In vitro culture and structural differences in the major immunoreactive protein gp36 of geographically distant Ehrlichia canis isolates.
Ehrlichia canis, the etiologic agent of canine ehrlichiosis, is an obligate intracytoplasmic Gram-negative tick-borne bacterium belonging to the Anaplasmataceae family. E. canis is distributed worldwide and can cause serious and fatal infections in dogs. Among strains of E. canis, the 16S rRNA gene DNA sequences are highly conserved. Using this gene to genetically differentiate isolates is therefore difficult. As an alternative, the gene gp36, which encodes for a major immunoreactive protein in E. canis, has been successfully used to characterize the genetic diversity of this pathogen. The present study describes the isolation and continuous propagation of a Spanish and 2 South African isolates of E. canis in IDE8 tick cells. Subsequently, canine DH82 cell cultures were infected using initial bodies obtained from infected IDE8 cultures. It was possible to mimic the life cycle of E. canis in vitro by transferring infection from tick cells to canine cells and back again. To characterize these E. canis strains at the molecular level, the 16S rRNA and gp36 genes were amplified by PCR, sequenced, and aligned with corresponding sequences available in GenBank. All 16S rRNA sequences amplified in this study were identical to previously reported E. canis strains. Maximum likelihood analysis based on the gp36 amino acid sequences showed that the South African and Spanish strains fall into 2 well-defined phylogenetic clusters amongst other E. canis strains. The members of these 2 phylogenetic clusters shared 2 unique molecular properties in the gp36 amino acid sequences: (i) deletion of glycine 117 and (ii) the presence of an additional putative N-linked glycosylation site. We further show correlation between the putative secondary structure and the theoretical isoelectric point (pI) of the gp36 amino acid sequences. A putative role of gp36 as an adhesin in E. canis is discussed. Overall, we report the successful in vitro culture of 3 new E. canis strains which present different molecular properties in their gp36 sequences.
Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-Universität München, Leopoldstrasse 5, 80802 Munich, Germany. Electronic address: ZweygarthE@gmail.com.
Erich
E
Zweygarth
University of South Bohemia, Faculty of Science and Biology Centre of the ASCR, Parasitology Institute, České Budějovice, Czech Republic.
Alejandro
A
Cabezas-Cruz
Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort 0110, South Africa.
Antoinette I
AI
Josemans
Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X4, Onderstepoort 0110, South Africa.
Marinda C
MC
Oosthuizen
Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X4, Onderstepoort 0110, South Africa; Department of Life and Consumer Sciences, College of Agriculture and Environmental Sciences, Unisa, Private Bag X06, Florida 1710, South Africa.
Paul T
PT
Matjila
Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-Universität München, Leopoldstrasse 5, 80802 Munich, Germany.
Katarzyna
K
Lis
Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-Universität München, Leopoldstrasse 5, 80802 Munich, Germany.
Marzena
M
Broniszewska
Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-Universität München, Leopoldstrasse 5, 80802 Munich, Germany.
Heidrun
H
Schöl
The Pirbright Institute, Pirbright Laboratory, Ash Road, Pirbright, Surrey GU24 0NF, UK.
Joana
J
Ferrolho
University of South Bohemia, Faculty of Science and Biology Centre of the ASCR, Parasitology Institute, České Budějovice, Czech Republic.
Libor
L
Grubhoffer
Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-Universität München, Leopoldstrasse 5, 80802 Munich, Germany; Departamento de Medicina Veterinária Preventiva, Escola de Veterinária-UFMG, Belo Horizonte, Minas Gerais, Brazil.
Lygia M F
LM
Passos
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004283:Dog Diseases / Q000382:microbiology*; D004285:Dogs; D041061:Ehrlichia canis / Q000235:genetics / Q000302:isolation & purification*; D016873:Ehrlichiosis / Q000382:microbiology / Q000662:veterinary*; D014644:Genetic Variation; D005843:Geography; D018884:Ixodes / Q000382:microbiology*; D008969:Molecular Sequence Data; D010802:Phylogeny; D017433:Protein Structure, Secondary; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D016415:Sequence Alignment / Q000662:veterinary; D017422:Sequence Analysis, DNA / Q000662:veterinary; D020080:Tandem Repeat Sequences / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
16S rRNA; DH82; Ehrlichia canis; IDE8 tick cells; In vitro culture; gp36