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.     

Phylogenetic Analysis of Coccidia Based on 18S rDNA Sequence Comparison Indicates that Isospora Is Most Closely Related to Toxoplasma and Neospora

The phylogenetic relationships and taxonomic affinities of coccidia with isosporan-type oocysts have been unclear as overlapping characters, recently discovered life cycle features, and even recently discovered taxa. continue to be incorporated into biological classifications of the group. We determined the full or partial 18S ribosomal RNA gene sequences of three mammalian Isospora spp., Isospora felis, Isospora ohioensis and Isospora suis , and a Sarcocystis sp. of a rattlesnake, and used these sequences for a phylogenetic analysis of the genus Isospora and the cyst-forming coccidia. Various alveolate 18S rDNA sequences were aligned and analyzed using maximum parsimony to obtain a phylogenetic hypothesis for the group. The three Isospora spp. were found to be most closely related to Toxoplasma gondii and Neospora caninum. This clade in turn formed the sister group to the Sarcocystis spp. included in the analysis. The results confirm that the genus Isospora does not belong to the family Eimeriidae, but should be classified together with the cyst-forming coccidia in the family Sarcocystidae. Furthermore, there appear to be two lineages within the Sarcocystidae. One lineage comprises Isospora and the Toxoplasma/Neospora clade which share the characters of having a proliferative phase of development preceding gamogony in the definitive host and an exogenous phase of sporogony. The other lineage comprises the Sarcocystis spp. which have no proliferative phase in the definitive host and an endogenous phase of sporogony.     

Phylogenetic analysis of Cystoisospora species at the rRNA ITS1 locus and development of a PCR-RFLP assay

The ITS1 sequences for C. suis, C. belli, C. rivolta, C. felis, and C. ohioensis-like oocysts were determined and a diagnostic PCR-RFLP assay specific for Cystosisopora species was developed. Phylogenetic analysis of ITS1 sequences of Cystosisopora species along with ITS1 sequences for Toxoplasma, Neospora, Sarcocystis and Eimeria spp. using distance, minimum evolution and parsimony-based methods confirmed previous studies, which suggested that the genus Cystoisospora does not belong to the family Eimeriidae, but should be classified together with the cyst-forming coccidia in the family 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.     

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.     

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.     

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.     

Myosin diversity in Apicomplexa

A polymerase chain reaction (PCR) screen was used to examine the diversity of myosins in 7 Apicomplexan parasites: Toxoplasma gondii, Plasmodium falciparum, Neospora caninum, Eimeria tenella, Sarcocystis muris, Babesia bovis, and Cryptosporidium parvum. Using degenerate PCR primers compatible with the majority of known myosin classes, putative myosin sequences were obtained from all of these species. All of the sequences obtained showed greatest similarity to previously identified apicomplexan myosins, suggesting that the diversity of myosins in these parasites is limited. Myosin classes that are known to be widespread across the phylogenetic spectrum, e.g., the myosins I, II, and V, were not seen in the Apicomplexa. Thus, like the plants, the Apicomplexa may have evolved their own unique cohort of myosins that are responsible for the myosin-driven cellular functions observed in these parasites.     

Phylogenetic relationships of the genus Frenkelia: a review of its history and new knowledge gained from comparison of large subunit ribosomal ribonucleic acid gene sequences.

The different genera currently classified into the family Sarcocystidae include parasites which are of significant medical, veterinary and economic importance. The genus Sarcocystis is the largest within the family Sarcocystidae and consists of species which infect a broad range of animals including mammals, birds and reptiles. Frenkelia, another genus within this family, consists of parasites that use rodents as intermediate hosts and birds of prey as definitive hosts. Both genera follow an almost identical pattern of life cycle, and their life cycle stages are morphologically very similar. However, the relationship between the two genera remains unresolved because previous analyses of phenotypic characters and of small subunit ribosomal ribonucleic acid gene sequences have questioned the validity of the genus Frenkelia or the monophyly of the genus Sarcocystis if Frenkelia was recognised as a valid genus. We therefore subjected the large subunit ribosomal ribonucleic acid gene sequences of representative taxa in these genera to phylogenetic analyses to ascertain a definitive relationship between the two genera. The full length large subunit ribosomal ribonucleic acid gene sequences obtained were aligned using Clustal W and Dedicated Comparative Sequence Editor secondary structure alignments. The Dedicated Comparative Sequence Editor alignment was then split into two data sets, one including helical regions, and one including non-helical regions, in order to determine the more informative sites. Subsequently, all four alignment data sets were subjected to different tree-building algorithms. All of the analyses produced trees supporting the paraphyly of the genus Sarcocystis if Frenkelia was recognised as a valid genus and, thus, call for a revision of the current definition of these genera. However, an alternative, more parsimonious and more appropriate solution to the Sarcocystis/Frenkelia controversy is to synonymise the genus Frenkelia with the genus Sarcocystis.     

