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Molecular Evidence of Infections with Babesia gibsoni Parasites in Japan and Evaluation of the Diagnostic Potential of a Loop-Mediated Isothermal Amplification Method

Department of Veterinary Parasitology,¹ Department of Small Animal Medicine,² Veterinary Teaching Hospital, School of Veterinary MedicineAnimal Sciences, Kitasato University, Towada, Aomori 034-8628,³ Kamome Veterinary Clinic, Hashikami, Sannohe, Aomori 039-1208,⁴ Yanbaru Animal Clinic, Nago, Okinawa 905-0019,⁵ National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan⁶

Received 2 September 2003/ Returned for modification 3 November 2003/ Accepted 6 February 2004

Detection and analysis of Babesia gibsoni infection were performed with whole-blood samples collected between July 2002 and July 2003 from 945 and 137 dogs from the Aomori and Okinawa Prefectures of Japan, respectively, by PCR and loop-mediated isothermal amplification (LAMP). On the basis of the criterion for positivity by PCR, 3.9% (37 of 945) and 10.9% (15 of 137) of the dogs had B. gibsoni DNA. All 37 positive animals from Aomori Prefecture were male Tosa dogs (Japanese mastiff). The 15 dogs from Okinawa Prefecture with positive PCR assay results were of various breeds, ages, and sexes. The 18S ribosomal DNA (18S rDNA) sequences from all samples showed 100% homology to each other and to published B. gibsoni sequences. The limits of detection of B. gibsoni parasitemia by the PCR and LAMP methods with an 18S rDNA-based primer set were 0.0005% each. A comparison of the PCR and LAMP methods with microscopic examination for the detection of B. gibsoni infections in blood samples from 945 field dogs in Aomori Prefecture and 137 field dogs in Okinawa Prefecture showed that 37 and 15 dogs, respectively, were positive by the PCR and LAMP methods and that 16 and 12 dogs, respectively, were positive by light microscopic examination. All samples found to be positive by microscopic examination were also positive by the PCR and LAMP methods. The results of the PCR and LAMP methods agreed for samples with positive results by either method. Moreover, nonspecific reactions were not observed by the LAMP method. These results suggest that the LAMP method provides a useful tool for the detection of B. gibsoni infections in dogs.

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