Diaphanocephalus galeatus (Nematoda: Diaphanocephalidae), a poorly known parasite of lizards: redescription, first genetic characterization and a revision of its congeners from Brazil.

Diaphanocephalus galeatus collected from the small intestine of the lizard Dracaena paraguayensis in the Pantanal wetlands, State of Mato Grosso do Sul, Brazil, is redescribed. Genetic characterization and observations using scanning electron microscopy (SEM) were performed for the first time. The vouchers of D. galeatus and the type specimens of its congeners, deposited in the Coleção Helmintológica do Instituto Oswaldo Cruz (CHIOC), were consulted. Light and SEM observations revealed several undescribed features of D. galeatus, i.e. structure of the cephalic end and of the buccal capsule, position and morphology of deirids, presence of phasmids in females and presence of unpaired papilla on the membranous projection that covers the genital cone in males. After observation of the specimens deposited in the helminthological collection, D. jacuruxi is considered a synonym of D. galeatus, and D. diesingi, despite its incomplete description, is tentatively retained as valid due to the poor condition of the type material. The results also indicated low host specificity of D. galeatus, contradicting previous assertions. Genetic comparisons using patristic distances and phylogenetic trees generated from sequences of the 28S rRNA nuclear gene indicated that D. galeatus is closer to the taxa within Ancylostomatoidea and Strongyloidea than any lineage of Metastrongyloidea or Trichostrongyloidea. However, most of the nodal supports were low. Based on the genetic and morphological characterization, the validity of D. galeatus was confirmed. These data may serve for further comparative approaches for different populations of the parasite, from different hosts in different geographical areas, mitigating taxonomic confusions.

A new species of Raphidascaris (Nematoda: Raphidascarididae) infecting the fish Gymnogeophagus balzanii (Cichlidae) from the Pantanal wetlands, Brazil and a taxonomic update of the subgenera of Raphidascaris based on molecular phylogeny and morphology.

Raphidascaris (Sprentascaris) andersoni n. sp. (Nematoda: Raphidascarididae) collected in the intestine of the humphead cichlid Gymnogeophagus balzanii (Perugia) from the Pantanal wetlands, State of Mato Grosso do Sul (Brazil) is described and genetically characterized. The new species differs from its congeners mainly by having a conspicuous papilla-like formation slightly anterior to the cloacal aperture. Furthermore, males of R. (S.) lanfrediae and R. (S.) mahnerti have caudal alae, and R. (S.) hypostomi and R. (S.) pimelodi lack lateral alae, whereas in the new species caudal alae are absent and lateral alae present. The remaining congeners, namely, R. (S.) marano and R. (S.) saltaensis differ from Raphidascaris (Sprentascaris) andersoni n. sp. mainly because males have three pairs of postcloacal papillae (vs five pairs). In the phylogenetic reconstructions, using three nuclear genetic markers (18S, ITS1-5.8S-ITS2 and 28S rDNA) and one mitochondrial (cox1 mtDNA), the new species was separated from other representatives of Raphidascarididae, and the absence of monophyly in Hysterothylacium and Raphidascaroides was confirmed. Moreover, the subgenera Sprentascaris and Ichthyascaris appeared to be monophyletic. Therefore, even though Raphidascaris (Raphidascaris) was apparently not monophyletic, the subgenera of Raphidascaris should be re-erected as valid genera. The updated diagnoses of Ichthyascaris, Raphidascaris and Sprentascaris are given. The present study represents the first parasitological survey in G. balzanii.

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.