Detection and differentiation of coccidian oocysts by real-time PCR and melting curve analysis

Rapid and reliable detection and identification of coccidian oocysts are essential for animal health and foodborne disease outbreak investigations. Traditional microscopy and morphological techniques can identify large and unique oocysts, but they are often subjective and require parasitological expertise. The objective of this study was to develop a real-time quantitative PCR (qPCR) assay using melting curve analysis (MCA) to detect, differentiate, and identify DNA from coccidian species of animal health, zoonotic, and food safety concern. A universal coccidia primer cocktail was designed and employed to amplify DNA from Cryptosporidium parvum, Toxoplasma gondii, Cyclospora cayetanensis, and several species of Eimeria, Sarcocystis, and Isospora using qPCR with SYBR Green detection. MCA was performed following amplification, and melting temperatures (T(m)) were determined for each species based on multiple replicates. A standard curve was constructed from DNA of serial dilutions of T. gondii oocysts to estimate assay sensitivity. The qPCR assay consistently detected DNA from as few as 10 T. gondii oocysts. T(m) data analysis showed that C. cayetanensis, C. parvum, Cryptosporidium muris, T. gondii, Eimeria bovis, Eimeria acervulina, Isospora suis, and Sarcocystis cruzi could each be identified by unique melting curves and could be differentiated based on T(m). DNA of coccidian oocysts in fecal, food, or clinical diagnostic samples could be sensitively detected, reliably differentiated, and identified using qPCR with MCA. This assay may also be used to detect other life-cycle stages of coccidia in tissues, fluids, and other matrices. MCA studies on multiple isolates of each species will further validate the assay and support its application as a routine parasitology screening tool.     

Specific detection of Neospora caninum oocysts in fecal samples from experimentally-infected dogs using the polymerase chain reaction

Neospora caninum oocysts, passed in the feces of a definitive host (dog), were isolated, and genomic DNA was extracted. A polymerase cahin reaction (PCR) targeting the N. caninum-specific Nc 5 genomic sequence was performed using the isolated DNA. A synthesized competitor molecule containing part of the Nc 5 sequence was included in the assay as a check against false-negative PCR results and to quantify N. caninum oocyst DNA in fecal samples. A standard curve of the ratio of fluorescence intensity of PCR-amplified competitor to that of oocyst DNA was constructed to compare oocyst equivalents from fecal samples containing unknown numbers of N. caninum oocysts and to assess the sensitivity of the assay. The specificity of the assay was determined using the Nc 5-specific primers in PCR assays against other parasites likely to be found in canine feces. Genomic DNA sequences from the canine coccidians Hammondia heydorni, Cryptosporidium parvum, Sarcocystis cruzi, S. tenella, and Isospora ohioensis and the canine helminth parasites Strongyloides stercoralis, Toxocara canis, Dipylidium caninum, and Ancylostoma caninum were not amplified. In addition, genomic DNA sequences from oocysts of coccidian parasites that might contaminate dog feces, such as Hammondia hammondi, Toxoplasma gondii, or Eimeria tenella, were not amplified in the PCR assay. The assay should be useful in epidemiological surveys of both domestic and wild canine hosts and in investigations of oocyst biology in experimental infections.     

Evolutionary complication of life cycles in Coccidea (Sporozoa: Coccidea)

Similar strategies to preserve a species were evolved independently in different groups of Coccidea. Polyenergid oocysts and tissue cysts are found in representatives of the orders Protococcidiida and Eimeriida. Hypnozoits are found in Karyolysus lacerate and Plasmodium vivax; transovarial transmission of parasites occurs in life cycles of Coccidea of the genera Karyolysis and Babesia. Formation of heteroxenity in groups of Coccidea apparently was developed by different ways and in different periods. In some groups (Cystoisospora, Toxoplasma, Aggregata, Atoxoplasma, Schelackia, Lankesterella, and Calyptospora), recent definitive hosts were initial hosts; in other groups (Sarcocystis, Karyolysus, Haemogregarina, Hepatozoon, Plasmodium, Haemoproteus, Leucytozoon, Babesiosoma, Theileria, and Babesia), intermediate hosts were initial hosts.     

