Formation of the food vacuole in Plasmodium falciparum: a potential role for the 19 kDa fragment of merozoite surface protein 1 (MSP1(19))

Plasmodium falciparum Merozoite Surface Protein 1 (MSP1) is synthesized during schizogony as a 195-kDa precursor that is processed into four fragments on the parasite surface. Following a second proteolytic cleavage during merozoite invasion of the red blood cell, most of the protein is shed from the surface except for the C-terminal 19-kDa fragment (MSP1(19)), which is still attached to the merozoite via its GPI-anchor. We have examined the fate of MSP1(19) during the parasite's subsequent intracellular development using immunochemical analysis of metabolically labeled MSP1(19), fluorescence imaging, and immuno-electronmicroscopy. Our data show that MSP1(19) remains intact and persists to the end of the intracellular cycle. This protein is the first marker for the biogenesis of the food vacuole; it is rapidly endocytosed into small vacuoles in the ring stage, which coalesce to form the single food vacuole containing hemozoin, and persists into the discarded residual body. The food vacuole is marked by the presence of both MSP1(19) and the chloroquine resistance transporter (CRT) as components of the vacuolar membrane. Newly synthesized MSP1 is excluded from the vacuole. This behavior indicates that MSP1(19) does not simply follow a classical lysosome-like clearance pathway, instead, it may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase.     

Localization of protective 143/140 kDa antigens of Plasmodium knowlesi by the use of antibodies and ultracryomicrotomy

Immune sera from mice immunized with the 143/140 kDa protein have been shown to partially block erythrocyte invasion by P. knowlesi merozoites. Therefore, immunoelectron microscopy utilizing ultracryomicrotomy, antibody to 143/140 kDa protein, and protein A gold particles were used to determine the precise localization of this protein in malarial parasites. Gold particles were not seen associated with young trophozoites but appeared in the parasite cytoplasm as the parasites grew to multi-nucleate schizonts. In presegmenter-schizonts, gold particles were associated with the well-developed endoplasmic reticulum, the parasite plasma membrane, and the parasitophorous vacuole membrane. The surface of merozoites was covered with gold particles. Maurer's clefts, which appeared in Plasmodium infected erythrocytes, were also associated with gold particles. These observations suggest that 143/140 kDa protective malarial proteins may be synthesized in the endoplasmic reticulum of P. knowlesi schizonts before being transported to the surface of the schizonts and merozoites. Shedding of the merozoite surface coat may be responsible for the presence of the 143/140 kDa proteins in the parasitophorous vacuole and Maurer's clefts.     

An electron microscopic study of Babesia microti invading erythrocytes

Summary Intracellular sporozoan parasites invade the host cell through the invagination of the plasma membrane of the host and a vacuole is formed which accommodates the entering parasite. The vacuole may disappear and the invaginated membrane of the host then becomes closely apposed to that of the parasite's own membrane. As a result the parasite is covered by two membranes. Members of the class Piroplasmea differ from other Sporozoa in that their trophozoites are covered by a single membrane. By screening numerous sections of intraerythrocytic Babesia microti belonging to the class Piroplasmea, it was found that merozoites of Babesia enter the erythrocytes of hamsters in the same way as those of other Sporozoa. When a merozoite touches the red blood cell with its anterior end it becomes attached to the membrane of the host, which starts to invaginate and a parasitophorous vacuole is formed. The vacuolar space disappears rapidly and the membrane of the vacuole and that of the parasite become closely adjacent. At this stage the parasite is surrounded by two plasma membranes. The outer membrane derived from the invaginated host membrane disintegrates quickly and the parasite is left with a single membrane throughout its life span.Supported by Grant AI 08989 from the U.S. Public Health Service. The excellent technical assistance of Ms. Renata Klatt is gratefully acknowledged     

Selective inhibition of a two-step egress of malaria parasites from the host erythrocyte

