Molecular survey of Apicomplexa in Podarcis wall lizards detects Hepatozoon, Sarcocystis, and Eimeria species

The occurrence of apicomplexan parasites in Podarcis sp. wall lizards from the Iberian Peninsula and Balearic islands was studied by amplification and sequencing of the 18S rRNA gene. Species from 3 genera, Hepatozoon , Sarcocystis , and Eimeria , were found. The phylogenetic analysis of the 18S rRNA gene provides unexpected insights into the evolutionary history of these parasites. All Hepatozoon spp. specimens were recovered as part of a clade already identified in lizards from North Africa. The Sarcocystis species, detected in Podarcis lilfordi from Cabrera Island in the Balearic Islands, appears related to Sarcocystis gallotiae , known only from endemic Gallotia sp. lizards from the Canary Islands. Based on the lack of snake predators on this island, this parasite presumably presents an atypical transmission cycle that uses the same host species as both intermediate and final host through cannibalism, like S. gallotiae . Eimeria sp. is reported for the first time from Podarcis spp. lizards. This study shows the power of detecting multiple different apicomplexan parasites through screening of tail tissue samples and blood drops that are often collected in reptiles for other purposes.     

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.     

Evolutionary relationships among cyst-forming coccidia Sarcocystis spp. (Alveolata: Apicomplexa: Coccidea) in endemic African tree vipers and perspective for evolution of heteroxenous life cycle

Cyst-forming coccidia of the genus Sarcocystis (Alveolata: Apicomplexa: Coccidea) parasitize vertebrates worldwide. Data from the small subunit rRNA genes (SSU) and the D2 domain of the large subunit rRNA genes were used to reconstruct phylogeny for all species in the Sarcocystidae for which sequences are currently available. We have focused on the evolutionary history of species that circulate between snakes as definitive hosts and rodents as intermediate hosts. Trees were reconstructed using maximum parsimony, minimum evolution, maximum likelihood and the bayesian phylogenetics. Our reconstructions support monophyly of Sarcocystidae but fail to robustly resolve the relationship within clades. Using a concatenated dataset of available rDNAs, the "isosporoid" coccidia Neospora, Toxoplasma, Besnoitia, Isospora and Hyaloklossia form a sister group to the monophyletic Sarcocystis. Moreover, we show that Sarcocystis from arboreal vipers of the genus Atheris, which are endemic to the mountain rain forests of the Equatorial Africa, are monophyletic, with sister species parasitizing the desert viper Pseudocerastes persicus from the Near East. We report the co-evolution of Sarcocystis spp. with their final snake hosts. The geological history of the African continent, mountain ranges, forests and general SSU rDNA rates were used to construct a linearized tree. Possible origin of the heteroxenous life cycle of Sarcocystis is discussed.     

Sarcocystis stenodactylicolubris n. sp., a new sarcosporidian coccidium with a snake-gecko heteroxenous life cycle

Oocysts/sporocysts of Sarcocystis sp. measuring 9.7 (9-10) x 7.6 (7-8) microns were found in the intestinal contents of the Dahl's whip snake Coluber najadum. Of wide spectrum of experimentally inoculated hosts, only species of the family Gekkonidae--Ptyodactylus guttatus and Stenodactylus grandiceps--were found to be susceptible intermediate hosts. Transparent, barely visible sarcocysts found in tail, limbs and tongue striated muscles of the geckoes were 175-200 microns x 35-50 microns in size at 78 DPI. Ultrastructurally, the primary cyst wall was characteristic by spine-like villar protrusions up to 800 nm long, 200-250 nm in diameter at their base, tapering to thinner apex. Protrusions appear typically lobular or irregular in the cross-sections. Back-transmission from P. guttatus to Coluber rogersi leaded to oocysts/sporocysts excretion since 38 days post infection. Based on sarcocyst morphology and experimental data, Sarcocystis stenodactylicolubris is apparently a new species. Based on obtained and already published results, Sarcosporidia parasitising colubrid snakes as definitive hosts are suggested to be family specific on the level of their intermediate host.     

