Molecular detection and characterization of Anaplasma platys and Ehrlichia canis in dogs from the Caribbean

Abstract

Anaplasma platys is a tick-transmitted rickettsial pathogen, which is known to be the etiologic agent for cyclic thrombocytopenia in its primary canine host. Infections with this pathogen are also reported in cats, cattle and people. Similarly, Ehrlichia canis is another tick-borne rickettsial pathogen responsible for canine monocytic ehrlichiosis and is also reported to cause infections in people. We describe infections in dogs with these two pathogens on the Caribbean island of Grenada, West Indies by detection using molecular methods. We utilized a 16S rRNA gene-based PCR assay to detect both Ehrlichia and Anaplasma species by screening 155 canine blood samples from asymptomatic dogs. We found 18.7 % of the dogs to be positive for A. platys and 16.8 % for E. canis. Samples that tested positive for A. platys were further assessed by sequence analysis targeting 16S rRNA, 23S rRNA, citrate synthase (gltA) and heat shock protein (groEL) genes. Phylogenetic analysis revealed high correlation of A. platys 16S rRNA and gltA gene sequences with the geographic origins, while 23S rRNA and groEL gene sequences clustered independent of the geographic origins. This study represents an important step in defining the widespread distribution of active rickettsial infections in Caribbean dogs with no apparent clinical signs, thus posing a high risk for canine health and to a lesser extent to humans, as most dogs in the Caribbean are free-roaming.

Keywords: 16S rRNA; 23S rRNA; Anaplasma platys; Citrate synthase (gltA) and heat shock protein (groEL); Ehrlichia canis; PCR; Tick-borne diseases.

Fig. 1.

Maximum likelihood (ML) phylogenetic analysis of A. platys 16S rRNA gene sequences. Phylogenetic relationships between A. platys and homologous 16S sequences from A. marginale, A. phagocytophilum, E. canis, E. chaffeensis and E. ruminantium were established. Bootstrap values are shown by numbers at each internal node and these represent the percentage of 1000 replicates for which the same branching pattern was obtained. There were a total of 1456 positions in the dataset. Both NJ and UPGMA analyses for 16S sequences generated trees with similar topology with those derived from ML analysis (Supplementary Fig. 1a and b). A total of 29 sequences were used in the final analysis and the scale-bar represents a 1% nucleotide sequence divergence. Pairwise identity results of these data are given in Supplementary Table 1 and Data 1. (KSU*) = sample obtained from a dog tested positive in Florida, USA. GND = Grenada samples.

Fig. 2.

Maximum likelihood (ML) phylogenetic analysis of A. platys 23S rRNA gene sequences. Phylogenetic relationships between A. platys and homologous 23S sequences from A. marginale, A. phagocytophilum, E. canis, E. chaffeensis and E. ruminantium were established. Bootstrap values are shown by numbers at each internal node and represent the percentage of 1000 replicates for which the same branching pattern was obtained. There were a total of 2794 positions in the dataset. Both NJ and UPGMA analyses for 23S sequences generated trees with similar topology with those derived from ML analysis (Supplementary Fig. 2a and b). A total of 13 nucleotide sequences were used in the final analysis and the scale-bar represents a 2% nucleotide sequence divergence. Pairwise identity results of these data are given in Supplementary Table 2 and Data 2. (KSU*) = sample obtained from a dog tested positive in Florida, USA. GND = Grenada samples.

Fig. 3.

Phylogenetic analysis of A. platys gltA gene sequences. Phylogenetic relationships between A. platys and homologous gltA sequences from A. marginale, A. phagocytophilum, E. canis, E. chaffeensis and E. ruminantium were established. Bootstrap values are shown by numbers at each internal node and represent the percentage of 1000 replicates for which the same branching pattern was obtained. There were a total of 1287 positions in the dataset. The analysis was performed as in Fig. 2. For additional details, please refer to Supplementary Fig. 3a and b. A total of 21 nucleotide sequences were used in the final analysis and the scale-bar represents a 10 % nucleotide sequence divergence. Pairwise identity results are included in Supplementary Table 3 and Data 3.

Fig. 4.

Phylogenetic analysis of A. platys groEL gene sequences. Phylogenetic relationships between A. platys and homologous groEL sequences from A. marginale, A. phagocytophilum, E. canis, E. chaffeensis and E. ruminantium were established. Bootstrap values are shown by numbers at each internal node and represent the percentage of 1000 replicates for which the same branching pattern was obtained. There were a total of 1653 positions in the dataset. The analysis was performed as in the previous two figures. Additional details are provided in Supplementary Fig. 4a and b. A total of 19 nucleotide sequences were used in the final analysis and the scale-bar represents a 5% nucleotide sequence divergence. Pairwise identity results are included in Supplementary Table 4 and Data 4.