Investigations on the Host Specificity of Dihomoxenous Sarcosporidia in the Intermediate and Definitive Host

ABSTRACT. Cross-transmission experiments were performed in order to determine the host specificity in the intermediate and definitive hosts of the four described dihomoxenous Sarcocystis species, S. gallotiae, S. stehlinii, S. simonyi , and S. dugesii from lacertid lizards of the genera Gallotia and Podarcis from the Macaronesian Islands. Sarcocysts of either species from experimentally infected lizards were fed to a variety of laboratory-bred lizard species of the genera Gallotia, Lacerta , and Podarcis . These sarcocysts proved to be infectious to all examined animals, showing no definitive host specificity in the tested genera. Lizards of the genera Chalcides and Tarentola , however, were not susceptible definitive hosts for S. gallotiae . The inoculation of experimentally obtained sporocysts of each of the four Sarcocystis species to various lacertid lizard species revealed varying degrees of intermediate host specificity, generally demonstrating each native host to be the most susceptible.     

Molecular survey of Hepatozoon species in lizards from North Africa

The prevalence of Hepatozoon parasites in 460 lizards from North Africa was studied by amplification and sequencing of the 18S rRNA gene. The phylogenetic analysis of the 18S rRNA gene provides new insights into the phylogeny of these parasites with multiple genetically distinct lineages recovered. Parasite prevalence differed significantly between lacertid lizards and geckos. Our results show that there is limited host specificity and no clear relation to the geographical distribution of Hepatozoon parasites.     

Multiple origin of the dihomoxenous life cycle in sarcosporidia

Although their ssrRNA gene sequences are closely related, the lizard sarcosporidia (Apicomplexa, Sarcocystidae) Sarcocystis lacertae and Sarcocystis gallotiae posses heteroxenous and dihomoxenous life cycles, respectively. When aligned with available sarcosporidian ssrRNA genes, both species constitute a monophyletic clade that is only distantly related with sarcosporidia that have a viperid snake as their definitive host (Sarcocystis sp., Sarcocystis atheridis). To test the phyletic status of the dihomoxenous life style, Sarcocystis rodentifelis and Sarcocystis muris, two dihomoxenous parasites of mammals were included into this study. All studied species group together with former Frenkelia spp., Sarcocystis neurona and related marsupial and bird sarcosporidia in a monophyletic clade. However, the available dataset supports independent appearance of the dihomoxenous life cycle at least twice during the evolution of the Sarcocystidae.     

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.     

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.     

Molecular characterization of Hepatozoon species in reptiles from the Seychelles

Hepatozoon parasites were examined for the first time in reptiles from the Seychelles Islands. Although both prevalence and intensity were low, Hepatozoon species were detected in individuals from 2 endemic species, the lizard Mabuya wrightii and the snake Lycognathophis seychellensis. This was confirmed using visual identification and through sequencing part of the 18s rRNA gene. Phylogenetic analysis indicates that the Hepatozoon on the Seychelles form a monophyletic lineage, although more data are clearly needed to stabilize estimates of relationships based on this marker.     

Phylogeny of nuclear small subunit rRNA genes of hemogregarines amplified with specific primers

Hemogregarines, apicomplexan intracellular blood parasites, are cosmopolitan in distribution and infect a broad range of vertebrate and invertebrate hosts. Molecular phylogenetic studies have been hampered by lack of hemogregarine-specific polymerase chain reaction primers that would allow amplification of parasite, but not host, DNA. A novel method for separating parasite and host 18S rRNA genes has been developed, and new primers that are specific for hemogregarine rRNA genes have been designed. These primers were used to obtain sequences from 4 isolates of hemogregarines of lizards from California, the Caribbean island of Grenada, eastern Australia, and Israel. Combining these results with already published sequences, a preliminary phylogeny of hemogregarines and several other apicomplexan taxa has been created. The hemogregarines form a monophyletic group and appear to be more closely related to coccidia than to Plasmodium species. The difficulty of using 18S genes that have multiple copies in some apicomplexan parasites was explored for systematic studies.     

