Diversity of haemogregarine parasites infecting Brazilian snakes from the Midwest and Southeast regions with a description of two new species of Hepatozoon (Apicomplexa: Adeleorina: Hepatozoidae)

Although Brazil is a hotspot for snake species, there is a lack of information on the biodiversity of haemoparasites infecting these hosts. Thus, the present study aimed to bring new insights on the diversity of species of Hepatozoon (Apicomplexa: Adeleorina: Hepatozoidae) infecting Brazilian snakes from the Midwest and Southeast regions. The snakes were captured from 2018 to 2020 from the states of Mato Grosso, Mato Grosso do Sul, Goiás, and São Paulo. Three to five blood smears were made and the remaining blood sample was stored for further molecular analysis. Moreover, histopathological slides of the organs were stained with haematoxylin-eosin. Regarding molecular diagnosis, PCR was performed targeting different regions of the 18S rRNA gene of apicomplexan parasites. From the 13 free-living snakes screened, ten (76.92%) were found infected with Hepatozoon spp. Based on morphological and morphometric tools, five different morphotypes of species of Hepatozoon gamonts were detected. Molecular data and phylogenetic analysis support the morphological data, identifying five species of Hepatozoon from snakes, of which three species belong to previously described species, Hepatozoon cevapii, Hepatozoon cuestensis, and Hepatozoon quagliattus, with a genetic similarity of 100% (based on the 18S rRNA genetic marker). The present study identifies and describes two new species of Hepatozoon, Hepatozoon annulatum sp. nov. infecting the snake Leptodeira annulata and Hepatozoon trigeminum sp. nov. infecting the snake Oxyrhopus trigeminus. Thus, based on morphological and molecular data the present study provides new insights on haemogregarine diversity infecting Brazilian snakes from the Midwest and Southeast regions.     

Molecular detection and characterization of Hepatozoon canis in stray dogs from Cuba

Canine hepatozoonosis caused by Hepatozoon canis is a worldwide distributed tick-borne disease of domestic and wild canids that is transmitted by ingestion of Rhipicephalus sanguineus sensu lato (s.l.) ticks. The present study was aimed to determine the prevalence of Hepatozoon infections in 80 stray dogs from Havana Province in Cuba, and to confirm the species identity and phylogenetic relationships of the causative agent. Samples were screened by microscopical examination of thin blood smears for the presence of Hepatozoon spp. gamonts and by genus-specific SYBR green-based real-time PCR assay targeting the 18S rRNA gene. Direct microscopy examination revealed Hepatozoon gamonts in the peripheral blood of 8 dogs (10.0%; 95% CI: 4.80–18.0%), while 38 animals (47.5%; 95% CI: 36.8–58.4%) were PCR-positive, including all microscopically positive dogs. Hence, the agreement between the two detection methods was ‘poor’ (κ = 0.20). Hematological parameters did not differ significantly between PCR-positive and PCR-negative dogs (p > 0.05). The DNA sequences of the 18S rRNA gene of the Hepatozoon spp. from Cuban dogs showed a nucleotide identity >99% with those of 18S rRNA sequences of Hepatozoon canis isolates from Czech Republic, Brazil and Spain. Phylogenetic analysis revealed that obtained sequences clustered within the Hepatozoon canis clade, different from the Hepatozoon felis or Hepatozoon americanum clades. The present study represents the first molecular characterization of Hepatozoon canis in stray dogs within Cuba.     

Morphological and molecular characteristics of a species of Hepatozoon Miller, 1908 (Apicomplexa: Adeleorina) from the blood of Isoodon obesulus (Marsupialia: Peramelidae) in Western Australia

Examination of blood films as part of a study to assess the health status of the southern brown bandicoot Isoodon obesulus (Shaw) in Western Australia revealed the gamonts of a haemogregarine parasite in some samples, the first to be recognised in a bandicoot in this state. Light microscope morphological characteristics and partial sequence of the 18S rRNA gene were used to describe these organisms. Morphological characters did not differentiate the organism in the current study from previously reported Hepatozoon peramelis (Welsh & Dalyell, 1909). Phylogenetic analysis has not previously been reported for any species of Hepatozoon from Australian marsupials and consequently could not be used to confirm the identity of the organism in the current study as that described in the 1900s. If this organism is H. peramelis, then it has a wide distribution, being found in three species of bandicoot, in western and eastern Australia and the in island state of Tasmania.     

