Molecular evidence of infections with Babesia gibsoni parasites in Japan and evaluation of the diagnostic potential of a loop-mediated isothermal amplification method

Abstract

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.

FIG. 1.

Sensitivities of the PCR methods determined by agarose gel electrophoresis of the PCR products from samples serially diluted 10-fold and ethidium bromide staining. Primers Bg.Pd3 and Bg.Pd4 (A) and primers B18S-F and B18S-R (B) were used in PCRs to detect B. gibsoni. Lanes 1, dilution of 10⁻¹ with 0.5% parasitemia; lanes 2, dilution of 10⁻² with 0.05% parasitemia; lanes 3, dilution of 10⁻³ with 0.005% parasitemia; lanes 4, dilution of 10⁻⁴ with 0.0005% parasitemia; lanes 5, dilution of 10⁻⁵ with 0.00005% parasitemia; lanes 6, dilution of 10⁻⁶ with 0.000005% parasitemia; lane M (A), 100-bp DNA ladder marker; lane M (B), HindIII-digested bacteriophage lambda marker.

FIG. 2.

(A) Agarose gel electrophoresis of LAMP products from B. gibsoni-infected dog blood (lane 1) and leukocytes from healthy dogs (lane 2) stained with ethidium bromide. (B) Sensitivity of the LAMP method determined by agarose gel electrophoresis of LAMP products from 10-fold serial diluted samples and ethidium bromide staining. Target B. gibsoni DNA was detection by the LAMP reaction. Lane 1, dilution of 10⁻¹ with 0.5% parasitemia; lane 2, dilution of 10⁻² with 0.05% parasitemia; lane 3, dilution of 10⁻³ with 0.005% parasitemia; lane 4, dilution of 10⁻⁴ with 0.0005% parasitemia; lane 5, dilution of 10⁻⁵ with 0.00005% parasitemia; lane 6, dilution of 10⁻⁶ with 0.000005% parasitemia; lanes M, 100-bp DNA ladder marker.