Escape from the host erythrocyte by the invasive stage of the malaria parasite Plasmodium falciparum is a fundamental step in the pathogenesis of malaria of which little is known. Upon merozoite invasion of the host cell, the parasite becomes enclosed within a parasitophorous vacuole, the compartment in which the parasite undergoes growth followed by asexual division to produce 16-32 daughter merozoites. These daughter cells are released upon parasitophorous vacuole and erythrocyte membrane rupture. To examine the process of merozoite release, we used P. falciparum lines expressing green fluorescent protein-chimeric proteins targeted to the compartments from which merozoites must exit: the parasitophorous vacuole and the host erythrocyte cytosol. This allowed visualization of merozoite release in live parasites. Herein we provide the first evidence in live, untreated cells that merozoite release involves a primary rupture of the parasitophorous vacuole membrane followed by a secondary rupture of the erythrocyte plasma membrane. We have confirmed, with the use of immunoelectron microscopy, that parasitophorous vacuole membrane rupture occurs before erythrocyte plasma membrane rupture in untransfected wild-type parasites. We have also demonstrated selective inhibition of each step in this two-step process of exit using different protease inhibitors, implicating the involvement of distinct proteases in each of these steps. This will facilitate the identification of the parasite and host molecules involved in merozoite release.     

Interaction between Karyolysus sp. and rock lizard liver cells during hibernation]

During the rock lizard hibernation, no nuclear division occurs in the exoerythrocytar trophozoites of Karyolysus sp., found in hepatocytes or Kupffer's cells. In addition, organelles of the apical complex are seen persisting in these trophozoites, unlike the situation routinely observed in the majority of other intracellular sporozoans. Thus, the parasites under study can be compared with hypnozoites of other coccidia. The material being examined from natural rather than experimental conditions, during lizards' hibernation, the dynamics of host-parasite interrelations can be followed that involves the appearance in the infected cell of autophagous vacuoles, changes in mitochondrial structure and pattern of endoplasmic reticulum, the connection of the system of lamellar channels with the space of the parasitophorous vacuole. The results of the present observations may suggest, first, that during the host hibernation, exoerythrocytar trophozoites of Karyolysus being intracellular in their spatial distribution, are able to obtain nutrients from the outer, extracellular space, through the system of lamellar channels; second, that these channels can represent intercellular connections, which makes one consider the exoerythrocytar trophozoites as intercellular rather than intracellular parasites.     

Laser-induced inactivation of Plasmodium falciparum

ABSTRACT: BACKGROUND: Haemozoin crystals, produced by Plasmodium during its intra-erythrocytic asexual reproduction cycle, can generate UV light via the laser-induced, non-linear optical process of third harmonic generation (THG). In the current study the feasibility of using haemozoin, constitutively stored in the parasite's food vacuole, to kill the parasite by irradiation with a near IR laser was evaluated. METHODS: Cultured Plasmodium parasites at different stages of development were irradiated with a pulsed NIR laser and the viability of parasites at each stage was evaluated from their corresponding growth curves using the continuous culture method. Additional testing for germicidal effects of haemozoin and NIR laser was performed by adding synthetic haemozoin crystals to Escherichia coli in suspension. Cell suspensions were then irradiated with the laser and small aliquots taken and spread on agar plates containing selective agents to determine cell viability (CFU). RESULTS: Parasites in the late-trophozoites form as well as trophozoites in early-stage of DNA synthesis were found to be the most sensitive to the treatment with ~4-log reduction in viability after six passes through the laser beam; followed by parasites in ring phase (~2-log reduction). A ~1-log reduction in E. coli viability was obtained following a 60 min irradiation regimen of the bacteria in the presence of 1 muM synthetic haemozoin and a ~2-log reduction in the presence of 10 muM haemozoin. Minimal ([less than or equal to]15%) cell kill was observed in the presence of 10 muM haemin. CONCLUSIONS: Laser-induced third-harmonic generation by haemozoin can be used to inactivate Plasmodium. This result may have clinical implications for treating severe malaria symptoms by irradiating the patient's blood through the skin or through dialysis tubing with a NIR laser.     

A role of falcipain-2, principal cysteine proteases of Plasmodium falciparum in merozoite egression

The process of merozoite release in Plasmodium falciparum involves rupture of the parasitophorous vacuole membrane and erythrocyte plasma membrane. Through the use of protease inhibitors that halt the merozoite release, a number of parasite proteases, especially serine, aspartic, and cysteine proteases, have been implicated in the schizont rupture. To understand the precise role of cysteine proteases in the merozoite release, in the present study, we treated P. falciparum cultures with siRNAs corresponding to falcipain-1, falcipain-2, and falcipain-3, the three papain-family proteases of the parasite. Treatment of malaria parasites with either of the falcipain siRNAs considerably reduced parasite growth. Morphological examination of the siRNA treated parasite cultures revealed that most of the parasites in falcipain-2 siRNA treated cultures were arrested at schizont stage. Analysis of a transgenic P. falciparum line expressing chimeric-GFP upon treatment with falcipain-2 siRNA revealed block in the rupture of erythrocyte membrane at the time of merozoite egression. These results suggest that falcipain-2 is an important parasitic protease that participates in hemoglobin degradation and in the merozoite release.     

