J Vet Sci.  2014 Jun;15(2):241-248. 10.4142/jvs.2014.15.2.241.

Isolation, in vitro propagation, genetic analysis, and immunogenic characterization of an Ehrlichia canis strain from southeastern Brazil

• Alves RN
• Rieck SE
• Ueira-Vieira C
• Labruna MB
• Beletti ME

• KMID: 1784645
• DOI: http://doi.org/10.4142/jvs.2014.15.2.241

Abstract

Amplification of the 16S rRNA gene from a blood sample obtained from a dog in southeastern Brazil was used to confirm a naturally acquired Ehrlichia (E.) canis infection. Following isolation and culturing of the new bacterial strain called Uberlandia, partial sequences of the dsb and p28 genes were obtained. The dsb partial sequence of the novel strain was 100% similar to dsb gene sequences of E. canis obtained from different geographic areas around the world. Conversely, the p28 partial sequence for the E. canis Uberlandia strain differed at several nucleotides from other sequences available in GenBank. To confirm the antigenic profile of the Uberlandia strain, an indirect immunofluorescence assay against E. canis antigens was performed using dog sera collected from two different areas in Brazil (Uberlandia and Sao Paulo). The results suggest that both antigens were able to identify animals seropositive for E. canis in Brazil since these Brazilian strains appear to be highly conserved.

Keyword

Brazil; Ehrlichia canis; genetic characterization; isolation; serology

MeSH Terms

Animals
Antigens, Bacterial/blood/*diagnostic use
Bacterial Outer Membrane Proteins/genetics/metabolism
Bacterial Proteins/*genetics/metabolism
Base Sequence
Brazil
Dog Diseases/diagnosis/*microbiology
Dogs
Ehrlichia canis/*genetics/*immunology/isolation & purification
Ehrlichiosis/diagnosis/microbiology/*veterinary
Fluorescent Antibody Technique, Indirect/veterinary
Male
Molecular Sequence Data
Polymerase Chain Reaction/veterinary
RNA, Ribosomal, 16S/genetics/metabolism
Sequence Alignment/veterinary
Antigens, Bacterial
Bacterial Outer Membrane Proteins
Bacterial Proteins
RNA, Ribosomal, 16S

Figure

• 

  Fig. 1 Photomicrograph of an Ehrlichia (E.) canis strain isolated from the leukocytes of a dog in Uberlândia, Brazil. Note the multiple round, large morulae in the DH82 cell cytoplasm (arrows). Diff-Quik staining was performed. Scale bar = 10 µm.

• 

  Fig. 2 Clustal alignment of p28 gene sequences from E. canis VHE (Venezuelan human ehrlichia), São Paulo, Jake, Oklahoma, Jaboticabal, and Uberlândia strains. Boxed nucleotides are bases that differ from the Uberlândia strain sequence, and a dash represents a gap or nucleotide that was unidentified.

• 

  Fig. 3 Comparison of the antigenic indices for the predicted partial proteins translated from the p28 gene of the following strains: E. canis Uberlândia, São Paulo, Jaboticabal, Jake, Oklahoma, and VHE. Regions with values above zero are possible antigenic determinants. Boxed sequences are potential epitopes that differ from the Uberlândia strain sequence. The scale indicates amino acid positions.

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