Sarcocystis spp. in white-tailed deer. I. Definitive and intermediate host spectrum with a description of Sarcocystis odocoileocanis n. sp

Sporocysts containing four sporozoites and measuring (avg.) 15.2 micrometers X 10.7 micrometers (N = 195) were shed in the feces of dogs (Canis familiaris) 8 to 16 days (avg. 11.6 days) after the first feeding of venison infected with Sarcocystis sp. Sporocysts containing four sporozoites and measuring (avg.) 11.5 micrometers X 8.1 micrometers (N = 35) were shed by a cat (Felis catus) 14 days after ingesting Sarcocystis-infected venison. Statistical (pooled t-test) comparison of the mean measurements of the sporocysts passed by the dog and cat demonstrated a significant difference (P less than .01). The raccoon (Procyon lotor) and opossum (Didelphis virginiana) could not be infected with Sarcocystis from white-tailed deer (Odocoileus virginianus). The name, Sarcocystis odocoileocanis, is proposed for the species transmitted from white-tailed deer to dogs. Sarcocystis odocoileocanis is differentiated from S. hemionilatrantis Hudkins and Kistner, 1977 of mule deer (Odocoileus hemionus), S. ovicanis Heydorn, Gestrich, Mehlhorn and Rommel, 1975 of sheep (Ovis aries) and S. cruzi Hasselmann, 1926 (=S. bovicanis Heydorn, Gestrich, Mehlhorn and Rommel, 1975) of cattle (Bos taurus) because S. odocoileocanis has (1) low infectivity for calves and sheep and (2) apparent insignificant pathogenicity for its intermediate host.     

Ultrastructure of the cyst and life cycle of Sarcocystis sp. from wild sheep (Ovis musimon)

Sarcocystis sp. (Eimeriina: Sarcocystidae) is described as a heteroxenous coccidian with domestic dogs as an experimental definitive host and wild sheep (Ovis musimon) as natural intermediate hosts. Mature sarcocysts of this Sarcocystis sp. were examined by transmission electron microscopy. Sarcocysts in various muscle tissues were microscopic, had a thin primary cyst wall and septa and measured 81.0 x 30.5 microns. The cysts were located within muscle cells and were limited by a primary cyst wall (PCW). The cyst surface was highly folded forming densely packed projections. Between the PCW projections the surface of the cyst was marked with pit-like invaginations. The ground substance of the cyst formed a layer at the periphery of the cyst, filled the projections and formed septa which divided the cyst into compartments. Sarcocysts contained numerous bradyzoites that were 15.2 x 3 microns and few metrocytes 11.5 x 3.5 microns. Twelve days after ingesting Sarcocystis sp.-infected wild sheep meat, four dogs began passing sporocysts in their feces: two domestic cats did not pass oocysts or sporocysts after ingesting meat from the same animals. Sporocysts measured 14.8 x 9.9 microns.     

Comparative review of the developmental biology of the genera Sarcocystis, Frenkelia, Isospora, Cystoisospora, Hammondia, Toxoplasma and Besnoitia (author's transl)]

