Identification of Genotypes of Giardia intestinalis Isolates from Dogs in Japan by Direct Sequencing of the PCR Amplified Glutamate Dehydrogenase Gene.

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Abstract
Giardia has been detected in domestic dogs in Japan, but the genotype of isolates has remained unclear because identification has relied on conventional microscopy. Here we tried to identify the genotypes of four isolates from dogs in Japan by direct sequencing of the PCR amplified Giardia glutamate dehydrogenase (GDH) gene. The primer pair GDHF3 and GDHB5, targeting the GDH gene, was designed to prime a region of the GDH gene sequence conserved in the strains found to have the dog-specific genotype. The specific PCR product (approximately 220 bp), amplified with this primer pair, was only observed when Giardia DNA was used as the template. The sequences of the diagnostic fragments were identical among the isolates from dogs, and were differed by 15 bp or 1 bp from the strains, which were found to be the dog-specific genotypes, Assemblage C or D respectively. To verify the identity of the amplified DNA, a phylogenetic analysis was performed. Consequently, the sequence of the isolates from dogs clearly clustered with the strain found to be Assemblage D with neighbor-joining analyses. Therefore, all the isolates from dogs examined were identified as the dog-specific genotype, Assemblage D. In the present study, we revealed the genotype of Giardia isolates in Japan, and showed that direct sequencing of the PCR product amplified with the primer pair GDHF3 and GDHB5 was a useful tool for distinguishing between the zoonotic and dog-specific genotypes.