Characterization of Sarcocystis from four species of hawks from Georgia, USA

During 2001 to 2004, 4 species of hawks (Buteo and Accipiter spp.) from Georgia were surveyed for Sarcocystis spp. infections by examining intestinal sections. In total, 159 of 238 (66.8%) hawks examined were infected with Sarcocystis spp. Samples from 10 birds were characterized by sequence analysis of a portion of the 18S rRNA gene (783 base pairs). Only 3 of the 10 sequences from the hawks were identical; the remainder differed by at least 1 nucleotide. Phylogenetic analysis failed to resolve the position of the hawk Sarcocystis species, but they were closely related several Sarcocystis species from raptors, rodents, and Sarcocystis neurona. The high genetic diversity of Sarcocystis suggests that more than 1 species infects these 4 hawk species; however, additional molecular or experimental work will be required to determine the speciation and diversity of parasites infecting these avian hosts. In addition to assisting with determining species richness of Sarcocystis in raptors, molecular analysis should be useful in the identification of potential intermediate hosts.     

Protozoan parasites of British small rodents

Thirty-five species of protozoan parasites belonging to thirteen genera have now been recorded for British small rodents. These include species of Entamoeba, Giardia, Spironucleus, Trichomonas, Chilomastix, Eimeria and Cryptosporidium in the gut; Trypanosoma, Hepatozoon and Babesia in the blood; and Toxoplasma, Frenkelia and Sarcocystis in the tissues. Recent advances have progressed along two lines, the elucidation of the life-cycles of the species of Frenkelia and Sarcocystis , which are now known to involve a carnivore as the final host, and laboratory studies on those parasites that can be maintained in laboratory animals. It is now possible to draw up a definitive list of hosts and parasites and this should serve as a basis for studies on the epidemiology of these parasites and their possible effects on their hosts.     

Molecular and morphological description of a Hepatozoon species in reptiles and their ticks in the Northern Territory, Australia

Ticks, representing 3 species of Amblyomma, were collected from the water python (Liasis fuscus) and 3 additional reptile species in the Northern Territory, Australia, and tested for the presence of Hepatozoon sp., the most common blood parasites of snakes. In addition, blood smears were collected from 5 reptiles, including the water python, and examined for the presence of the parasite. Hepatozoon sp. DNA was detected in all tick and reptile species, with 57.7% of tick samples (n = 187) and 35.6% of blood smears (n=35) showing evidence of infection. Phylogenetic analysis of the 18S rRNA gene demonstrated that half of the sequences obtained from positive tick samples matched closest with a Hepatozoon species previously identified in the water python population. The remaining sequences were found to be more closely related to mammalian and amphibian Hepatozoon species. This study confirms that species of Amblyomma harbor DNA of the same Hepatozoon species detected in the water pythons. The detection of an additional genotype suggests the ticks may be exposed to 2 Hepatozoon species, providing further opportunity to study multiple host-vector-parasite relationships.     

Phylogenetic position of the adeleorinid coccidia (Myzozoa, Apicomplexa, Coccidia, Eucoccidiorida, Adeleorina) inferred using 18S rDNA sequences

Investigating the evolutionary relationships of the major groups of Apicomplexa remains an important area of study. Morphological features and host-parasite relationships continue to be important in the systematics of the adeleorinid coccidia (suborder Adeleorina), but the systematics of these parasites have not been well-supported or have been constrained by data that were lacking or difficult to interpret. Previous phylogenetic studies of the Adeleorina have been based on morphological and developmental characters of several well-described species or based on nuclear 18S ribosomal DNA (rDNA) sequences from taxa of limited taxonomic diversity. Twelve new 18S rDNA sequences from adeleorinid coccidia were combined with published sequences to study the molecular phylogeny of taxa within the Adeleorina and to investigate the evolutionary relationships of adeleorinid parasites within the Apicomplexa. Three phylogenetic methods supported strongly that the suborder Adeleorina formed a monophyletic clade within the Apicomplexa. Most widely recognized families within the Adeleorina were hypothesized to be monophyletic in all analyses, although the single Hemolivia species included in the analyses was the sister taxon to a Hepatozoon sp. within a larger clade that contained all other Hepatozoon spp. making the family Hepatozoidae paraphyletic. There was an apparent relationship between the various clades generated by the analyses and the definitive (invertebrate) host parasitized and, to lesser extent, the type of intermediate (vertebrate) host exploited by the adeleorinid parasites. We conclude that additional taxon sampling and use of other genetic markers apart from 18S rDNA will be required to better resolve relationships among these parasites.     

