J. W., Corstvet, R. E., Gaunt, S. D., Boudreaux, C., Guedry, T., Walker, D. H. (2003). Kinetics of Antibody Response to Ehrlichia canis Immunoreactive Proteins. Infect. Immun. 71: 2516-2524 [Abstract] [Full Text]  
  • Felek, S., Greene, R., Rikihisa, Y. (2003). Transcriptional Analysis of p30 Major Outer Membrane Protein Genes of Ehrlichia canis in Naturally Infected Ticks and Sequence Analysis of p30-10 of E. canis from Diverse Geographic Regions. J. Clin. Microbiol. 41: 886-888 [Abstract] [Full Text]  
  • Cheng, C., Paddock, C. D., Reddy Ganta, R. (2003). Molecular Heterogeneity of Ehrlichia chaffeensis Isolates Determined by Sequence Analysis of the 28-Kilodalton Outer Membrane Protein Genes and Other Regions of the Genome. Infect. Immun. 71: 187-195 [Abstract] [Full Text]  
  • Belanger, M., Sorenson, H. L., France, M. K., Bowie, M. V., Barbet, A. F., Breitschwerdt, E. B., Alleman, A. R. (2002). Comparison of Serological Detection Methods for Diagnosis of Ehrlichia canis Infections in Dogs. J. Clin. Microbiol. 40: 3506-3508 [Abstract] [Full Text]  
  • Jiggins, F. M., Hurst, G. D. D., Yang, Z. (2002). Host-Symbiont Conflicts: Positive Selection on an Outer Membrane Protein of Parasitic but not Mutualistic Rickettsiaceae. Mol Biol Evol 19: 1341-1349 [Abstract] [Full Text]  
  • Stich, R. W., Rikihisa, Y., Ewing, S. A., Needham, G. R., Grover, D. L., Jittapalapong, S. (2002). Detection of Ehrlichia canis in Canine Carrier Blood and in Individual Experimentally Infected Ticks with a p30-Based PCR Assay. J. Clin. Microbiol. 40: 540-546 [Abstract] [Full Text]  
  • Gusa, A. A., Buller, R. S., Storch, G. A., Huycke, M. M., Machado, L. J., Slater, L. N., Stockham, S. L., Massung, R. F. (2001). Identification of a p28 Gene in Ehrlichia ewingii: Evaluation of Gene for Use as a Target for a Species-Specific PCR Diagnostic Assay. J. Clin. Microbiol. 39: 3871-3876 [Abstract] [Full Text]  
  • Allsopp, M. T. E. P., Dorfling, C. M., Maillard, J. C., Bensaid, A., Haydon, D. T., van Heerden, H., Allsopp, B. A. (2001). Ehrlichia ruminantium Major Antigenic Protein Gene (map1) Variants Are Not Geographically Constrained and Show No Evidence of Having Evolved under Positive Selection Pressure. J. Clin. Microbiol. 39: 4200-4203 [Abstract] [Full Text]  
  • Unver, A., Ohashi, N., Tajima, T., Stich, R. W., Grover, D., Rikihisa, Y. (2001). Transcriptional Analysis of p30 Major Outer Membrane Multigene Family of Ehrlichia canis in Dogs, Ticks, and Cell Culture at Different Temperatures. Infect. Immun. 69: 6172-6178 [Abstract] [Full Text]  
  • Unver, A., Perez, M., Orellana, N., Huang, H., Rikihisa, Y. (2001). Molecular and Antigenic Comparison of Ehrlichia canis Isolates from Dogs, Ticks, and a Human in Venezuela. J. Clin. Microbiol. 39: 2788-2793 [Abstract] [Full Text]  
  • Lodes, M. J., Mohamath, R., Reynolds, L. D., McNeill, P., Kolbert, C. P., Bruinsma, E. S., Benson, D. R., Hofmeister, E., Reed, S. G., Houghton, R. L., Persing, D. H. (2001). Serodiagnosis of Human Granulocytic Ehrlichiosis by Using Novel Combinations of Immunoreactive Recombinant Proteins. J. Clin. Microbiol. 39: 2466-2476 [Abstract] [Full Text]  
  • Ohashi, N., Rikihisa, Y., Unver, A. (2001). Analysis of Transcriptionally Active Gene Clusters of Major Outer Membrane Protein Multigene Family in Ehrlichia canis and E. chaffeensis. Infect. Immun. 69: 2083-2091 [Abstract] [Full Text]  
  • Brayton, K. A., Knowles, D. P., McGuire, T. C., Palmer, G. H. (2001). Efficient use of a small genome to generate antigenic diversity in tick-borne ehrlichial pathogens. Proc. Natl. Acad. Sci. USA 98: 4130-4135 [Abstract] [Full Text]  
  • McBride, J. W., Corstvet, R. E., Breitschwerdt, E. B., Walker, D. H. (2001). Immunodiagnosis of Ehrlichia canis Infection with Recombinant Proteins. J. Clin. Microbiol. 39: 315-322 [Abstract] [Full Text]  
  • Alleman, A. R., Barbet, A. F., Bowie, M. V., Sorenson, H. L., Wong, S. J., Bélanger, M. (2000). Expression of a Gene Encoding the Major Antigenic Protein 2 Homolog of Ehrlichia chaffeensis and Potential Application for Serodiagnosis. J. Clin. Microbiol. 38: 3705-3709 [Abstract] [Full Text]  
  • Tajima, T., Zhi, N., Lin, Q., Rikihisa, Y., Horowitz, H. W., Ralfalli, J., Wormser, G. P., Hechemy, K. E. (2000). Comparison of Two Recombinant Major Outer Membrane Proteins of the Human Granulocytic Ehrlichiosis Agent for Use in an Enzyme-Linked Immunosorbent Assay. CVI 7: 652-657 [Abstract] [Full Text]  
  • Unver, A., Rikihisa, Y., Ohashi, N., Cullman, L. C., Buller, R., Storch, G. A. (1999). Western and Dot Blotting Analyses of Ehrlichia chaffeensis Indirect Fluorescent-Antibody Assay-Positive and -Negative Human Sera by Using Native and Recombinant E. chaffeensis and E. canis Antigens. J. Clin. Microbiol. 37: 3888-3895 [Abstract] [Full Text]  
  • French, D. M., Brown, W. C., Palmer, G. H. (1999). Emergence of Anaplasma marginale Antigenic Variants during Persistent Rickettsemia. Infect. Immun. 67: 5834-5840 [Abstract] [Full Text]  
  • Harrus, S., Waner, T., Bark, H., Jongejan, F., Cornelissen, A. W. C. A. (1999). Recent Advances in Determining the Pathogenesis of Canine Monocytic Ehrlichiosis. J. Clin. Microbiol. 37: 2745-2749 [Full Text]  
  • Buller, R. S., Arens, M., Hmiel, S. P., Paddock, C. D., Sumner, J. W., Rikihisa, Y., Unver, A., Gaudreault-Keener, M., Manian, F. A., Liddell, A. M., Schmulewitz, N., Storch, G. A. (1999). Ehrlichia ewingii, a Newly Recognized Agent of Human Ehrlichiosis. NEJM 341: 148-155 [Abstract] [Full Text]  
  • Zhi, N., Ohashi, N., Rikihisa, Y. (1999). Multiple p44 Genes Encoding Major Outer Membrane Proteins Are Expressed in the Human Granulocytic Ehrlichiosis Agent. J. Biol. Chem. 274: 17828-17836 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»