Brazilian Journal of Microbiology

, Volume 50, Issue 1, pp 1–12 | Cite as

Molecular characterization of Ehrlichia canis from naturally infected dogs from the state of Rio de Janeiro

  • Renata Lins da CostaEmail author
  • Patrícia Gonzaga Paulino
  • Claudia Bezerra da Silva
  • Gabriela Lopes Vivas Vitari
  • Maristela Peckle Peixoto
  • Ana Paula Martinez de Abreu
  • Huarrisson Azevedo Santos
  • Carlos Luiz Massard
Bacterial, Fungal and Virus Molecular Biology - Research Paper


The aim of the present study was to evaluate the genetic diversity of Ehrlichia canis in naturally infected dogs from six mesoregions of Rio de Janeiro state. E. canis was diagnosed with a real-time polymerase chain reaction (qPCR) targeting a 93 base pair (bp) fragment of the 16S rDNA gene. To evaluate the genetic diversity of the parasite, we amplified a positive sample from each mesoregion by distinct conventional PCR assays with targets in the gp19 (414 bp), gp36 (814 bp), and p28 (843 bp) genes. A total of 267 samples were collected from dogs in Rio de Janeiro state. Among the samples analyzed, 42.3% (n = 113/267) were 16S rDNA-qPCR positive. When performing PCR for the gp19 and gp36 genes, 100% (n = 113/113) and 5.3% (n = 6/113) of the samples amplified fragments of 414 bp and 814 bp, respectively. The six PCR-positive samples for the gp36 gene also amplified the p28 gene fragment. The characterization based on the gp19 gene demonstrated that it is highly conserved. In protein analysis (TRP36), all samples showed a tandem repeat protein (TRP) that comprised 11 replicates. Seven high-entropy amino acid sites were distributed throughout the gp36 gene. Eleven high-entropy amino acid sites were found throughout the p28 gene. There is a positive selection pressure in both genes (p ≤ 0.05). Comparing and characterizing an organism are useful for providing important information about the host’s immune response and identifying new antigenic targets, as well as essential characteristics for the development of vaccines and new diagnostic tools.


Genetic diversity Molecular markers Epitopes Canine monocytic ehrlichiosis 



This study was financially supported by the National Council for Scientific and Technological Development (CNPq) of Brazil, the “Carlos Chagas Filho” Foundation for Research Support of the state of Rio de Janeiro (FAPERJ) and Coordination of Improvement of Higher Education Personnel (CAPES) funds.

Compliance with ethical standards

Procedures performed on animals in this study were approved by the Ethics Committee on Animal Use of the UFRRJ (CEUA/UFRRJ) under procedure number 072/2014. These procedures comply with the basic and ethical principles for research involving the use of animals. All procedures were performed by a team of trained veterinarians.

Conflict of interest

The author(s) declared that there is no conflict of interest.