Defining the role of PfCRT in Plasmodium falciparum chloroquine resistance

Recent studies have highlighted the importance of a parasite protein referred to as the chloroquine resistance transporter (PfCRT) in the molecular basis of Plasmodium falciparum resistance to the quinoline antimalarials. PfCRT, an integral membrane protein with 10 predicted transmembrane domains, is a member of the drug/metabolite transporter superfamily and is located on the membrane of the intra-erythrocytic parasite's digestive vacuole. Specific polymorphisms in PfCRT are tightly correlated with chloroquine resistance. Transfection studies have now proven that pfcrt mutations confer verapamil-reversible chloroquine resistance in vitro and reveal their important role in resistance to quinine. Available evidence is consistent with the view that PfCRT functions as a transporter directly mediating the efflux of chloroquine from the digestive vacuole.     

Investigation of the Plasmodium falciparum food vacuole through inducible expression of the chloroquine resistance transporter (PfCRT)

Haemoglobin degradation during the erythrocytic life stages is the major function of the food vacuole (FV) of Plasmodium falciparum and the target of several anti-malarial drugs that interfere with this metabolic pathway, killing the parasite. Two multi-spanning food vacuole membrane proteins are known, the multidrug resistance protein 1 (PfMDR1) and Chloroquine Resistance Transporter (PfCRT). Both modulate resistance to drugs that act in the food vacuole. To investigate the formation and behaviour of the food vacuole membrane we have generated inducible GFP fusions of chloroquine sensitive and resistant forms of the PfCRT protein. The inducible expression system allowed us to follow newly-induced fusion proteins, and corroborated a previous report of a direct trafficking route from the ER/Golgi to the food vacuole membrane. These parasites also allowed the definition of a food vacuole compartment in ring stage parasites well before haemozoin crystals were apparent, as well as the elucidation of secondary PfCRT-labelled compartments adjacent to the food vacuole in late stage parasites. We demonstrated that in addition to previously demonstrated Brefeldin A sensitivity, the trafficking of PfCRT is disrupted by Dynasore, a non competitive inhibitor of dynamin-mediated vesicle formation. Chloroquine sensitivity was not altered in parasites over-expressing chloroquine resistant or sensitive forms of the PfCRT fused to GFP, suggesting that the PfCRT does not mediate chloroquine transport as a GFP fusion protein.     

Ultrastructure of cytoplasmic and nuclear changes in Eimeria tenella during first-generation schizogony in cell culture.

Eimeria tenella sporozoites were inoculated into primary cultures of chick kidney cells. Cells fixed from 1 1/2 to 54 hr later were examined with the electron microscope. At 1 1/2 and 24 hr, most intracellular sporozoites were fusiform and retained organelles typical of extracellular sporozoites. However, at 35 hr, rounded trophozoites were present without these structures; only a refractile body, nucleus, mitochondria, and endoplasmic reticulum remained. Binucleate parasites were also present at that time, but at 48 hr many multinucleate schizonts were present. Nuclei, with adjacent conoids, were at the periphery of these schizonts. Partly developed merozoites, each containing a conoid and a nucleus, protruded into the parasitophorous vacuole. At 54 hr, fully developed merozoites were separated from the residual body. Merozoites resembled sporozoites but lacked the large refractile bodies seen in sporozoites. Linear inclusions were present near the merozoite nucleus and in the residual body. Round vacuoles and ribosomes were also found in the residuum. Nucleoli were first seen in sporozoite nuclei at 1 1/2 hr. They were also present in merozoites but were more prominent in trophozoites and schizonts. Peripheral and scattered nuclear heterochromatins were prominent in intracellular sporozoites and diminished in trophozoites, but increased after several nuclear divisions and were again prominent in the merozoite. Small, distinct interchromatin granules were found in all stages. Intranuclear spindles, centrocones, and centrioles were found in connection with nuclear divisions. Ultrastructure of first-generation schizogony in cell culture was similar to that described for second-generation E. tenella in the chicken and to schizogony of other species of Eimeria.     

