Anaplasma platys in Dogs, Chile

We conducted a 16S rRNA nested PCR for the genus Ehrlichia and Ehrlichia spp. with blood samples from 30 ill dogs in Chile. Phylogenetic analysis was performed by using groESL gene amplification. We identified Anaplasma platys as 1 of the etiologic agents of canine ehrlichiosis.

We conducted a 16S rRNA nested PCR for the genus Ehrlichia and Ehrlichia spp. with blood samples from 30 ill dogs in Chile. Phylogenetic analysis was performed by using groESL gene amplifi cation. We identifi ed Anaplasma platys as 1 of the etiologic agents of canine ehrlichiosis. E hrlichioses are recognized as important emerging tickborne diseases in humans and wild and domestic animals. The brown dog tick, Rhipicephalus sanguineus, is the main tick that infests dogs in Chile (1). This tick species is a vector of Ehrlichia canis and has been implicated, but not confi rmed, as a vector of Anaplasma platys (2). Serologic and clinical evidence of canine ehrlichiosis and serologic evidence of human ehrlichiosis have been reported in Chile (3,4). The purpose of this study was to identify the etiologic agent of canine ehrlichiosis in Chile.

The Study
Blood samples were obtained from 30 pet dogs seen in a private veterinary clinic in Santiago, Chile, with tick infestation and clinical signs compatible with ehrlichiosis (hemorrhagic manifestations and thrombocytopenia). We performed a nested PCR to amplify a portion of the 16S rRNA gene by using specifi c primers for the genus Ehrlichia and for Ehrlichia spp. DNA was extracted from 300 μL of whole blood by using the Wizard Genomic DNA Purifi cation kit (Promega, Madison, WI, USA). For Ehrlichia genus-specifi c PCR, 2.5 μL of DNA was amplifi ed by using outer primers EHR-OUT1 and EHR-OUT2 and inner primers GE2F and EHRL3-IP2 in 1 reaction with a fi nal volume of 25 μL (5) ( Table 1).
The fi rst-round amplifi cation included 20 cycles of denaturation at 94°C for 45 s, annealing at 72°C for 1.5 min, and chain extension at 72°C for 1.5 min. The second-round amplifi cation included 50 cycles of denaturation at 94°C for 45 s, annealing at 50°C for 1 min, and chain extension at 72°C for 1 min, followed by a fi nal extension at 72°C for 5 min. Amplifi cation products were analyzed by agarose gel electrophoresis. The expected size of the amplifi cation product was 120 bp. A. phagocytophilum DNA was used as a positive control (provided by Didier Raoult). For Ehrlichia spp.-specifi c amplifi cation, we used the same set of outer primers for Anaplasmataceae and specifi c inner primers for A. phagocytophilum (6) The Ehrlichia genus PCR resulted in the expected DNA band in 6 of 30 dogs (dogs 7, 12, 17, 19, 23, and 25). These 6 samples were positive only for A. platys, showing the expected 151-bp product, and negative for other species tested ( Figure 1, panel A). A. platys PCR was also conducted on the remaining 24 Ehrlichia-negative samples; none were positive. DNA obtained from 3 16S rRNA PCR products (dogs 7, 17, and 25) was purifi ed by using a commercial kit (Rapid Gel Extraction System; Marligen Biosciences, Ljamsville, Germany) and sequenced twice with an ABI 3100 genetic analyzer (Model 3100; Applied Biosystems, Foster City, CA, USA). The 16S rRNA sequences obtained were compared by using BLAST (www.ncbi.nlm.nih.gov/blast) with sequences available at GenBank. Sequences obtained were similar to that of A. platys strain Okinawa 1 (Gen-Bank accession no. AF536828), with similarities of 98%, 95%, and 98%, respectively. GenBank accession nos. for 16S rRNA sequences of A. platys strains obtained in this study are DQ125260 and DQ125261, which correspond to strains from dogs 7 and 17, respectively.
For phylogenetic analysis, the groESL gene of A. platys was amplifi ed from samples positive for A. platys 16S rRNA that had suffi cient amounts of DNA (dogs 17, 23, and 25) and from 1 negative sample (dog 13). Reactions contained 2 μL of purifi ed DNA as template in a total volume of 25 μL. Amplifi cations contained 1.25 U Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA), 3 mmol/L MgCl 2 , 2.5 mmol/L deoxynucleotide triphosphates (Invitrogen), and 0.2 pmol/L of primers EEgro1F and EEgro2R (8) ( Table 1). DNA was denatured by heating at 95°C for 10 min. PCR amplifi cation included 40 cycles of denaturation at 95°C for 1.5 min, annealing at 52°C for 2 min, and extension at 72°C for 1.5 min, followed by a fi nal extension at 72°C for 10 min. For nested amplifi cations, 1 μL of primary PCR products was used as the template in a total volume of 25 μL. Reaction conditions were the same as for primary amplifi cations. The primers used were SQ3F, SQ5F, SQ4R, and SQ6R (9) ( Table 1). PCR products were analyzed by 1.5% agarose gel electrophoresis.
We amplifi ed 3 overlapping fragments (790, 1,170, and 360 bp) in 3 16S rRNA-positive samples (Figure 1, panel  B). These DNAs were purifi ed by using a commercial kit (Rapid Gel Extraction System; Marligen), sequenced, and analyzed for phylogenetic relationships. Multiple alignment and ‡Alcántara Veterinary Clinic, Santiago, Chile analysis was performed with the ClustalW program (www. ebi.ac.uk/clustalw). Calculation of distance matrices and construction of a phylogenetic tree were made with MEGA 3.1 software (www.megasoftware.net). A phylogenetic tree was constructed by the neighbor-joining method and distance matrices for the aligned sequences were calculated by using the Kimura 2-parameter method. Stability of the tree was estimated by bootstrap analysis of 1,000 replications. A fi nal sequence of 686 bp obtained from the overlapping fragments was used for comparison and showed 100% identity between the 3 Chilean sequences and 99.8% similarity with sequences of the A. platys groESL gene deposited in GenBank (Table 2). Phylogenetic relationships of Chilean A. platys strains with other Anaplasmataceae species are shown in Figure 2. GenBank accession no. for the groESL gene sequence of A. platys is EF201806 (corresponding to dogs 17, 23, and 25).