A review is given of the advances in our knowledge of the developmental biology of the so-called cyst-forming coccidia in the years from 1974 to 1978. Until 1970 only 6 Isospora species were known to occur in cats, dogs and men. After the discovery of the coccidian nature of the genera Toxoplasma, Sarcocystis, Besnoitia and Frenkelia, and after the discovery of the new genus Hammondia the number of known species rose to over 30. In addition it could be shown that also birds of prey, owls and reptiles serve as final hosts for several Sarcocystis and Frenkelia species. The coccidia with isosporoid oocysts can be classified into two major groups: Species with gamogony and sporogony in the final host (Sarcocystis, Frenkelia) and species with schizogony and gamogony in the final host and sporogony on the ground (Isospora, Cystoisospora, Hammondia, Toxoplasma, Besnoitia). The subdivision of the first group into the genera Sarcocystis and Frenkelia based on the localization of their cysts in the musculature and in the brain, respectively, cannot be upheld in the future. Their classification into organisms with small cystozoites of about 7 microm with birds or reptiles as final hosts (Sarcocystis and Frenkelia species of rodents) and those with large cystozoites of about 15 microm and mammals as final hosts (Sarcocystis spp. of domestic animals and rodents) would be more significative. The second group can be subdivided into monoxenous species (Isospora), species with an optional intermediate host in which no or only slight multiplication occurs (Cystoisospora) and in genera with a multiplication in two phases in the intermediate host (Hammondia, Toxoplasma, Besnoitia). The nomenclature of single species is very controversial. As an example the controversial apprehension of the taxonomy of the Sarcocystis species of cattle is discussed. An application has been submitted to the International Commission for the Zoological Nomenclature to delcare a number of names as nomina dubia and to introduce unambiguous names for those organisms for which type specimens are available.     

Sarcocystis speeri N. sp. (Protozoa: Sarcocystidae) from the opossum (Didelphis virginiana)

The North American opossum (Didelphis virginiana) is host to at least 3 species of Sarcocystis: Sarcocystisfalcatula, Sarcocystis neurona, and a recently recognized Sarcocystis sp. A new name, Sarcocystis speeri, is proposed for the third unnamed Sarcocystis. Immunodeficient mice are an experimental intermediate host for S. speeri. Sarcocystis speeri sporocysts are 12-15 x 8-10 microm in size, and its schizonts are found in many organs of mice. Sarcocysts of S. speeri are found in skeletal muscles and they are up to 5 mm long and filiform. By light microscopy, the sarcocyst wall is thin (<1 microm thick); ultrastructurally, the cyst wall is up to 1.8 microm thick and has characteristic steeple-shaped villar protrusions surmounted by a spire. Sarcocystis speeri schizonts are morphologically and antigenically distinct from schizonts of S. neurona, and S. speeri sporocysts were not infective to budgerigars (Melopsittacus undulatus).     

The life-cycle and ultrastructure of Sarcocystis ameivamastigodryasi n. sp., in the lizard Ameiva ameiva (Teiidae) and the snake Mastigodryas bifossatus (Colubridae)

Sarcocysts in muscles of the teiid lizard Ameiva ameiva from north Brazil were fed to the colubrid snake Mastigodryas bifossatus, the faeces of which had been shown to be devoid of coccidial oocysts or sporocysts. When necropsied 16 days later the snake was shown to have developed a massive intestinal infection of Sarcocystis. Mature sporocysts from another, naturally infected M. bifossatus were fed to juvenile specimens of A. ameiva in which no sarcocysts could be detected in tail muscle biopsies. When examined 30 and 47 days later they had very large numbers of sarcocysts in their tail and tongue muscles. The parasite is given the name of Sarcocystis ameivamastigodryasi n. sp. An ultrastructural study has been made of the sarcocyst and of the parasite's sporulation in the lamina propria of the snake: the latter provides details of the wall formation process in developing sporocysts. Attempts to infect a specimen of the boid Boa constrictor constrictor by feeding it with infected Ameiva failed, suggesting that sporocysts previously recorded in genera of the family Boidae may be those of a different species of Sarcocystis.     

Sarcocystis in muscles of raccoons (Procyon lotor L.)

Sarcocysts of Sarcocystis sp. were found in 26 (50%) of 52 raccoons (Procyon lotor) from Ohio, Pennsylvania, Florida, and Maryland. Although only 4 (7.7%) of 52 cardiac muscles specimens were found to contain sarcocysts, 25% to 36.5% of tongue, diaphragm, masseter muscle, and esophagus specimens were found infected. By light microscopy, sarcocyst walls were less than 3 micron thick and had no conspicuous projections; interior septa were indistinct. By transmission electron microscopy, sarcocyst walls had short (mean = 2.7 micron), villus-like protrusions; thin septa were seen within the sarcocysts. The raccoon may be an intermediate host for a Sarcocystis sp. that completes its life cycle in an unidentified, wild carnivore.     