Plasmodium vivax Tryptophan Rich Antigen PvTRAg36.6 Interacts with PvETRAMP and PvTRAg56.6 Interacts with PvMSP7 during Erythrocytic Stages of the Parasite

Plasmodium vivax is most wide spread and a neglected malaria parasite. There is a lack of information on parasite biology of this species. Genome of this parasite encodes for the largest number of tryptophan-rich proteins belonging to ‘Pv-fam-a’ family and some of them are potential drug/vaccine targets but their functional role(s) largely remains unexplored. Using bacterial and yeast two hybrid systems, we have identified the interacting partners for two of the P. vivax tryptophan-rich antigens called PvTRAg36.6 and PvTRAg56.2. The PvTRAg36.6 interacts with early transcribed membrane protein (ETRAMP) of P.vivax. It is apically localized in merozoites but in early stages it is seen in parasite periphery suggesting its likely involvement in parasitophorous vacuole membrane (PVM) development or maintenance. On the other hand, PvTRAg56.2 interacts with P.vivax merozoite surface protein7 (PvMSP7) and is localized on merozoite surface. Co-localization of PvTRAg56.2 with PvMSP1 and its molecular interaction with PvMSP7 probably suggest that, PvTRAg56.2 is part of MSP-complex, and might assist or stabilize the protein complex at the merozoite surface. In conclusion, the PvTRAg proteins have different sub cellular localizations and specific associated functions during intra-erythrocytic developmental cycle.     

Genetic linkage of pfmdr1 with food vacuolar solute import in Plasmodium falciparum

The P-glycoprotein homolog of the human malaria parasite Plasmodium falciparum (Pgh-1) has been implicated in decreased susceptibility to several antimalarial drugs, including quinine, mefloquine and artemisinin. Pgh-1 mainly resides within the parasite's food vacuolar membrane. Here, we describe a surrogate assay for Pgh-1 function based on the subcellular distribution of Fluo-4 acetoxymethylester and its free fluorochrome. We identified two distinct Fluo-4 staining phenotypes: preferential staining of the food vacuole versus a more diffuse staining of the entire parasite. Genetic, positional cloning and pharmacological data causatively link the food vacuolar Fluo-4 phenotype to those Pgh-1 variants that are associated with altered drug responses. On the basis of our data, we propose that Pgh-1 imports solutes, including certain antimalarial drugs, into the parasite's food vacuole. The implications of our findings for drug resistance mechanisms and testing are discussed.     

Description of Babesia duncani n.sp. (Apicomplexa: Babesiidae) from humans and its differentiation from other piroplasms

The morphologic, ultrastructural and genotypic characteristics of Babesia duncani n.sp. are described based on the characterization of two isolates (WA1, CA5) obtained from infected human patients in Washington and California. The intraerythrocytic stages of the parasite are morphologically indistinguishable from Babesia microti, which is the most commonly identified cause of human babesiosis in the USA. Intraerythrocytic trophozoites of B. duncani n.sp. are round to oval, with some piriform, ring and ameboid forms. Division occurs by intraerythrocytic schizogony, which results in the formation of merozoites in tetrads (syn. Maltese cross or quadruplet forms). The ultrastructural features of trophozoites and merozoites are similar to those described for B. microti and Theileria spp. However, intralymphocytic schizont stages characteristic of Theileria spp. have not been observed in infected humans. In phylogenetic analyses based on sequence data for the complete18S ribosomal RNA gene, B. duncani n.sp. lies in a distinct clade that includes isolates from humans, dogs and wildlife in the western United States but separate from Babesia sensu stricto, Theileria spp. and B. microti. ITS2 sequence analysis of the B. duncani n.sp. isolates (WA1, CA5) show that they are phylogenetically indistinguishable from each other and from two other human B. duncani-type parasites (CA6, WA2 clone1) but distinct from other Babesia and Theileria species sequenced. This analysis provides robust molecular support that the B. duncani n.sp. isolates are monophyletic and the same species. The morphologic characteristics together with the phylogenetic analysis of two genetic loci support the assertion that B. duncani n.sp. is a distinct species from other known Babesia spp. for which morphologic and sequence information are available.     

