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.

Diagnosis of Hepatozoon spp. in Amblyomma ovale and its experimental transmission in domestic dogs in Brazil.

Transmission of Hepatozoon spp. to dogs was investigated using four species of ixodid ticks: Rhipicephalus sanguineus, Amblyomma aureolatum, Amblyomma ovale and Amblyomma cajennense. We collected completely or partially engorged adult ticks of these species from dogs that were naturally infested and positive for Hepatozoon spp. We selected some of these ixodids and inoculated them orally in four negative dogs. The other ticks were dissected and examined for oocysts. Of all dogs inoculated orally with R. sanguineus, A. aureolatum, A. cajennense and A. ovale, only the animal that received the macerate of A. ovale was positive; evidence (gametocytes in peripheral blood) of infection was found 63 days after inoculation. Among all dissected ticks, we found only two oocysts; these were similar to those of Hepatozoon canis, and both were recovered from a single A. ovale specimen. We inoculated sporozoites recovered from the oocysts intraperitoneally into a Hepatozoon spp. negative dog, and circulating gametocytes were detected 84 days later. Our study demonstrated that A. ovale can be a vector of Hepatozoon spp. in Brazil.

Incidence and intensity of Babesia spp. sporokinetes in engorged Boophilus microplus from a dairy herd in Venezuela.

The objective of this study was to determine the incidence and intensity of Babesia spp. sporokinetes in hemolymph of engorged females (EF) Boophilus microplus monthly detached from two herds of a local dairy breed of cattle named Carora breed. The experimental herds were located in 2 ranches, 45 km apart, having different management of tick control (TC). Proper methods of TC were commonly used at ranch "A," whereas ineffective methods were used at ranch "B," Babesia spp. sporokinetes were diagnosed in EF B. microplus by microscopic exam of tiny smears of hemolymph stained with Giemsa. A total of 537 smears were checked, and 108 were positive to Babesia spp. (20.1%). The incidence of Babesia spp. was higher (p < 0.01) in ranch "B," 22.3%, compared with the value detected in ranch "A," 6.5%. Incidence of Babesia spp. was greater in the dry season (Dec-Apr) compared with the values detected during the rainy season (May-Nov). The intensity of Babesia spp. was 3.43 at ranch "B" (p < 0.01), and 0.32 at ranch "A" during the rainy season. The values detected for intensity kept no correlation with those two seasons, although it is known that the incidence and seroprevalence of bovine babesiosis are greater during the dry season. The importance of these parameters in the epizootiology of bovine babesiosis in Venezuela is discussed.

Perinatal infection with Trypanosoma vivax in a calf in Venezuela.

A case of perinatal infection by Trypanosoma vivax is reported in a 5-hr-old Carora local dairy breed calf. The levels of parasitemia were followed during 15 mo in the infected animal. Similarly, its immune response was monitored during 4 mo by the indirect fluorescent assay. Trypanosoma vivax was observed in Giemsa-stained blood films collected during the first 4 mo of life from the infected calf. Positivity was detected on days 0, 35, 80, and 125 after first sampling. Antibodies against T. vivax were detected each time the indirect fluorescent assay was performed, showing low titers (1:80-1:160). The importance of a congenital route of transmission for this Trypanosoma species in cattle herds, on a continent where Glossina spp. flies are absent, is discussed.