Conclusions
We identifi ed A. platys DNA in the blood of 6 dogs with clinical signs indicative of ehrlichiosis. These fi ndings support the conclusion that A. platys is an etiologic agent of canine ehrlichiosis in Chile.
Since its fi rst report in the United States in 1978 (10), A. platys has been described in several countries as the etiologic agent of cyclic thrombocytopenia in dogs. A tick vector of A. platys has not been determined, although R. sanguineus is the most suspected species (2). Because R. sanguineus is the only tick species that infests dogs in Santiago (1), our results support the conclusion that this species is the vector of A. platys in Chile.
A wide range of clinical manifestations of canine cyclic thrombocytopenia has been described. Cases from the United States have been described as mild or asymptomatic (10), and cases from Spain have more severe symptoms (11), which also seems to be the case in Chile. This variability in clinical symptoms of infection has not been clearly associated with strain variations (11)(12)(13).
Low diversity was observed when groESL gene sequences of Chilean strains were compared with other A. platys strains available in GenBank. This fi nding has also been observed in strains from different geographic origins (13).  Recent studies have shown more genetic variability when sequences of the gltA gene were used (11,12). Evidence of the zoonotic potential of A. platys is scarce. In Venezuela, a few symptomatic human cases have been diagnosed since 1992 by the presence of platelet morulae in blood smears (14). Monocytic and platelet morulae were reported in a 17-month-old girl with fever and rash (15). However, none of these cases have been confi rmed by mo-lecular assays. Further studies that investigate the pathogenic and zoonotic role of A. platys should be conducted.