A new species of <Emphasis Type="Italic">Hepatozoon</Emphasis> Miller, 1908 (Apicomplexa: Adelerina) from the snake <Emphasis Type="Italic">Philodryas nattereri</Emphasis> Steindachner (Squamata: Dipsadidae) in northeastern Brazil

Based on both unique morphological characteristics of the gamont, distinct changes caused to the host erythrocyte and analysis of partial 18S rRNA gene sequences, a new parasite of the genus Hepatozoon Miller, 1908 is described from the snake Philodryas nattereri Steindachner (Squamata: Dipsadidae) in northeastern Brazil. The new species, Hepatozoon musa n. sp., is characterized by large and curved mature gamonts (18.9 ± 0.9 μm in length and 3.8 ± 0.3 μm in width) that considerably engorge infected host erythrocytes and displace the nucleus laterally, which become longer and thinner. Phylogenetic estimates indicate the new species is more closely related to the recently described Hepatozoon cuestensis O’Dwyer, Moço, Paduan, Spenassatto, Silva & Ribolla, 2013, from Brazilian rattlesnakes. These recent findings highlight the need for further studies of Hepatozoon to better determine the biodiversity of this common but poorly-studied parasite group.     

Sexual Stages in the Life History of the Hemogregarine Hepatozoon rarefaciens (Sambon and Seligmann, 1907)*

SYNOPSIS. Sexual stages of a reptilian hemogregarine Hepatozoon rarefaciens are described from the hemocoele of a mosquito vector Culex tarsalis. The gametocytes associate within 0.5 hr after biting. Most of the gametocytes then pair off and begin to differentiate. After about 26 hr at 22–23 C, the macrogametocyte usually becomes crescent-shaped with the microgametocyte lying in the concavity. The nucleus of the macrogametocyte enlarges considerably, while the microgametocyte divides into 2 and eventually into 4 spindle-shaped microgametes which are biflagellated. The macrogametocyte increases in size and rounds up to form a macrogamete. Gamete formation is usually completed by 60 hr and fertilization begins soon thereafter. The zygotes develop into the uninucleate oocysts described previously. In some other species of Hepatozoon, gametogenesis has been described as taking place in the stomach of the arthropod vector, with the microgametocytes transforming without cytoplasmic division into non-flagellated microgametes. The occurrence of 2 distinct types of gametogenesis may possibly be a basis for separating Hepatozoon into 2 genera.     

Morphologic and morphometric analysis of Hepatozoon spp. (Apicomplexa,Hepatozoidae) of snakes

Hepatozoon species are the most abundant hemoparasites of snakes. Its identification has been based mainly on the morphologic characterization of the gamonts in the peripheral blood of the vertebrate host and also of the cysts found in the internal organs of the vertebrate and invertebrate hosts. Using a computerized image analysis system, we studied five species of Hepatozoon from recently captured snakes in Botucatu, State of S o Paulo, Brazil, to evaluate the importance of the morphology and morphometry of the gamonts for the characterization of Hepatozoon species and to analyze the morphologic changes induced in the erythrocytes by the parasite. The studied species were H. terzii of Boa constrictor amarali, Hepatozoon sp. of Crotalus durissusterrificus, H. philodryasi of Philodryas patagoniensis, and H. migonei and H. cyclagrasi of Hydrodynastes gigas. We observed three different groups, one of them including the species H. terzii, H. philodryasi and Hepatozoon sp. of C. durissus terrificus; and the other two consisting of H. migonei and H. cyclagrasi. Degree of alterations in the erythrocytes was variable and it may be useful for characterization of Hepatozoon species.     

An annotated checklist of the valid avian species of Haemoproteus,Leucocytozoon (Apicomplexa: Haemosporida) and Hepatozoon (Apicomplexa: Haemogregarinidae)

The valid avian species of the apicomplexan blood parasite genera Haemoproteus, Hepatozoon and Leucocytozoon are arranged according to host family, assuming host familial specificity; salient points of the parasite morphology are recorded where appropriate.     

Larval feeding performance of two Neotropical Ornithodoros ticks (Acari: Argasidae) on reptiles

This paper determines the feeding performance of the larvae of two Neotropical soft tick species namely Ornithodoros rostratus Aragão, 1911 and O. puertoricensis Fox, 1947 on reptiles (Gekkonidae) using rabbits, mice and guinea pigs to provide comparisons with feeding features on mammals. O. puertoricensis produced a larval feeding rate of 63% on reptiles, while that of O. rostratus was only 20%. But the final success (attaching + feeding) was similar, 12.4% for O. puertoricensis and 10.4% for O. rostratus. The feeding time was also very different for both species. In O. puertoricensis, detachment begins at 16th day and lasts until day 27. In O. rostratus detachment begins at 1.5 h and lasts until day 10. These values of feeding on reptiles are different from those obtained on mammals (average 5.6 days for O. puertoricensis and 2.9 for O. rostratus).     

