Molecular diagnosis of Hepatozoon canis in symptomatic dogs in the city of Goiania, Goiás, Brazil / Diagnóstico molecular de Hepatozoon canis em cães sintomáticos do município de Goiânia, Goiás, Brasil

More than 300 species have been described in the genus Hepatozoon, occurring in different vertebrates. Among these, only Hepatozoon canis and Hepatozoon americanum are seen in dogs. Different methods may be used for laboratory diagnosis. The most common of these is direct parasitological examination of parasite stages in blood smears. The aim of this investigation was to conduct a phylogenetic study on Hepatozoon isolates from symptomatic dogs in the city of Goiânia, Goiás, Brazil. Blood samples were obtained from 40 symptomatic dogs that had been referred to the Veterinary Hospital of the Federal University of Goiás. Among these, only two samples were positive for Hepatozoon spp. using the direct parasitological method. These samples were then subjected to a DNA extraction process and amplification of a fragment of the 18S rRNA by means of PCR. Subsequently, the PCR products from each sample were purified and sequenced. The sequences obtained were then analyzed using the BLASTn algorithm, which identified both sequences of this study as Hepatozoon canis. By applying the Mega4 software, it was confirmed that these isolates of H. canis from dogs in Goiânia are similar to other reference isolates of the same species from other regions of Brazil and worldwide.(AU)

São descritas mais de 300 espécies do gênero Hepatozoon que acometem diferentes vertebrados. Entre estas, apenas Hepatozoon canis e Hepatozoon americanum são descritas em cães. Diferentes métodos podem ser utilizados para o diagnóstico laboratorial. O mais empregado é o exame parasitológico direto do parasito em esfregaços sanguíneos. O objetivo deste trabalho foi realizar um estudo filogenético em Hepatozoon isolados de cães sintomáticos de Goiânia, Goiás. As amostras de sangue foram obtidas de 40 cães sintomáticos encaminhados ao Hospital Veterinário da Universidade Federal de Goiás. Entre essas, duas únicas amostras foram positivas para Hepatozoon spp. pelo método parasitológico direto. Estas amostras foram, então, submetidas ao processo de extração de DNA e de amplificação de um fragmento de 18S rRNA por PCR. Ambas as amostras foram positivas na PCR. Posteriormente, os produtos de PCR de cada amostra foram purificados e sequenciados. As sequências obtidas foram analisadas pelo algoritmo BLASTn, sendo identificadas como Hepatozoon canis. Por meio do software Mega4 foi confirmado que estes isolados de H. canis de cães de Goiânia são semelhantes a outros isolados de referência da mesma espécie de outras regiões do Brasil e do mundo.(AU)

Molecular characterization of Hepatozoon sp. from Brazilian dogs and its phylogenetic relationship with other Hepatozoon spp.

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 600bp 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.