Sarcocystis infections among snakes in Israel

Natural infection with Sarcocystis has been recovered from 11 species of snakes from Israel and the adjacent territories (Cisjordan), infection was most abundant in subadults. Sporocyst dimensions obtained from the different snakes overlapped in size, but even in a same host species formed distinct size cohorts clustering either around 8.5 x 6.5 microns and 11.0 x 8.5 microns or around 11.0 x 8.5 microns and 12 x 10 microns. Sporocyst-size distribution was, however, altered following cross infections or in consecutively emitted feces. The largest cohort size conformed with the sporocyst dimensions of S. murivipera Matuscka, Heydorn, Mehlhorn, Abd-Al-Az, Diesing, Bichler 1987, described from Vipera palaestinae, one of the snake species included in the present study. Among infected snakes were rodent predators but also species feeding on smaller prey (reptiles). Sporocysts from eight of these species including some small-prey feeders, were available for infecting rodents, all, like S. murivipera, developed into sarcocysts in laboratory white mice (Mus musculus), but in none of the inoculated rodents from other genera, or reptiles. Sarcocysts found in free-ranging house mice infected V. palaestinae. Despite of an apparent conspecificity, Sarcocystis from the different snake species demonstrated a pronounced variation in their virulence to mice. Primary wall was identical in all ultrastructurally studied sarcocysts found in both house mice and laboratory mice fed on sporocysts from the diverse snake species.     

Sarcocystis transmitted by blood transfusion.

Merozoites were found in blood smears from calves, lambs, and pigs exhibiting signs of acute sarcocystosis after oral infection with sporocysts of Sarcocystis bovicanis, Sarcocystis ovicanis, and Sarcocystis suihominis. In each of 3 experiments, whole blood containing merozoites was transfused from an infected host to 2 uninfected recipients of the same species; 2 additional animals of the same species served as uninfected nontransfused controls. The bovine, ovine, and porcine donors all died of acute sarcocystosis. Clinical signs of sarcocystosis were not seen in any transfusion recipients or controls. All recipients and controls were killed and histologic specimens of esophagus, diaphragm, heart, skeletal muscle, and tongue were examined microscopically. Large numbers of intramuscular cysts were present in transfusion recipients, whereas few or no cysts were present in controls, indicating that S. bovicanis, S. ovicanis, and S. suihominis had been transmitted between intermediate hosts of the same species by blood transfusion.     

The taxonomy of Sarcocystis (Protozoa, Apicomplexa) species

The taxonomy of the heteroxenous apicomplexan protozoan genus Sarcocystis was reviewed, and a list of 122 species with their synonyms and hosts given. Both definitive and intermediate hosts are known for only 56 species. The fine structure of the sarcocyst wall may change with age and is not considered necessarily satisfactory for separating species. Specificity for the intermediate host is not narrow for all species. Earlier work on transmission of the parasite from one intermediate host to another should be repeated in the light of present knowledge of the life cycle of species of Sarcocystis.     

The nine-banded armadillo (Dasypus novemcinctus) is an intermediate host for Sarcocystis neurona

The nine-banded armadillo (Dasypus novemcinctus) is an intermediate host of at least three species of Sarcocystis, Sarcocystis dasypi, Sarcocystis diminuta, and an unidentified species; however, life cycles of these species have not been determined. Following feeding of armadillo muscles containing sarcocysts to the Virginia opossum (Didelphis virginiana), the opossums shed sporulated Sarcocystis sporocysts in their faeces. Mean dimensions for sporocysts were 11.0x7.5 microm and each contained four sporozoites and a residual body. Sporocysts were identified as Sarcocystis neurona using PCR and DNA sequencing. A 2-month-old foal that was negative for S. neurona antibodies in the CSF was orally inoculated with 5x10(5) sporocysts. At 4 weeks post-infection, the foal had a 'low positive' result by immunoblot for CSF antibodies to S. neurona and by week 6 had a 'strong positive' CSF result and developed an abnormal gait with proprioceptive deficits and ataxia in all four limbs. Based on the results of this study, the nine-banded armadillo is an intermediate host of S. neurona.     