Phylogenetic relationships of Hepatozoon (Apicomplexa: Adeleorina) based on molecular, morphologic, and life-cycle characters

To evaluate higher-level affinities of Hepatozoon species within Apicomplexa, we sequenced the 18S rRNA gene from 2 parasites (Hepatozoon americanum and Hepatozoon canis) of dogs and 1 (Hepatozoon catesbianae) of bullfrogs. Sequences from other apicomplexans among the Sarcocystiidae, Eimeriidae, Theileriidae, Plasmodiidae, Cryptosporiidae, and Babesiidae, a Perkinsus species and 2 dinoflagellates were obtained from GenBank. Phylogenetic analysis indicated that Plasmodium, Cryptosporidium, and Hepatozoon form a monophyletic group distinct from representatives of other apicomplexan families. Although equivocal, our analysis indicated that Plasmodium and Cryptosporidium are sister taxa and that Hepatozoon is basal to them. To evaluate phylogenetic affinities among H. americanum, H. canis, and other species of Hepatozoon, we examined 18 morphologic and life-cycle features of 13 species currently assigned to Hepatozoon. This analysis indicates paraphyly of Hepatozoon (as currently arranged) because Hepatozoon lygosomarum was found most closely related to Hemolivia mauritanicum. These results, combined with results of previous studies, support elevating Hepatozoon to familial level (Hepatozoidae) as originally suggested by Wenyon in 1926. Both DNA sequence data and morphologic and life-cycle characters support a sister-group relationship between H. americanum and H. canis.     

Diagnoses of and illustrated key to the species of <Emphasis Type="Italic">Ixodes</Emphasis> Latreille, 1795 (Acari: Ixodidae) from Brazil

The current Brazilian Ixodes fauna is composed of the following eight species: I. amarali Fonseca, 1935; I. aragaoi Fonseca, 1935; I. auritulus Neumann, 1904; I. fuscipes Koch, 1844; I. loricatus Neumann, 1899; I. luciae Sénevet, 1940; I. paranaensis Barros-Battesti, Arzua, Pichorim &; Keirans, 2003; and I. schulzei Aragão &; Fonseca, 1951. Further studies are needed to establish the taxonomic status of I. serrafreirei Amorim, Gazeta, Bossi &; Linhares, 2003, a recently proposed species based solely on the nymphal stage. We present an up-to-date key to adults of the currently valid Brazilian species of Ixodes based on scanning electron microscopy. The relationships between Brazilian and other Neotropical Ixodes are also discussed.     

Redescription of Brochopeltis mjoebergi Verhoeff, 1924 and description of a second Brochopeltis species from Australia (Diplopoda,Polydesmida, Paradoxosomatidae)

Brochopeltis mjoebergi Verhoeff, 1924 is redescribed from type and new material, a lectotype is designated and Brochopeltis mjoebergi queenslandica Verhoeff, 1924 is synonymised with Brochopeltis mjoebergi. Brochopeltis mediolocus sp. n. is the first native paradoxosomatid described from Australia’s Northern Territory.     

Molecular epizootiology of canine hepatozoonosis in Croatia

An epizootiological survey was conducted to investigate the prevalence of hepatozoonosis in a population of 924 apparently asymptomatic dogs from different regions of Croatia. DNA was isolated from canine blood and screening PCR on the 666 bp fragment of 18S rRNA revealed that 108 (11.8%) of dogs were infected. Positive samples were confirmed by partial sequencing of the 18S rRNA gene. The consensus sequences, derived from various sequence data sets, were compared with sequences of 18S ssrRNA of Hepatozoon spp. available in GenBank. The alignments revealed 106 Hepatozoon canis and two Hepatozoon sp. sequences. Among H. canis isolates, we found a certain amount of heterogeneity, while both Hepatozoonsp. isolates were identical to the Spanish isolate (Accession No. AY600625) from Clethrionomys glareolus. On the basis of eight commonly mutated nucleotide positions in the partial 18S rRNA gene sequence, we divided the H. canis isolates into five groups. The results obtained indicate a higher prevalence and significance of hepatozoonosis in Croatia than previously believed and demonstrate that the organisms belonging to H. canis that infect European dogs are genetically very heterogeneous.     

