Morphological detection and molecular characterization of Hepatozoon spp. from venomous terrestrial snakes in Iran

We announce the release of an advanced version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis. In version 6.0, MEGA now enables the inference of timetrees, as it implements the RelTime method for estimating divergence times for all branching points in a phylogeny. A new Timetree Wizard in MEGA6 facilitates this timetree inference by providing a graphical user interface (GUI) to specify the phylogeny and calibration constraints step-by-step. This version also contains enhanced algorithms to search for the optimal trees under evolutionary criteria and implements a more advanced memory management that can double the size of sequence data sets to which MEGA can be applied. Both GUI and command-line versions of MEGA6 can be downloaded from www.megasoftware.net free of charge.

Hemogregarines of the genus Hepatozoon are intraerythrocytic apicomplexan parasites that have been described from all groups of tetrapod vertebrates. Gametogenesis, fertilization, and sporogonic development, which culminates in the formation of polysporocystic oocysts, occur in the gut or hemocoel of a hematophagous arthropod definitive host. Merogonic development occurs in the internal organs of vertebrate hosts after they ingest these infected arthropods. The presence of cystic stages, observed for many Hepatozoon species, increases life cycle complexity and exploits the feeding behavior of vertebrate hosts. The inconsistency of morphological characteristics of these parasites, especially those associated with gamont structure, coupled with low host specificity of the parasites for their invertebrate and vertebrate hosts, have rendered species differentiation difficult. A systematic review of the hemogregarine complex has resulted in the expansion of the genus Hepatozoon to include all members of the genus Haemogregarina that infect amphibians, snakes, lizards, crocodilians, birds, and mammals.

Molecular techniques were used to examine the phylogenetic relationships among Hepatozoon species isolated from 13 foxes and 15 opossums from Brazil, and from 15 dogs, 20 foxes, 45 rodents, and 330 domestic cats from Spain. Hemogregarine infection was confirmed by amplification of the 18S rRNA gene and later sequencing. No hemogregarine infections were found in opossums. The prevalence of Hepatozoon in canids ranged from 26.6% (symptomatic domestic dogs) to 90% (Spanish foxes). Four different H. canis genotypes were detected, as well as an H. americanum-related protozoan (97% identical to the USA strain). Two Spanish cats were parasitized by a Hepatozoon species (0.6% prevalence) that showed 96% sequence identity to H. canis. DNA amplification assays performed on Spanish rodents showed 2 bank voles (Clethrionomys glareolus) to be infected by a Hepatozoon species (4.44% prevalence) with 95% sequence identity to Hepatozoon sp. from cats. Phylogenetic analysis showed Hepatozoon to be a monophyletic genus, in which species from carnivorous mammals (Hepatozoon sp. from cats, H. americanum and H. canis) appear as a sister lineage of that of lower vertebrates and rodents. This association suggests that H. americanum evolved in ticks and carnivores (either canids, or felids, or both) rather than in other ectoparasites and other types of mammal.