Plasmodium simium: Ultrastructure of erythrocytic phase

An electron microscopic study of Plasmodium simium infections in the squirrel monkey has supplied information on the ultrastructure of erythrocytic trophozoites, schizonts, merozoites, and gametocytes, in addition to an unusual form of host cell pathology. In general, the structural features, as well as certain specialized functions, e.g., hemoglobin ingestion and utilization, nuclear and cytoplasmic division, were found to be similar to those described for other malarial parasites. Some striking features were noted, however. A highly asynchronous mode of merozoite production was observed within single segmenting parasites in spite of the overall developmental synchrony displayed by the population as a whole. Secondly, during parasite segmentation, newly formed merozoites are connected to one another, as well as to the parasitophorous membrane, by periodic surface strands. It is speculated that these interparasite bridges serve as structural support to the segmenting parasite. When merozoites are matured fully, these interconnections break, leaving a uniform array of short surface bristles. In addition, a number of different pathological changes in host cell structure have been noted. Localized surface discontinuities appear in region of infected cells where apical regions of developing or fully mature merozoites are abutted against the plasma membrane. These profiles suggest that these specialized apical regions of the merozoite function in release as well as in host cell penetration. More generalized surface pathology occurs within parasitized erythrocytes in the form of surface blebs, surface clefts, and associated cytoplasmic microvesicles. The severity of this pathology increases as the intraerythrocytic parasite matures. Topographically these altered cells have a “berry-like” surface texture which makes them quite distinctive when viewed by scanning electron microscopy.     

The malaria parasite's chloroquine resistance transporter is a member of the drug/metabolite transporter superfamily

The malaria parasite's chloroquine resistance transporter (CRT) is an integral membrane protein localized to the parasite's acidic digestive vacuole. The function of CRT is not known and the protein was originally described as a transporter simply because it possesses 10 transmembrane domains. In wild-type (chloroquine-sensitive) parasites, chloroquine accumulates to high concentrations within the digestive vacuole and it is through interactions in this compartment that it exerts its antimalarial effect. Mutations in CRT can cause a decreased intravacuolar concentration of chloroquine and thereby confer chloroquine resistance. However, the mechanism by which they do so is not understood. In this paper we present the results of a detailed bioinformatic analysis that reveals that CRT is a member of a previously undefined family of proteins, falling within the drug/metabolite transporter superfamily. Comparisons between CRT and other members of the superfamily provide insight into the possible role of the protein and into the significance of the mutations associated with the chloroquine resistance phenotype. The protein is predicted to function as a dimer and to be oriented with its termini in the parasite cytosol. The key chloroquine-resistance-conferring mutation (K76T) is localized in a region of the protein implicated in substrate selectivity. The mutation is predicted to alter the selectivity of the protein such that it is able to transport the cationic (protonated) form of chloroquine down its steep concentration gradient, out of the acidic vacuole, and therefore away from its site of action.     

Effects of chloroquine on the feeding mechanism of the intraerythrocytic human malarial parasite Plasmodium falciparum

Ultrastructural investigations of P. falciparum cultivated in vitro in human erythrocytes revealed new features of the feeding mechanism of the parasite. Mature trophozoites and schizonts take up a portion of the host cytosol by endocytosis which is restricted to cytostomes and which involves the invagination of both parasitophorous and parasite membranes. The resulting endocytic vesicles, surrounded by two concentric membranes, migrate towards the central food vacuole membrane. The external membrane of the endocytic vesicles apposes that of the food vacuole, leading to the internalization of vesicles bounded by a single membrane into the vacuole space where they are rapidly degraded. We conclude from this sequence of events that endocytic vesicles fuse with the food vacuole. Treatment of infected cells with therapeutic concentrations of chloroquine inhibited the last step of the feeding process, i.e. vacuolar degradation. This was manifested by the accumulation within the vacuolar space of intact vesicles bounded by single membranes. The implications of these findings for the antimalarial activity of chloroquine are discussed.     

