Recombinant Ehrlichia canis GP19 Protein as a Promising Vaccine Prototype Providing a Protective Immune Response in a Mouse Model.

The intracellular bacterium Ehrlichia canis is the causative pathogen of canine monocytic ehrlichiosis (CME) in dogs. Despite its veterinary and medical importance, there is currently no available vaccine against this pathogen. In this study, the recombinant GP19 (rGP19) was produced and used as a recombinant vaccine prototype in a mouse model against experimental E. canis infection. The efficacy of the rGP19 vaccine prototype in the part of stimulating B and T cell responses and conferring protection in mice later challenged with E. canis pathogen were evaluated. The rGP19-specific antibody response was evaluated by ELISA after E. canis challenge exposure (on days 0, 7, and 14 post-challenge), and demonstrated significantly higher mean antibody levels in rGP19-immunized mice compared with adjuvant-immunized and naive mice. Significantly lower ehrlichial loads in blood, liver, and spleen DNA samples were detected in the immunized mice with rGP19 by qPCR. The up-regulation of IFNG and IL1 mRNA expression were observed in mice immunized with rGP19. In addition, this study detected IFN-γ-producing memory CD4+ T cells in the rGP19-immunized mice and later infected with E. canis on day 14 post-infection period using flow cytometry. The present study provided a piece of evidence that rGP19 may eliminate E. canis by manipulating Th1 and B cell roles and demonstrated a promising strategy in vaccine development against E. canis infection in the definitive host for further study.

Effect of GP19 Peptide Hyperimmune Antiserum on Activated Macrophage during Ehrlichia canis Infection in Canine Macrophage-like Cells.

In terms of its veterinary importance, vaccine development against Ehrlichia canis is needed. However, the effect of developing vaccines on humoral immune response against E. canis infection is still unknown. Novel GP194-43 was synthesized according to E. canis GP19 epitope prediction. To restrict any loss and/or illness in the host animal, rabbits were used in this study to produce GP194-43 hyperimmune sera. The effect of GP194-43 hyperimmune sera on neutralization was examined in vitro by determining the inhibition of E. canis infection of the macrophage-like cell line (DH82) in the presence of the sera. Four groups of DH82 cells received differing treatments. These included E. canis experimentally infected DH82 cells, E. canis-infected DH82 cells with control rabbit serum (untreated group), E. canis-infected DH82 cells with GP194-43 rabbit antiserum (treated group) and uninfected cells (negative control group), respectively. The treated group developed a decrease (p < 0.01) in the percentage of E. canis infected cells after 3 days post-infection at 48.57 ± 1.28. In addition, real-time PCR analyses of cytokine mRNA expression involved with the macrophage, humoral, and cellular immune responses were conducted. The findings revealed an upregulated expression of IFNG in the treated group during the infection. This study demonstrated neutralization in the GP194-43 peptide hyperimmune sera of immunized rabbits. Notably, IFN-γ production could be effectively promoted in canine macrophages in relation to the activation of macrophages and adaptive immune responses. The results of this study indicate the potential for the use of this immunogen in further investigations involving immunized and infected dogs as E. canis host species.

Two different genogroups of Ehrlichia canis from dogs in Thailand using immunodominant protein genes.

Ehrlichia canis is the causative agent of canine monocytic ehrlichiosis (CME). While there is a high prevalence of CME in Thailand, genetic diversity of E. canis is still poorly defined. This study examined the molecular characteristics of E. canis using PCR and phylogenetic analysis of the dsb, gp19 and gp36 genes. DNA was extracted from 220 whole blood samples of naturally infected dogs, and all had clinical signs compatible with tick-borne diseases. Of these, 16.4% (36/220) provided positive E. canis DNA via the dsb and gp19 genes. However, only 13 out of the 36 samples (36.1%) were positive for the gp36 gene. Sequences of the dsb gene had very high identity (99-100%) with previously deposited E. canis sequences. Sequences of the gp19 gene were similar to those from US and Taiwanese genogroups (98.8-99.5% identity). Elucidation of genetic characteristics of E. canis based on the gp36 gene displayed 91.4-99.1% shared identity. There were 426-429 bp of a 5' end pre-repeat tandem region, a 27 bp repetition with variable numbers of a tandem repeat (TR) region of 9 amino acid sequences (TEDSVSAPA), and a variable 3' end region with sequence length depending on the isolate (72-93 bp). Phylogenetic trees of E. canis, particularly using the gp36 amino acid sequences, showed that the Thai strains fell into two phylogenetic clades contained within other worldwide E. canis strains. Alignment and phylogenetic analysis suggested that E. canis strains from Thailand could be divided into two genogroups, the US and Taiwanese genogroups. This study provides the first characterization of the dsb and gp19 genes of E. canis in Thailand, the results support the conclusion that the gp36 is a potential target for genotyping and elucidation of phylogenetic relationships among E. canis strains.