  1. 1.
    Donatien A, Lestoquard F (1935) Existence En Algerie D’une Rickettsia Du Chein. Bull Soc Pathol Exot 28:418–419Google Scholar
  2. 2.
    Huxsoll DL, Hildebrandt PK, Nims RM, Walker JS (1970) Tropical canine pancytopenia. Journal of American Veterinary Medical Association 157(11):1627–1632Google Scholar
  3. 3.
    Macieira DB, Messick JB, Cerqueira AM, Linhares GFC, Freire IMA, Almeida NKO, Almosny NRP (2005. In press) Prevalence of Ehrlichia canis infection in thrombocytopenic dogs from Rio de Janeiro. Brazil Veterinary Clinical Pathology 34:44–48CrossRefGoogle Scholar
  4. 4.
    Ferreira RF, AMF C, Castro TX, Ferreira EO, FPG N, Barbosa AV, Macieira DB, NRP A (2014) Genetic diversity of Ehrlichia canis strains from naturally infected dogs in Rio de Janeiro. Rev Bras Parasitol Vet 23:301–308CrossRefGoogle Scholar
  5. 5.
    Nakaghi ACH, Machado RZ, Ferro J, Labruna MB, Chryssafidis AL, André MR, Baldani CD (2010) Sensitivity evaluation of a single-step PCR assay using Ehrlichia canis p28 gene as a target and its application in diagnosis of canine ehrlichiosis. Rev Bras Parasitol Vet 19:75–79CrossRefGoogle Scholar
  6. 6.
    Aguiar DM, Zhang X, Melo ALT, Pacheco TA, Meneses AMC, Zanutto MS, Horta MC, Santarém VA, Camargo LMA, Mcbride JW, Labruna MB (2013) Genetic diversity of Ehrlichia canis in Brazil. Vet Microbiol 164:315–321CrossRefGoogle Scholar
  7. 7.
    Eiras DF, Craviotto MB, Vezzani D, Eyal O, Baneth G (2013) First description of natural Ehrlichia canis and Anaplasma platys infections in dogs from Argentina. Comp Immunol Microbiol Infect Dis 36(2):169–173CrossRefGoogle Scholar
  8. 8.
    Mittal M, Kundu K, Chakravarti S, Mohapatra JK, Nehra K, Sinha VK, Sanjeeth BS, Churamani CP, Kumar A (2017) Canine Monocytic Ehrlichiosis among working dogs of organised kennels in India: a comprehensive analyses of clinico-pathology, serological and molecular epidemiological approach. Prev Vet Med 1(147):26–33CrossRefGoogle Scholar
  9. 9.
    Kamani J, Lee CC, Haruna AM, Chung PJ, Weka PR, Chung YT (2013) First detection and molecular characterization of Ehrlichia canis from dogs in Nigeria. Res Vet Sci 94(1):27–32CrossRefGoogle Scholar
  10. 10.
    Maazi N, Malmasi A, Shayan P, Nassiri SM, Salehi TZ, Fard MS (2014) Molecular and serological detection of Ehrlichia canis in naturally exposed dogs in Iran: an analysis on associated risk factors. Braz J Vet Parasitol 23(1):16–22CrossRefGoogle Scholar
  11. 11.
    Murphy GE, Ewing SA, Whitworth LC, Fox JC, Kocan AAA (1998) A molecular and serologic survey of Ehrlichia canis, E. chaffensis, and E. ewingii in dogs from Oklahoma. Vet Parasitol 79:325–339CrossRefGoogle Scholar
  12. 12.
    Baneth G, Harrus S, Ohnona FS, Schlesinger Y (2008) Longitudinal quantification of Ehrlichia canis in experimentel infection with comparison to natural infection. Vet Microbiol 136:321–325CrossRefGoogle Scholar
  13. 13.
    Chen YH, Lee CC, Tsang CL, Chung YT (2010) Detection and characterization of four novel genotypes of Ehrlichia canis from dogs. Vet Microbiol 146:70–75CrossRefGoogle Scholar
  14. 14.
    Zhang X, Luo T, Keysary A (2008) Genetic and antigenic diversities of major immunoreactive proteins in globally distributed Ehrlichia canis strains. Clin Vaccine Immunol 15:1080–1088CrossRefGoogle Scholar
  15. 15.
    Cárdenas AM, Doyle CK, Zhang X, Nethery K, Corstvet RE, Walker DH, Mcbride JW (2007) Enzyme-linked Immunosorbent assay with conserved immunoreactive glycoproteins TRP36 and gp19 has enhanced sensitivity and provides species-specific immunodiagnosis of Ehrlichia canis infection. Clin Vacc Immun 14:123–128CrossRefGoogle Scholar
  16. 16.
    Doyle CK, Nethery KA, Popov VL, McBride JW (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. Infec Immun 74:711–720CrossRefGoogle Scholar
  17. 17.
    Mcbride JW, Walker DH (2011) Molecular and cellular pathobiology of Ehrlichia infection: targets for new therapeutics and immunomodulation strategies. Expert Rev Mol Med 31:1–3Google Scholar
  18. 18.
    Long W, Zhang XF, Qi H, Standaert S, Walker D, Yu XJ (2002) Antigenic variation of Ehrlichia chaffeensis resulting from differential expression of the 28-Kilodalton protein gene family. Infect Immun 70:1824–1831CrossRefGoogle Scholar