Recognition of cyclic transmission of Sarcocystis stehlinii n. sp. in the Gran Canarian giant lizard

The transmission of Sarcocystis species via cannibalism seems to be common among lizards of the genus Gallotia that are endemic to the Canary Islands. Gran Canarian giant lizards, Gallotia stehlini, were screened for the presence of sarcosporidian parasites. Sarcocysts, measuring 90-400 micrometers in length and 60-160 micrometers in width, were found in the musculature of the lizards' tails. In their feces the lizards passed sporocysts of 8.5(8.2-9.4) x 6.5(5.9-7.0) micrometers. A series of laboratory infections was carried out to shed light on the life cycle of Sarocystis stehlinii n.sp., proving it to be another dihomoxenous Sarcocystis species.     

Phylogenetic analysis of Sarcocystis spp. of mammals and reptiles supports the coevolution of Sarcocystis spp. with their final hosts.

Sequences of the small subunit rRNA genes were obtained for two coccidians, Sarcocystis dispersa and an unnamed Sarcocystis sp. which parasitise the European barn owl and an African viperid snake as their final host, respectively, and share mouse as their intermediate host. Phylogenetic analysis of the sequence data showed that Sarcocystis sp. from the viperid snake is most closely related to another Sarcocystis sp. isolated from an American crotalid snake, while S. dispersa grouped with other bird-transmitted species. The available dataset failed to resolve the evolutionary relationships among four major branches into which all Sarcocystidae and Isospora spp. were split. However, within these branches, the phylogenetic relationships of the majority of analysed members of the genus Sarcocystis reflected coevolution with their final, rather than intermediate hosts.     

The first detection of Babesia species DNA from Japanese black bears (Ursus thibetanus japonicus) in Japan

In this study, we tried to detect protozoan blood parasites from the liver or blood of 156 Japanese black bears (Ursus thibetanus japonicus) in Iwate Prefecture of Japan by polymerase chain reaction. Two amplicons (approximately 540 bp and 480 bp) were detected by amplification for V4 hyper-variable regions of the 18S rRNA gene. Approximately 540-bp products were obtained in 119 samples (76.3%) and were considered to be DNA of Hepatozoon ursi. Approximately 480-bp products were obtained in 22 samples (14.1%) and were considered to be DNA of Babesia species. The nucleotide sequences (1635 bp) of the 18S rRNA gene of Babesia sp. were very similar (99.3%) to those (AY190123, AY190124) of Babesia sp. detected previously from Ixodes ovatus. Phylogenetic analysis showed that Babesia sp. detected in this study closely related to Babesia sp. derived from raccoons in Japan and the U.S.A. This is the first report of Babesia species detected from Japanese black bears.     

The toxoplasma micronemal protein MIC4 is an adhesin composed of six conserved apple domains

The initial stage of invasion by apicomplexan parasites involves the exocytosis of the micronemes-containing molecules that contribute to host cell attachment and penetration. MIC4 was previously described as a protein secreted by Toxoplasma gondii tachyzoites upon stimulation of micronemes exocytosis. We have microsequenced the mature protein, purified after discharge from micronemes and cloned the corresponding gene. The deduced amino acid sequence of MIC4 predicts a 61-kDa protein that contains 6 conserved apple domains. Apple domains are composed of six spacely conserved cysteine residues which form disulfide bridges and are also present in micronemal proteins from two closely related apicomplexan parasites, Sarcocystis muris and Eimeria species, and several mammalian serum proteins, including kallikrein. Here we show that MIC4 localizes in the micronemes of all the invasive forms of T. gondii, tachyzoites, bradyzoites, sporozoites, and merozoites. The protein is proteolytically processed both at the N and the C terminus only upon release from the organelle. MIC4 binds efficiently to host cells, and the adhesive motif maps in the most C-terminal apple domain.     

Molecular phylogeny of the other tissue coccidia: Lankesterella and Caryospora

Nearly complete sequences were obtained from the 18S rDNA genes of Eimeria falciformis (the type species of the genus), Caryospora bigenetica, and Lankesterella minima. Two clones of the rDNA gene from C. higenetica varied slightly in primary structure. Parsimony-based and maximum likelihood phylogenetic reconstructions with a number of other apicomplexan taxa support 2 major clades within the Eucoccidiorida, i.e., the isosporoid coccidia (consisting of Toxoplasma, Neospora, Isospora [in part], and Sarcocystis spp.) and a second clade containing Lankesterella and Caryospora spp., as well as the eimeriid coccidia (Cyclospora, Isospora [in part], and Eimeria spp.). Our observations suggest that Caryospora spp. may not belong in the family Eimeriidae but rather may be allied with the family Lankesterellidae with which they share molecular and life history similarities. This may be a third lineage of coccidian parasites that has independently evolved a unique heteroxenous transmission strategy.     

