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An important group of animal and human pathogens, belonging to the phylum Apicomplexa, employs a novel form of motility, known as gliding, to move on solid substrates and to enter host cells. Gliding is dependent on the parasite cytoskeleton and involves a conserved family of secretory adhesins.
Apicomplexa are a phylum of obligate intracellular parasites critically dependent on invasion of a host cell. An in vitro assay for erythrocyte invasion by Babesia bovis was established, employing free merozoites obtained after the application of high-voltage to the parasitised erythrocytes. The invasion proceeds efficiently in phosphate-buffered saline solution without the requirement for any serum or medium components. The kinetics of invasion can be measured over a time span of 5-60 min after which invasion is completed at an average efficiency of 41%. The fast kinetics and high efficiency exceed those of most previously established apicomplexan invasion assays. The manipulation of intracellular calcium concentration inhibits invasion. Preincubation of merozoites at 37 degrees C also reduces invasion, possibly by the premature secretion of protein. Proteins that are shed into the environment during invasion were directly detectable by protein staining after 2-D gel electrophoresis. The limitations posed by the immunological detection of proteins released during in vitro invasion by other apicomplexan parasites can, therefore, be avoided by this method. A unique feature of the assay is the reversible uncoupling of invasion and intracellular development, the latter taking place only under serum-rich medium conditions. In addition, host cell attachment is uncoupled from invasion by cytochalasin B.
Apicomplexan parasites possess a highly specialized secretory apparatus. The timed secretion of proteins from three different organelles--micronemes, rhoptries and dense granules--serves to establish and maintain a parasitophorous vacuole inside the host cell in which the parasites can divide. Recent efforts have identified components that sort apicomplexan proteins to these unusual secretory organelles and have shown that this machinery is evolutionarily conserved across species. Concise amino acid sequences (e.g. tyrosine-based motifs) within the targeted protein determine their destination in Apicomplexa in a way similar to mammalian cells. Additionally, the parasite exploits new or unusual mechanisms of protein targeting (e.g. post-secretory membrane insertion).
Transmission of haemospororin parasites (phylum Apicomplexa) needs the fertilization of at least one female by one male gamete within the bloodmeal of a suitable vector. Male and female gamete precursors (gametocytes) in Plasmodium and Haemoproteus parasites are normally alone inside the erythrocytes of the vertebrate host, but they also occur in male-female pairs in single erythrocytes. These paired gametocytes could enhance transmission success by facilitating the encounter between the female and male gametes when inside the midgut of the vector. Further study of these particular infections could provide new insights into the biology of and control strategies for haemospororin parasites.
Phylogenetic studies of the genus Plasmodium have been performed using sequences of the nuclear, mitochondrial and plastid genes. Here we have analyzed the adenylosuccinate lyase (ASL) gene, which encodes an enzyme involved in the salvage of host purines needed by malaria parasites for DNA synthesis. The ASL gene is present in several eukaryotic as well as prokaryotic organisms and does not have repeat regions, which facilitates the accuracy of the alignment. Furthermore, it has been shown that ASL is not subject to positive natural selection. We have sequenced the ASL gene of several different Plasmodium species infecting humans, rodents, monkeys and birds and used the obtained sequences along with the previously known P. falciparum ASL sequence, for structural and phylogenetic analysis of the genus Plasmodium. The genetic divergence of ASL is comparable with that observed in other nuclear genes such as cysteine proteinase, although ASL cannot be considered conserved when compared to aldolase or superoxide dismutase, which exhibit a slower rate of evolution. Nevertheless, a protein like ASL has a rate of evolution that provides enough information for elucidating evolutionary relationships. We modeled 3D structures of the ASL protein based on sequences used in the phylogenetic analysis and obtained a consistent structure for four different species despite the divergence observed. Such models would facilitate alignment in further studies with a greater number of plasmodial species or other Apicomplexa.
The rediscovery of Elleipsisoma thomsoni Franca 1912 from Talpa europaea is reported. This organism, whose identity as a parasite was initially doubted, is shown to be an intra-erythrocytic protozoan parasite. Light microscope observation confirmed the original description and 22/53 (42.5%) moles examined from a locality near Blackpool were positive. Organ impression smears demonstrated that E. thomsoni was concentrated in the heart and lungs, occasionally in the kidneys, liver and spleen. Electron microscope studies conclusively showed that the parasite was a member of the phylum Apicomplexa. A typical apical complex was present with rhoptries, micronemes, polar ring and microtubules but, in contrast to the genera Plasmodium, Babesia and Theileria, a conoid was also present. The parasitized red cells were larger than normal mature erythrocytes and the remaining cytoplasm of the erythrocyte was less dense than that of non-infected red cells. The problem of studying this enigmatic parasite and its life-cycle are described.