Morphological and biochemical correlates in the characterization of Sarcocystis spp

Isoenzyme electrophoretic techniques were applied to the characterization of seven Sarcocystis spp. that had been identified by conventional morphological studies. Cystozoites were harvested from macroscopic cysts from sheep, cattle, and mice and from microscopic cysts from sheep, cattle, and goats. Soluble cystozoite extracts were subjected to cellulose acetate gel electrophoresis and characterized at 15 of the 39 enzyme loci examined. Genetic relationships among isolates were examined by simple phenetic clustering. Two different morphological types of macroscopic cysts from sheep, identified as S. gigantea (syn. S. ovifelis) and S. medusiformis, consistently differed at 40% of the loci examined. Such genetic divergence confirms their separate morphotypic classification. Both differed from microscopic cyst isolates from sheep at 87% of the loci examined; however, two different morphotypes of microscopic cysts were found in the sheep sampled (thick-walled and thin-walled cysts). Until sufficient numbers of each type can be isolated and examined separately, both were regarded as belonging to the species S. tenella (syn. S. ovicanis). Macroscopic and microscopic cysts from cattle consistently differed at 80% of the loci thereby supporting their separate classification as S. hirsuta (syn. S. bovifelis) and S. cruzi (syn. S. bovicanis), respectively. Isolates from goats (microscopic cysts identified as S. capracanis) differed from S. tenella and S. cruzi at 20% and 47% of the loci, respectively. All macroscopic cyst isolates from the various host animal species (including S. muris from mice) differed from each other at nearly all loci. Isoenzyme electrophoretic techniques therefore provided genetic evidence supporting the classification of these various Sarcocystis spp. by their morphological characteristics.     

Sarcocystis dubeyella n. sp. and Sarcocystis phacochoeri n. sp. (Protozoa: Sarcocystidae) from the Warthog (Phacochoerus aethiopicus) in South Africa

ABSTRACT Sarcocystis dubeyella n. sp. and S. phacochoeri n. sp. from muscle fibers of the skeletal musculature of two warthogs in South Africa are described by light and and electron microscopy. Sarcocystis dubeyella sarcocysts are macroscopic (up to 12 mm long and 1 mm wide), with a parasite-induced encapsulation of the host muscle fiber in which the plasma membrane of the latter remained unaltered. The sarcocyst wall is characterized by evenly arranged, irregularly semicircular or rectangular villar protrusions (5.0 T. 2.8-11.0 μm) with indented margins and no specific content. Sarcocystis phacochoeri formed filiform microcysts (up to 4 mm long and 0.13 mm wide). Its cyst wall is provided with tightly packed, molarlike villar protrusions (1.6-3.3 T. 1.7-3.3 μm), with smooth margins, hollow on one side, and with longitudinal condensations of the fine granular matrix at various locations in the interior.     

Identification of Sarcocystis hominis-like (Protozoa: Sarcocystidae) cyst in water buffalo (Bubalus bubalis) based on 18S rRNA gene sequences

DNA templates were extracted from isolates of Sarcocystis hominis-like cysts collected from cattle and water buffalo, as well as from Sarcocystis fusiformis cysts and Sarcocystis suihominis cysts. The 18S rRNA genes were amplified using DNA from a single cyst as the templates. Approximately 1,367-1,440 bp sequences were obtained. The sequence difference in isolates of Sarcocystis hominis-like cysts from water buffaloes, and isolates of S. hominis cysts from cattle were very low, only about 0.1%, much lower than the lowest value (1.7%) among different species. Combined with their morphological structure, these sequence data indicate that the 4 isolates from cattle and water buffalo might be the same species, i.e., S. hominis, suggesting that both cattle and water buffalo may serve as the intermediate hosts for this parasite. Apparently, this is the first report using a single cyst to do such work and is a useful way to distinguish the Sarcocystis cyst in an intermediate host that may be simultaneously infected by several different Sarcocystis species.     