  19. 19.
    Mcbride JW, Yu XJ, Walker DH (1999) Molecular cloning of the gene for a conserved major immunoreactive 28-Kilodalton protein of Ehrlichia canis: a potential serodiagnosis antigen. Clin Diagn Lab Immun 6:392–399Google Scholar
  20. 20.
    Mcbride JW, YU X, Walker DH (2000) A conserved, transcriptionally active p28 multigene lócus of Ehrlichia canis. Gene 254:245–252CrossRefGoogle Scholar
  21. 21.
    IBGE (2010) Municípios. Disponível em: Último acesso em: 14 de dezembro de 2017
  22. 22.
    Sampaio IBM (2002) Estatística aplicada à experimentação Animal, 2th edn. Belo Horizonte, BrazilGoogle Scholar
  23. 23.
    Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55(4):611–622CrossRefGoogle Scholar
  24. 24.
    Svec AC, Tichopa DB, Novosadova V, Michael W, Pfaffl MW, Kubistaa M (2015) How good is a PCR efficiency estimate: recommendations for precise and robust qPCR efficiency assessments. Biomolecular Detection and Quantification 3:9–16CrossRefGoogle Scholar
  25. 25.
    Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74:5463–5467CrossRefGoogle Scholar
  26. 26.
    Posada D, Crandall KA (1998) Modeltest: testing the model of DNA substitution. Bioinformatics 14:817–818CrossRefGoogle Scholar
  27. 27.
    Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 11(22):4673–4680CrossRefGoogle Scholar
  28. 28.
    Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 30:772CrossRefGoogle Scholar
  29. 29.
    Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  30. 30.
    Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA 6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729CrossRefGoogle Scholar
  31. 31.
    Brum FA (2010) Cloning and Expression of the gp19 protein of Ehrlichia canis. 2010. 10f. Dissertação (Mestrado: Biologia) Universidade Federal de Pelotas, PelotasGoogle Scholar
  32. 32.
    Mcbride JW, Doyle CK, Zhang X, Cardenas AM, Popov VL, Nethery KA, Woods ME (2007) Identification of a glycosylated Ehrlichia canis 19-Kilodalton major immunoreactive protein with a species-specific serine-rich glycopeptide epitope. Infect Immun 75:74–82CrossRefGoogle Scholar
  33. 33.
    Zweygarth A, Cabezas-Cruz A, Marinda C, Oosthuizen P, Matjila KL, Marzena B, Schöl H, Ferrolho J, Grubhoffer L, Passos LMF (2014) In vitro culture and structural differences in the major immunoreactive protein TRP36 of geographically distant Ehrlichia canis sequences. Ticks and Tick-borne Diseases:1–9Google Scholar
  34. 34.
    Hasegawa MY (2005) Dinâmica da infecção experimental de cães por Ehrlichia canis: Aspectos clínicos, laboratoriais e resposta imune humoral e celular. 136f. Tese (Doutorado em Medicina Veterinária) Programa de Pós Graduação em Clínica Veterinária Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo-FMVZ, SPGoogle Scholar
  35. 35.
    Nakaghi ACH, Machado RZ, Costa MT, André MR, Baldani CD (2008) Canine Ehrlichiosis: clinical, hematological, serological and molecular aspects. Ciência Rural 38:766–770CrossRefGoogle Scholar
  36. 36.
    Alves RN, Rieck SE, Ueira-Vieira C, Labruna MB, Beletti ME (2014) Isolation, in vitro propagation, genetic analysis, and immunogenic characterization of an Ehrlichia canis strain from southeastern Brazil. J Vet Sci 15(2):241–248CrossRefGoogle Scholar
  37. 37.
    Aguiar DM, Hagiwara MK, Labruna MB (2008) In vitro isolation and molecular characterization of an Ehrlichia canis strain from São Paulo, Brazil. Braz J Microbiol 39(3):489–493CrossRefGoogle Scholar

Copyright information

© Sociedade Brasileira de Microbiologia 2018

Authors and Affiliations

  • Renata Lins da Costa
    • 1
    Email author
  • Patrícia Gonzaga Paulino
    • 2
  • Claudia Bezerra da Silva
    • 2
  • Gabriela Lopes Vivas Vitari
    • 1
  • Maristela Peckle Peixoto
    • 1
  • Ana Paula Martinez de Abreu
    • 1
  • Huarrisson Azevedo Santos
    • 2
  • Carlos Luiz Massard
    • 1
  1. 1.Department of Animal Parasitology, Veterinary InstituteFederal Rural University of Rio de JaneiroSeropédicaBrazil
  2. 2.Department of Epidemiology and Public Health, Veterinary InstituteFederal Rural University of Rio de JaneiroSeropédicaBrazil

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