The current taxonomy of parasites in the genus Sarcocystis is largely based on morphological characteristics as well as on host specificity and life-cycle structure. Recently, phylogenetic analyses of partial ribosomal RNA (rRNA) sequences provided support for paraphyly of Sarcocystis. We have tested the validity of this hypothesis by sequencing the complete 18S rRNA genes of Sarcocystis arieticanis, Sarcocystis gigantea and Sarcocystis tenella and comparing them with gene sequences derived from other taxa of the phylum Apicomplexa. The results obtained from this study do not reject the hypothesis of monophyly of Sarcocystis species, although the bootstrap data were inconclusive for some species.
Small subunit ribosomal RNA (srRNA) genes of three Theileria species, one Cytauxzoon and four Babesia species were amplified using the polymerase chain reaction (PCR), cloned and sequenced. Our sequences were aligned with srRNA sequences previously published for eight species of Apicomplexa, one ciliate and one dinoflagellate, the last two being included as free-living outgroup species. Phylogenetic relationships between the organisms were inferred by four independent methods of phylogenetic tree construction using the ciliate Oxytricha nova to root the trees. Our trees fail to show a consensus branching order. They do, however, clearly indicate that the theilerias form a monophyletic taxon derived from a paraphyletic group which includes the species B. equi, C. felis and B. rodhaini. The distance trees indicate that the babesias sensu stricto (B. canis, B. caballi, B. bigemina and B. bovis) form another monophyletic taxon which diverged before the theilerias separated from the above-mentioned paraphyletic group. The parsimony and maximum likelihood trees suggest that the babesias and theilerias are sister taxa, both of which were derived from the paraphyletic group.
Codon usage has been analysed in individual gene sequences, derived from a variety of parasitic protozoa in the class Sporozoa of the phylum Apicomplexa using metric multidimensional scaling. The two groups of codon usage patterns detected reflect the two main subgroups of organisms studied (the coccidia and the piroplasms), and it is the pattern of usage of synonymous codons that has the largest influence on overall codon usage in the individual genes, rather than being the pattern of amino acid composition of the gene product. The magnitude of the codon usage bias in the sequences was determined using three commonly used indices-NC, GC3S and B. In general, although relatively low levels of codon usage bias were detected in these gene sequences, codon usage bias does explain at least some of the codon usage patterns observed. Codon usage bias was observed to be dependent on the overall base composition of the genes analysed, which in turn was reflected in the types of codons that were either over- or under-represented in the nucleotide sequences. In keeping with observations on prokaryotic organisms, it is speculated that the codon usage patterns detected in these parasitic protozoa are the result of directional mutation pressure on the base composition of the genomic DNA.
Neospora caninum is a protozoan parasite that causes bovine abortion. The epidemiology of N. caninum is poorly understood and little is known about the genetic diversity of the parasite, or whether individual isolates differ in virulence. Such diversity may, among other factors, underlie the range of pathologies seen in cattle. In this study we analysed biological and genetic variation in 6 isolates of N. caninum originating from canine and bovine hosts by measurement of growth rate in vitro, Western blotting and random amplification of polymorphic DNA (RAPD). This comparative analysis of intra-species diversity demonstrated that heterogeneity exists within the species. The relative growth rate in vitro, as assessed by 3[H]uracil uptake, showed significant variation between isolates. However, no significant differences were detected between the antigenic profiles of each isolate by Western blotting. RAPD-PCR was performed on DNA from the 6 Neospora isolates; 3 strains of Toxoplasma gondii, Sarcocystis sp. and Cryptosporidium parvum were also analysed. Twenty-six RAPD primers gave rise to 434 markers of which 222 were conserved between all the Neospora isolates and distinguished them from the other Apicomplexa. An additional 54 markers were unique for Neospora but were polymorphic within the species and able to differentiate between the individual isolates. The RAPD data were subjected to pair-wise similarity and cluster analysis and showed that the Neospora isolates clustered together as a group, with T. gondii as their nearest neighbour. N. caninum isolates showed no clustering with respect either to host or geographical origin. The genetic similarity between Neospora isolates from cattle and dogs suggests that these hosts may be epidemiologically related, although further analysis of bovine and canine field samples are required. The genetic and biological diversity observed in this study may have important implications for our understanding of the pathology and epidemiology of neosporosis.