Evolution of ruminant Sarcocystis (Sporozoa) parasites based on small subunit rDNA sequences

We present an evolutionary analysis of 13 species of Sarcocystis, including 4 newly sequenced species with ruminants as their intermediate host, based on complete small subunit rDNA sequences. Those species with ruminants as their intermediate host form a well-supported clade, and there are at least two major clades within this group, one containing those species forming microcysts and with dogs as their definitive host and the other containing those species forming macrocysts and with cats as their definitive host. Those species with nonruminants as their intermediate host form the paraphyletic sister group to these clades. Most of the species have considerable genotypic differences (differing in more than 100 nucleotide positions), except for S. buffalonis and S. hirsuta. There is a large suite of genotypic differences indicating that those species infecting ruminant and nonruminant hosts have had very different evolutionary histories, and similarly for the felid- and canid-infecting species. Furthermore, the rDNA sequences that represent the different structural regions of the rRNA molecule have very different genotypic behavior within Sarcocystis. The evolution of these regions should be functionally constrained, and their differences can be explained in terms of the importance of the nucleotide sequences to their functions.     

Ultrastructure of Sarcocystis spp. (Protozoa: Apicomplexa) in rodents from North Sulawesi and West Java, Indonesia

Tissue cysts of the protozoan genus Sarcocystis were detected in the skeletal muscles of 16 (40%) of 40 wild rodents captured in North Sulawesi and West Java, Indonesia. Two types of cysts were found to differ in their morphological characteristics. Macroscopic and microscopic cysts bounded by thick radially-striated cyst walls were detected at both locations in a total of 13 rodents belonging to seven different species (Bunomys chrysocomus, B. fratrorum, Maxomys bartelsii, M. musschenbroekii, Paruromys dominator, Rattus xanthurus and R. exulans). The primary cyst walls contained numerous broad spatula-like protrusions and the cysts were identified as S. singaporensis Zaman and Colley, 1976. In contrast, microscopic cysts bounded by thin smooth cyst walls were detected in seven rodents belonging to three different species captured at Toraut in North Sulawesi (B. chrysocomus, B. fratrorum and P. dominator). Ultrastructural examination revealed numerous slender hair-like protrusions of their primary cyst walls. It is proposed that these cysts be named S. sulawesiensis sp. n. on the basis of their unique morphological characteristics, their intermediate host range and their limited geographic distribution. Mixed infections by both species were found in three rodent species (B. chrysocomus, B. fratrorum and P. dominator).     

Sarcocystis dubeyi (Huong and Uggla, 1999) infection in water buffaloes (Bubalus bubalis) from Egypt

Water buffaloes (Bubalus bubalis) are intermediate hosts for 4 species of Sarcocystis , i.e., Sarcocystis fusiformis and Sarcocystis buffalonis with cats as definitive hosts; Sarcocystis levinei with dogs as definitive hosts; and Sarcocystis dubeyi with an unknown definitive host but thought to be zoonotic. Currently, the latter species has been identified with certainty only from Vietnam. In the present study, sarcocysts of S. dubeyi are reported in 11 (30%) of 35 Egyptian water buffaloes from which the esophageal muscles were examined histologically. Sarcocysts were microscopic, measuring 180-250 × 70-110 μm in size. Ultrastructurally, the sarcocyst wall was 3.5-6.5 μm thick and had palisade-like villar protrusions which give it a striated appearance. The villar protrusions contained microtubules that were distributed along the whole villus. This is the first report of S. dubeyi from water buffaloes in Egypt.