Fine Structure of Schizonts and Merozoites of Eimeria nieschulzi*

SYNOPSIS. Schizonts of E. nieschulzi lie in a vacuole within the host cell. After nuclear division the cell membrane invaginates forming merozoites. Differentiation of the pellicle and other organelles occurs while merozoites are still attached to the schizont cytoplasm. Merozoites have a pellicle thickened at the anterior end to form a polar ring. Radiating posteriorly from the ring, directly beneath the pellicle, are about 25 microtubules. Within the polar ring is a dense conoid. Extending posteriorly from within the conoid is a paired organelle. The paired organelle varies in size and shape in each generation of merozoites. Numerous toxonemes occupy the anterior half of the merozoites. Two paranuclear bodies are present in 1st generation merozoites. One or 2 granular bodies were seen in the anterior end of 2nd generation merozoites. In 3rd generation merozoites 6 or more granular bodies were seen anterior to the nucleus. Each merozoite has a single nucleus containing diffuse chromatin material. Elongate mitochondria and glycogen granules are present. The vacuole surrounding mature merozoites contains residual cytoplasm of the schizont and some granular material. Microvilli project into the vacuole from the host cell membrane.     

Quantitative calcium measurements in subcellular compartments of Plasmodium falciparum-infected erythrocytes

The acidic food vacuole exerts several important functions during intraerythrocytic development of the human malarial parasite Plasmodium falciparum. Hemoglobin taken up from the host erythrocyte is degraded in the food vacuole, and the heme liberated during this process is crystallized to inert hemozoin. Several anti-malarial drugs target food vacuolar pathways, such as hemoglobin degradation and heme crystallization. Resistance and sensitization to some antimalarials is associated with mutations in food vacuolar membrane proteins. Other studies suggest a role of the food vacuole in ion homeostasis, and release of Ca2+ from the food vacuole may mediate adopted physiological responses. To investigate whether the food vacuole is an intracellular Ca2+ store, which in turn may affect other physiological functions in which this organelle partakes, we have investigated total and exchangeable Ca2+ within the parasite's food vacuole using x-ray microanalysis and quantitative confocal live cell Ca2+ imaging. Apparent free Ca2+ concentrations of approximately 90, approximately 350, and approximately 400 nM were found in the host erythrocyte cytosol, the parasite cytoplasm, and the food vacuole, respectively. In our efforts to determine free intracellular Ca2+ concentrations, we evaluated several Ca2+-sensitive fluorochromes in a live cell confocal setting. We found that the ratiometric Ca2+ indicator Fura-Red provides reliable determinations, whereas measurements using the frequently used Fluo-4 are compromised due to problems arising from phototoxicity, photobleaching, and the strong pH dependence of the dye. Our data suggest that the food vacuole contains only moderate amounts of Ca2+, disfavoring a role as a major intracellular Ca2+ store.     

Interaction of sporozoites of Theileria parva with bovine lymphocytes in vitro. I. Early events after invasion

Sporozoites of Theileria parva rapidly enter bovine lymphocytes by a mechanism of passive endocytosis that depends upon progressive circumferential binding of ligands on the parasite to receptors on the host-cell membrane. Within 10 min of entry, the micronemes of the sporozoite discharge their content and the enveloping host-cell membrane is lysed. The host cell responds within 30 min of invasion by polymerization of microtubules arrayed tangential to the sporozoite and converging upon the cytocentrum. Multivesicular bodies and lysosomes are generated and gather around the parasite but are ineffective in the absence of an endocytotic membrane with which they can fuse. Thus Theileria parva can be added to the category of obligate intracellular parasites that ensure their survival by lysis of the parasitophorous vacuole.     

Phylogenetic relationships of the benign Theileria species in cattle and Asian buffalo based on the major piroplasm surface protein (p33/34) gene sequences.

In order to examine the taxonomic relationship of Theileria sp. of Asian buffalo to the benign Theileria spp. of cattle, we sequenced and compared the major piroplasm protein (p33/34) genes of these parasites. The two consensus sequences determined for the buffalo parasite were of the same length (852 bp) and showed >80% identity with the sequences of the homologous genes (849 bp) in the cattle parasites. Alignment of the inferred aa sequences with those of Theileria sergenti and Theileria buffeli predicted that there is an insertion of a single residue at the N-terminus in the inferred polypeptide of the buffalo parasite. Phylogenetic analyses based on the aa sequences suggested that Theileria sp. of the Asian buffalo should be classified within the benign Theileria parasite group as a separate species from the cattle parasites. Based on this, we propose a rearrangement of the currently used classification for the benign Theileria species in cattle and Asian buffalo.