Babesia microti (Apicomplexa: Piroplasmida) has historically been considered a common parasite of Holarctic rodents. However, human babesiosis due to this species has generally been limited to the northeastern seaboard of the United States and Minnesota and Wisconsin. The absence of reports of B. microti babesiosis from sites where the agent is enzootic, such as in western Europe, remains unexplained. Previous work focusing on the 18S rDNA demonstrates little sequence diversity among samples from allopatric host populations across a wide geographical area. It may be that genetic diversity is underestimated due to sample size or the gene analysed. Accordingly, we collected blood or spleen samples from American or Eurasian animals with parasites that were morphologically consistent with B. microti, amplified the 18S rDNA and beta-tubulin gene, and conducted phylogenetic analysis. Surprisingly, what was considered to be 'B. microti' by microscopy appears to be a diverse species complex. We identify 3 distinct clades within this complex, including parasites from non-rodent hosts. Rodent parasites comprise 2 clades, one representing zoonotic isolates, and the other apparently maintained in microtine rodents, and therefore their morphological detection within animals from a site does not necessarily imply a risk to public health.
Cryptosporidium parvum is a protozoan parasite that causes widespread diarrhoeal disease in humans and other animals and is responsible for large waterborne outbreaks of cryptosporidiosis. Unlike many organisms belonging to the phylum Apicomplexa, such as Plasmodium spp. and Toxoplasma gondii, there is no clinically proven drug treatment against this parasite. Aspects of the basic biology of C. parvum remain poorly understood, including a detailed knowledge of key metabolic pathways, its genome organization and organellar complement. Previous studies have proposed that C. parvum lacks a relic plastid organelle, or 'apicoplast', but that it may possess a mitochondrion. Here we characterize a mitochondrion-like organelle in C. parvum by (i) ultrastructural and morphological description (ii) localization of heterologous mitochondrial chaperonin antibody probes (iii) phylogenetic analysis of genes encoding mitochondrial transport proteins (iv) identification and analysis of mitochondrion-associated gene sequences. Our descriptive morphological analysis was performed by energy-filtering transmission electron microscopy (EFTEM) of C. hominis and C. parvum. The 'mitochondrion-like' organelle was characterized by labelling the structure with a heterologous mitochondrial chaperonin probe (hsp60) both in immunoelectron microscopy (IMEM) and immunofluorescence (IMF). Phylogenetic analysis of the mitochondrial import system and housekeeping components (hsp60 and hsp70-dnaK) suggested that the C. parvum mitochondrion-like organelle is likely to have descended from a common ancestral apicomplexan mitochondrion. We also identified a partial cDNA sequence coding for an alternative oxidase (AOX) gene, a component of the electron transport chain which can act as an alternative to the terminal mitochondrial respiratory complexes III and IV, which has not yet been reported in any other member of this phylum. Degenerate primers developed to identify selected mitochondrial genes failed to identify either cytochrome oxidase subunit I, or cytochrome b. Taken together, our data aim to provide new insights into the characterization of this Cryptosporidium organelle and a logical framework for future functional investigation.
The human malarial parasite Plasmodium falciparum extensively modifies its host erythrocyte, and to this end, is faced with an interesting challenge. It must not only sort proteins to common organelles such as endoplasmic reticulum, Golgi and mitochondria, but also target proteins across the 'extracellular' cytosol of its host cell. Furthermore, as a member of the phylum Apicomplexa, the parasite has to sort proteins to novel organelles such as the apicoplast, micronemes and rhoptries. In order to overcome these difficulties, the parasite has created a novel secretory system, which has been characterized in ever-increasing detail in the past decade. Along with the 'hardware' for a secretory system, the parasite also needs to 'program' proteins to enable high fidelity sorting to their correct subcellular location. The nature of these sorting signals has remained until relatively recently, enigmatic. Experimental work has now begun to dissect the sorting signals responsible for correct subcellular targeting of parasite-encoded proteins. In this review we summarize the current understanding of such signals, and comment on their role in protein sorting in this organism, which may become a model for the study of novel protein trafficking mechanisms.
