Detection of Babesia Species from Infected Dog Blood by Polymerase Chain Reaction.

Abstract

ABSTRACT. Polymerase chain reaction (PCR) was first applied to diagnosis of canine babesiosis in Japan. Blood samples from 13 dogs suffering from canine babesiosis were used for examination of specificity and sensitivity of the PCR diagnosis. Of the 13 dogs, three were experimentally infected, and ten were naturally infected with Babesia species in west part of Japan. We designed a nested PCR to amplify the babesial small subunit ribosomal RNA gene and found that only the nested PCR produced a visual band, which were not apparent by the first-round PCR to the positive samples. Specificity of the nested PCR was confirmed by amplification after the secondround PCR. Sensitivity of the nested PCR was examined by diluting the blood samples from infected and uninfected dogs. The nested PCR was found to show positive results on the most diluted blood at 0.0001% parasitemia. These results indicate that the nested PCR is highly sensitive and useful for diagnosis of canine babesiosis. KEY WORDS: babesia, canine, PCR.J. Vet. Med. Sci. 63(1): 111-113, 2001 Among Babesia species, which infect wide variety of animals, Babesia canis (B. canis) and B. gibsoni have been known as causative agents of canine babesiosis [1,9,13,14]. Clinical findings of canine babesiosis are fever, anorexia, malaise, hemoglobinuria and hemolytic anemia [5,6,9,13], and B. canis develops more acute and rapid hemolysis than B. gibsoni [9]. The area of B. canis infection is reported in Southern Europe, North America, Africa and Asia, where B. gibsoni is found in Africa and Asia [6,9,13]. In Japan, B. gibsoni causes mainly canine babesiosis and B. canis was reported only in Okinawa [9]. Generally, Babesia species is identified by the demonstrating the organisms in blood smears under the light microscope or by serological examinations [5]. Although the detection of several Babesia species by polymerase chain reaction (PCR) and other DNAbased methods have been reported [2,4,[6][7][8][10][11][12], these approaches have never been applied on canine babesiosis in Japan. In the present study, we try to detect the small subunit ribosomal RNA (ss-rRNA) of Babesia species from canine blood by PCR. Sensitivity of this method was determined by using diluted blood of Babesia infection.Blood samples infected with Babesia species were collected from 10 naturally infected dogs in west part of Japan and 3 experimentally infected dogs. A blood sample from uninfected dog was used as a negative control. By microscopic examination of Giemsa-stained blood smears, these 10 dogs were considered infected with B. gibsoni because of their size and shape (Fig. 1). Ranges of their parasitemia were estimated from about 1.0% to 5.0%. DNA was isolated from 200 µl of whole blood. Briefly, blood was mixed with 500 µl of Tris-EDTA (TE) buffer (50 mM Tris, pH 8.0, 100 mM EDTA) containing 0.5% sodium dodecyl sulfate (SDS) and 25 µl of 10 mg/ml proteinase K (TaKaRa Shuzo Co., Ltd., Japan) and incubated at 42°C. After incubation at overnight, 500 µl of phenol-equilibrated with TE (10 mM T...

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