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.     

Infectivity of sarcocystis spp. from bison, elk, moose, and cattle for cattle via sporocysts from coyotes

Bison bison (bison), Cervus canadensis (elk), Alces alces (moose), and Bos taurus (cattle) musculature containing Sarcocystis spp. cysts was fed to laboratory raised Canis latrans (coyotes), Sporocysts collected from the feces of coyotes fed musculature of each of the ruminant species were fed to four groups of three laboratory-raised domestic calves, respectively, to determine if Sarcocystis spp. was transmissible from wild to domestic ruminants and if so, to compare clinical signs of infection and morphologic features of cysts with those resulting from infection with Sarcocystis bovicanis. All calves fed sporocysts of Sarcocystis from coyotes that ate bison or cattle muscle had similar clinical signs and harbored morphologically similar parasites, suggesting that both bison and cattle are intermediate hosts for S. bovicanis and that this species is transmissible between the two ruminant species. All calves fed sporocysts from coyotes that ate elk muscle or moose muscle remained asymptomatic but one calf in each group had intramuscular cysts. The finding of relatively large numbers of intramuscular cysts in one calf fed sporocysts of elk origin and smaller numbers in one calf fed sporocysts of moose origin could represent either spurious natural infections or indicate low infectivity of Sarcocystis spp. from elk and moose to cattle.     

Multiple DNA markers differentiate Sarcocystis neurona and Sarcocystis falcatula

Studies designed to investigate the causative agent of equine protozoal myeloencephalitis and its life cycle have been hampered by the marked similarity of Sarcocystis neurona to other Sarcocystis spp. present in the same definitive host. Random-amplified polymorphic DNA techniques were used to amplify DNA from isolates of S. neurona and Sarcocystis falcatula. DNA sequence analysis of polymerase chain reaction (PCR) products was then used to design PCR primers to amplify specific Sarcocystis spp. DNA products. The ribosomal RNA internal transcribed spacer was also amplified and compared between S. neurona and S. falcatula. Useful sequence heterogeneity between the 2 organisms was identified, creating potential markers to distinguish these Sarcocystis spp. These markers were used to characterize Sarcocystis isolates from opossum (Didelphis virginiana) feces. Our data suggest that S. neurona and S. falcatula can be differentiated with these markers and that multiple Sarcocystis spp., including S. neurona and S. falcatula, are shed by opossums.     

DNA barcoding identifies Eimeria species and contributes to the phylogenetics of coccidian parasites (Eimeriorina, Apicomplexa, Alveolata)

Partial (～ 780 bp) mitochondrial cytochrome c oxidase subunit I (COI) and near complete nuclear 18S rDNA (～ 1,780 bp) sequences were directly compared to assess their relative usefulness as markers for species identification and phylogenetic analysis of coccidian parasites (phylum Apicomplexa). Fifteen new COI partial sequences were obtained using two pairs of new primers from rigorously characterised (sensu Reid and Long, 1979) laboratory strains of seven Eimeria spp. infecting chickens as well as three additional sequences from cloned laboratory strains of Toxoplasma gondii (ME49 and GT1) and Neospora caninum (NC1) that were used as outgroup taxa for phylogenetic analyses. Phylogenetic analyses based on COI sequences yielded robust support for the monophyly of individual Eimeria spp. infecting poultry except for the Eimeria mitis/mivati clade; however, the lack of a phenotypically characterised strain of E. mivati precludes drawing any firm conclusions regarding this observation. Unlike in the 18S rDNA-based phylogenetic reconstructions, Eimerianecatrix and Eimeria tenella formed monophyletic clades based on partial COI sequences. A species delimitation test was performed to determine the probability of making a correct identification of an unknown specimen (sequence) based on either complete 18S rDNA or partial COI sequences; in almost all cases, the partial COI sequences were more reliable as species-specific markers than complete 18S rDNA sequences. These observations demonstrate that partial COI sequences provide more synapomorphic characters at the species level than complete 18S rDNA sequences from the same taxa. We conclude that COI performs well as a marker for the identification of coccidian taxa (Eimeriorina) and will make an excellent DNA 'barcode' target for coccidia. The COI locus, in combination with an 18S rDNA sequence as an 'anchor', has sufficient phylogenetic signal to assist in the resolution of apparent paraphylies within the coccidia and likely more broadly within the Apicomplexa.     

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.