Plasmodium and Theileria parasites are obligate intracellular protozoa of the phylum Apicomplexa. Theileria infection of bovine leukocytes induces transformation of host cells and infected leukocytes can be kept indefinitely in culture. Theileria-dependent host cell transformation has been the subject of interest for many years and the molecular basis of this unique phenomenon is quite well understood. The equivalent life cycle stage of Plasmodium is the infection of mammalian hepatocytes, where parasites reside for 2-7 days depending on the species. Some of the molecular details of parasite-host interactions in P. berghei-infected hepatocytes have emerged only very recently. Similar to what has been shown for Theileria-infected leukocytes these data suggest that malaria parasites within hepatocytes also protect their host cell from programmed cell death. However, the strategies employed to inhibit host cell apoptotic pathways appear to be different to those used by Theileria. This review discusses similarities and differences at the molecular level of Plasmodium- and Theileria-induced regulation of the host cell survival machinery.
We have investigated the influence of heteroxenous coccidia (Apicomplexa: Eimeriorina) on the predation risk of intermediate hosts. Voles infected with Frenkelia spp. were found more frequently in buzzards' (Buteo buteo) prey than among snap-trapped rodents. To eliminate the possibility of traps selecting for uninfected rodents, a laboratory experiment was performed. Mice experimentally infected with Sarcocystis dispersa seemed to be more likely caught by the final host, the long-eared owl (Asio otus); this result was confirmed by a mathematical model. Field data confirmed the adaptive value of parasite-induced changes. The increase of predation is directed towards the specific final host only or is non-specific. In the populations studied the probability of predation of parasitized individuals by the specific predator was increased.
The protozoan parasite Toxoplasma gondii is representative of a large group of parasites within the phylum Apicomplexa, which share a highly unusual motility system that is crucial for locomotion and active host cell invasion. Despite the importance of motility in the pathology of these unicellular organisms, the motor mechanisms for locomotion remain uncertain, largely because only limited data exist about composition and organization of the cytoskeleton. By using cytoskeleton stabilizing protocols on membrane-extracted parasites and novel imaging with high-resolution low-voltage field emission scanning electron microscopy (LVFESEM), we were able to visualize for the first time a network of actin-sized filaments just below the cell membrane. A complex cytoskeletal network remained after removing the actin-sized fibers with cytochalasin D, revealing longitudinally arranged, subpellicular microtubules and intermediate-sized fibers of 10 nm, which, in stereo images, are seen both above and below the microtubules. These approaches open new possibilities to characterize more fully the largely unexplored and unconventional cytoskeletal motility complex in apicomplexan parasites.
Cryptosporidium parvum (Apicomplexa, formerly Sporozoa) is the causative agent of cryptosporidiosis, an enteric disease of substantial medical and veterinary importance. C. parvum shows a number of unique features that differ from the rest of the class of coccidea in which it is currently grouped taxonomically. Differences occur in the overall structure of the transmission form and the invasive stages of the parasite, its intracellular location, the presence of recently described additional extracellular stages, the host range and target cell tropism, the ability to autoinfection, the nonresponsiveness to anticoccidial drugs, the immune response of the host, and immunochemical and genetic characteristics. These differences have an important impact on the infectivity, the epidemiology, the therapy, and the taxonomy of the parasite. The present article describes the structural analysis of the parasite using light and electron microscopy with an emphasis on structural details unique to C. parvum.
Neospora caninum is an apicomplexan parasite first mentioned in 1984 as a causative agent of neuromuscular disease in dogs. It is closely related to Toxoplasma gondii and Hammondia heydorni, and its subsequent description in 1988 has been, and still is, accompanied by discussions on the true phylogenetical status of the genus Neospora. N. caninum exhibits features that clearly distinguish this parasite from other members of the Apicomplexa, including distinct ultrastructural properties, genetic background, antigenic composition, host cell interactions, and the definition of the dog as a final host. Most importantly, N. caninum has a particular significance as a cause of abortion in cattle. In vitro culture has been indispensable for the isolation of this parasite and for investigations on the ultrastructural, cellular, and molecular characteristics of the different stages of N. caninum. Tissue culture systems include maintenance of N. caninum tachyzoites, which represent the rapidly proliferating stage in a large number of mammalian host cells, culture of parasites in organotypic brain slice cultures as a tool to investigate cerebral infection by N. caninum, and the use of techniques to induce the stage conversion from the tachyzoite stage to the slowly proliferating and tissue cyst-forming bradyzoite stage. This review will focus on the use of these tissue culture models as well as light- and electron-microscopical techniques for studies on N. caninum tachyzoites and bradyzoites, and on the physical interactions between parasites and host cells.