The fate of Hepatozoon species naturally infecting Florida black racers and watersnakes in potential mosquito and soft tick vectors, and histological evidence of pathogenicity in unnatural host species.

Haemogregarine parasites, derived from the Florida snakes Coluber constrictor and Nerodia fasciata and ingested by Aedes aegypti, completed sporogony within the hemocoeles of nearly all fed mosquitoes in 14-18 days, and produced oocysts typical of Hepatozoon. However, mortalities and morbidity were high in the Culex which had fed on the Coluber. Oocysts were not found in any Ornithodoros turicata (Argasidae) which fed upon either snake host, but many sections of fed ticks had gametocyte-like cells within the gut lumen. Most lizards, Anolis carolinensis and Anolis sagrei, infected per os with oocysts derived from both snake species developed infections. Infections in the lizards were largely confined to hepatic schizonts with few parasites found in erythrocytes. Unlike naturally infected snake hosts, Hepatozoon schizonts in livers of lizards were often either surrounded by an unidentified dark pigment or heavily infiltrated with mononuclear inflammatory cells.     

High prevalence of Hepatozoon spp. (Apicomplexa, Hepatozoidae) infection in water pythons (Liasis fuscus) from tropical Australia

Molecular methods were used to identify blood parasites frequently observed in blood smears of water pythons (Liasis fuscus) captured in our study area in the Northern Territory of Australia. A nested polymerase chain reaction (PCR) using primers amplifying the 18s ribosomal RNA (rRNA) nuclear gene resulted in a short PCR product (180 bp) matching this region in the genus Hepatozoon. However, because of the short sequence obtained. 2 new primers were designed based on 18s rRNA sequences of 3 Hepatozoon taxa available in GenBank. Using these primers, approximately 600 bp of the parasite's 18s rRNA gene was amplified successfully and sequenced from 2 water python samples. The new primers were used to investigate the prevalence of blood parasites in 100 pythons. In 25 of these samples we did not observe any blood parasites when examining stained slides. All the samples revealed a 600-bp PCR product, demonstrating that pythons in which we did not visually observe any parasites were infected by Hepatozoon spp. We also analyzed the nucleotide sequences of blood parasites in 4 other reptile taxa commonly encountered in our study area. The sequences obtained from water pythons and from 1 of these taxa were identical, suggesting that the parasite is capable of infecting hosts at different taxonomic levels.     

The flea, Megabothris abantis: an invertebrate host of Hepatozoon sp. and a likely definitive host in hepatozoon infections of the montane vole, Microtus montanus

In searching for an invertebrate host for Hepatozoon sp. infecting the montane vole (Microtus montanus), we collected fleas, ticks, and mites from live-trapped voles and searched squash preparations for Hepatozoon oocysts. From 1989 through 1996, we identified six species of fleas in Grand Teton National Park: Megabothris abantis, Megabothris asio megacolpus, Aetheca wagneri, Peromyscopsylla selenis, Peromyscopsylla. hesperomys, and Hystrichopsylla dippiei dippiei. We found Hepatozoon oocysts only in M. abantis; we found no oocysts in mites or ticks. We conclude that M. abantis is an invertebrate host of Hepatozoon sp. and is likely to be the definitive host for theHepatozoon spp. of M. montanus.     

黄牛、水牛体内人肉孢子虫18S rRNA基因研究及虫种鉴定

本文对自然感染的水牛源的人肉孢子虫以及黄牛源人孢子虫ＤＮＡ的１８Ｓ ｒＲＮＡ基因的ＰＣＲ扩增产物进行了测序。对年获的８６３ｂｐ的１８Ｓ ｒＲＮＡ基因分析比较表明,二者有较高的同源性,因此认为二者可能同是一种肉孢子虫－－人肉孢子虫（Ｓａｒｃｏｃｙｓｔｉｓ ｈｏｍｉｎｉｓ Ｒａｉｌｌｉｅｔ ａｎｄ Ｌｕｃｅｔ,１８９１）。由此推断,不仅黄牛可作为人肉孢子虫的中间宿主,水牛也可作为人肉孢子虫的中间宿主。