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To characterize phylogenetically the species which causes canine hepatozoonosis at two rural areas of Rio de Janeiro State, Brazil, we used universal or
Hepatozoon spp. primer sets for the 18S SSU rRNA coding region. DNA extracts were obtained from blood samples of thirteen dogs naturally infected, from four experimentally infected, and from five puppies infected by vertical transmission from a dam, that was experimentally infected. DNA of sporozoites of
Hepatozoon americanum was used as positive control. The amplification of DNA extracts from blood of dogs infected with sporozoites of
Hepatozoon spp. was observed in the presence of primers to 18S SSU rRNA gene of
Hepatozoon spp., whereas DNA of
H. americanum sporozoites was amplified in the presence of either universal or
Hepatozoon spp.-specific primer sets; the amplified products were approximately 600
bp in size. Cloned PCR products obtained from DNA extracts of blood from two dogs experimentally infected with
Hepatozoon sp. were sequenced. The consensus sequence, derived from six sequence data sets, were blasted against sequences of 18S SSU rRNA of
Hepatozoon spp. available at GenBank and aligned to homologous sequences to perform the phylogenetic analysis. This analysis clearly showed that our sequence clustered, independently of
H. americanum sequences, within a group comprising other
Hepatozoon canis sequences. Our results confirmed the hypothesis that the agent causing hepatozoonosis in the areas studied in Brazil is
H. canis, supporting previous reports that were based on morphological and morphometric analyses.