New molecular data on mammalian Hepatozoon species (Apicomplexa: Adeleorina) from Brazil and Spain

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.     

Some Corrections in Haemogregarine (Apicomplexa: Protozoa) Nomenclature1

The nomenclature of three genera in the family Haemogregarinidae (Haemogregarina, Karyolysus, and Hepatozoon) has been reviewed and the following new names are introduced to replace homonyms or for previously unnamed species: Haemogregarina carlosi n. nom., in the erythrocytes of the lizard Lacerta ocellata; Haemogregarina tincae n. nom., in the stomach and intestine of the tench Tinca tinca; Hepatozoon insectivorae n. sp., in the leucocytes of the shrews Sorex araneus and Crocidura leucodon; Hepatozoon krampitzi n. sp., in the leucocytes of the vole Microtus oeconomus; Hepatozoon peromysci n. sp., in the leucocytes of the deermice Peromyscus boylii and P. truei gilberti; and Hepatozoon pallida (Pessoa et al., 1971) n. comb., in the erythrocytes of the snake Thamnodynastes pallidus nattereri.     

Patterns of genetic diversity in Hepatozoon spp. infecting snakes from North Africa and the Mediterranean Basin

Species of Hepatozoon Miller, 1908 are blood parasites most commonly found in snakes but some have been described from all tetrapod groups and a wide variety of hematophagous invertebrates. Previous studies have suggested possible associations between Hepatozoon spp. found in predators and prey. Particularly, some saurophagous snakes from North Africa and the Mediterranean region have been found to be infected with Hepatozoon spp. similar to those of various sympatric lizard hosts. In this study, we have screened tissue samples of 111 North African and Mediterranean snakes, using specific primers for the 18S rRNA gene. In the phylogenetic analysis, the newly-generated Hepatozoon spp. sequences grouped separately into five main clusters. Three of these clusters were composed by Hepatozoon spp. also found in snakes and other reptiles from the Mediterranean Basin and North Africa. In the other two clusters, the new sequences were not closely related to geographically proximate known sequences. The phylogeny of Hepatozoon spp. inferred here was not associated with intermediate host taxonomy or geographical distribution. From the other factors that could explain these evolutionary patterns, the most likely seems series of intermediate hosts providing similar ribotypes of Hepatozoon and a high prevalence of host shifts for Hepatozoon spp. This is indicated by ribotypes of high similarity found in different reptile families, as well as by divergent ribotypes found in the same host species. This potentially low host specificity has profound implications for the systematics of Hepatozoon spp.     

A new species of Microlicia (Melastomataceae) from Brazil

A new species of the genusMicrolicia,M. flava, from the highland “campo rupestre” vegetation of Serra da Canastra National Park, São Roque de Minas, Minas Gerais, Brazil, is described and illustrated.     

Phylogenetic analysis of the genus Hepatozoon Miller, 1908 (Apicomplexa: Adeleorina)

A phylogenetic analysis of species of Hepatozoon Miller, 1908 was performed using 16 morphological, morphometric and developmental characters. An adeleorin parasite of gastropod molluscs, Klossia helicina Schneider, 1875 and four haemogregarines of other genera, Karyolysus lacertae (Danilewsky, 1886), Cyrilia lignieresi (Laveran, 1906), Desseria myoxocephali (Fantham, Porter & Richardson, 1942) and Haemogregarina balli Paterson & Desser, 1976 were used as the outgroup to 12 species of Hepatozoon. A single most parsimonious interpretation of the data was found, with the resulting cladogram having 28 transformations and a consistency index of 0.75. The proposed phylogeny revealed Hepatozoon as a paraphyletic group. One monophyletic lineage contained 10 of the 12 species of Hepatozoon, including all of those species that undergo gametogenesis and sporogony in the haemocoel of arthropods. Within this lineage, gamonts of those species found in vertebrate leucocytes instead of erythrocytes formed a clade basal to the remainder of the species. Species of Hepatozoon which undergo gametogenesis and sporogonic development in the gut epithelium of acarines, and which produce aflagellate microgametes, as well as the four outgroup taxa of haemogregarines, formed a monophyletic group and were the sister group to the remainder of the species of Hepatozoon. The biological and morphological diversity of these parasites suggests that species of the genus Hepatozoon, which are members of the paraphyletic haemogregarine complex, could be partitioned into at least two genera of adeleorin parasites.