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A two-step PCR method for diagnosis of canine infectious cyclic thrombocytopenia was established. Three primers derived from the 16S rRNA gene sequence were used to amplify genomic DNA specifically from Ehrlichia platys. Two-round amplification with DNA templates prepared from E. platys-infected blood specimens produced 742-and 385-bp fragments, but these products were not found when an Ehrlichia canis-infected blood sample and Escherichia coli were used. This method, for which the minimum detectable copy number in the blood specimen was estimated to be five ehrlichial inclusions within platelets, is more sensitive than single PCR amplification. These results demonstrate that this two-step PCR is highly sensitive and efficient for detecting the etiologic agent of canine infectious cyclic thrombocytopenia in blood. The same technique was applied to blood specimens collected from a dog inoculated with E. platys. Amplification of the target DNA fragments was observed with blood collected on the fifth day after inoculation, which indicates that this method is also feasible for early diagnosis of E. platys infection.

ABSTRACT. We evaluated the feasibility of using the two-step polymerase chain reaction (PCR) in determining the withdrawal time of antibiotic treatment for Ehrlichia platys infection. We also present experimental evidence of a dog remaining a carrier after treatment with tetracycline. Canine infectious cyclic thrombocytopenia (CICT) was induced in 3 dogs by intravenous inoculation of blood infected with E. platys. Tetracycline was administered to one of the dogs for 2 weeks when parasitemia appeared. Although the hematologic abnormality of cyclic thrombocytopenia soon disappeared, a few parasitized platelets reappeared after the withdrawal of treatment, and the dog thus remained as a carrier. The other dogs were treated with doxycycline when parasitemic episodes first developed. The durations of antibiotic regimens were determined by the results of two-step PCR in which the 16S rDNA of E. platys was amplified from blood samples. Doxycycline was withdrawn after 8 days of treatment, and the follow-up monitoring continued for 3 weeks. The platelet counts of the 2 dogs remained within the normal range, and the etiologic agent of CICT was not found either by Giemsa staining or by the twostep PCR, indicating complete elimination of the agent. -KEY WORDS: canine infectious cyclic thrombocytopenia, Ehrlichia platys, polymerase chain reaction.J. Vet. Med. Sci. 59(9): 849-851, 1997 examination.The efficacies of tetracycline for treatment of CICT have not been evaluated to date. In this communication, we demonstrate the ineffectiveness of tetracycline in completely eliminating E. platys infection in a dog. A healthy female crossbreed dog 6 months of age (dog A) was inoculated with E. platys infected blood [1]. Venous blood samples were collected on alternate days with ethylene diaminetetraacetic acid as an anticoagulant. Automated platelet counts (Sysmex K-1000; Toa Medical Electronics Co., Ltd., Japan) and Giemsa staining were carried out, and the infected platelet ratio was estimated by observing 200 platelets in one blood smear. Tetracycline was then administered orally at 22 mg/kg of body weight 3 times a day for 2 weeks after the day of the parasitemic episode. On the sixth day of treatment, the platelet count rose and remained within the normal range (Fig. 1). After completion of the therapeutic regimen, the platelet count and blood smear staining were continued for 2 weeks. Although thrombocytopenia was no longer observed, a few E. platys soon reappeared sporadically in the platelets after the termination of treatment (Fig. 1).Diagnosis of CICT could be performed either by demonstration of basophilic inclusions within the platelets in blood smears [5], by IFA [4], or by PCR [3]. Considering the fact that the parasitized platelets are cyclic in appearance and might be present only intermittently in the chronic stage, the determination of drug withdrawal by blood smear examination is not effective or reliable. Currently, the cultivation of E. platys has not been achieved, so the reisolation of etiologic agent as an...