Asexual replication in the apicomplexan Sarcocystis neurona involves two main developmental stages: the motile extracellular merozoite and the sessile intracellular schizont. Merozoites invade host cells and transform into schizonts that undergo replication via endopolygeny to form multiple (64) daughter merozoites that are invasive to new host cells. Given that the capabilities of the merozoite vary significantly from the schizont, the patterns of transcript levels throughout the asexual lifecycle were determined and compared in this study. RNA-Seq data were generated from extracellular merozoites and four intracellular schizont development time points. Of the 6,938 genes annotated in the S. neurona genome, 6,784 were identified in the transcriptome. Of these, 4,111 genes exhibited significant differential expression between the merozoite and at least one schizont development time point. Transcript levels were significantly higher for 2,338 genes in the merozoite and 1,773 genes in the schizont stages. Included in this list were genes encoding the secretory pathogenesis determinants (SPDs), which encompass the surface antigen and SAG-related sequence (SAG/SRS) and the secretory organelle proteins of the invasive zoite stage (micronemes, rhoptries, and dense granules). As anticipated, many of the S. neurona SPD gene transcripts were abundant in merozoites. However, several SPD transcripts were elevated in intracellular schizonts, suggesting roles unrelated to host cell invasion and the initial establishment of the intracellular niche. The hypothetical genes that are potentially unique to the genus Sarcocystis are of particular interest. Their conserved expression patterns are instructive for future investigations into the possible functions of these putative Sarcocystis-unique genes.
OBJECTIVE: The genus Sarcocystis is an expansive clade within the Apicomplexa, with the species S. neurona being an important cause of neurological disease in horses. Research to decipher the biology of S. neurona and its host-pathogen interactions can be enhanced by gene expression data. This study has identified conserved apicomplexan orthologs in S. neurona, putative Sarcocystis-unique genes, and gene transcripts abundant in the merozoite and schizont stages. Importantly, we have identified distinct clusters of genes with transcript levels peaking during different intracellular schizont development time points, reflecting active gene expression changes across endopolygeny. Each cluster also has subsets of transcripts with unknown functions, and investigation of these seemingly Sarcocystis-unique transcripts will provide insights into the interesting biology of this parasite genus.

A critical part of the malaria parasite\'s life cycle is invasion of red blood cells (RBCs) by merozoites. Inside RBCs, the parasite forms a schizont, which undergoes segmentation to produce daughter merozoites. These cells are released, establishing cycles of invasion. Traditionally, merozoites are represented as nonmotile, egg-shaped cells that invade RBCs \'narrower end\' first and pack within schizonts with this narrower end facing outwards. Here, we discuss recent evidence and re-evaluate previous data which suggest that merozoites are capable of motility and have spherical or elongated-teardrop shapes. Furthermore, merozoites invade RBCs \'wider end\' first and pack within schizonts with this wider end facing outwards. We encourage the field to review this revised model and consider its implications for future studies.

BACKGROUND: The erythrocytic stage of the life cycle of the malaria parasite, Plasmodium falciparum, consists of trophozoite, schizont and gametocyte stages in humans. Various anti-malarial agents target different stages of the parasite to produce treatment outcomes. This study reports on the stage-specific anti-malarial activity of heptaphylline and imperatorin against human P. falciparum in addition to their cytotoxicity and selectivity indices (SI).
METHODS: The compounds were isolated from Clausena anisata using column chromatography and their structures elucidated using NMR spectroscopy. The anti-malarial activity was determined by measuring the trophozoitocidal, schizonticidal and gametocytocidal activities of the compounds using the SYBR green assay. Cytotoxicity was evaluated using the tetrazolium-based colorimetric assay.
RESULTS: Heptaphylline and imperatorin produced trophozoitocidal, schizonticidal and gametocytocidal activities with IC50s of 1.57 (0.2317)-26.92 (0.3144) µM with those of artesunate (the standard drug) being 0.00024 (0.0036)-0.0070 (0.0013) µM. In the cytotoxicity assay, the compounds produced CC50S greater than 350 µM and SI of 13.76-235.90. Also, the trophozoitocidal and schizonticidal activities of the compounds were more pronounced than their gametocytocidal activity. Imperatorin was 42.04% more trophozoitocidal than hepthaphyline. However, hepthaphyline has more schizonticidal and gametocytocidal properties than imperatorin.
CONCLUSIONS: Heptaphylline and imperatorin are promising anti-malarial agents, since they possess potent anti-malarial activity with weak cytotoxicity on RBCs. However, imperatorin is a better anti-malarial prophylactic agent whereas heptaphylline is a better malaria treatment agent.

Two monoclonal in vitro isolates of Perkinsus beihaiensis were established from tissues of infected Mediterranean mussels Mytilus galloprovincialis, using a lipid concentrate and yeast extract medium (LpcYM) to induce enlarged prezoospoangia from histozoic trophozoites, and Perkinsus broth medium (PBM) to support subsequent zoosporulation and schizogonic proliferation from those cells. Zoospores of the isolates developed into uninucleate signet-ring trophozoites followed by schizonts typical of Perkinsus spp., but an additional type of unknown botryoidal cell clusters appeared and increased in density. These botryoidal cell clusters had second-generation daughter schizonts within which further internal cells were produced. Although botryoidal cell clusters in primary cultures rarely proliferated in PBM, cells contained by botryoidal clusters enlarged as prezoosporangia in LpcYM, and a large number of proliferative stages (i.e. trophozoites and schizonts typical for Perkinsus spp.) were obtained from these prezoosporangia in PBM. At lower cell density, trophozoites transformed into the botryoidal cell clusters in PBM, and the number of cells increased little, while trophozoites rapidly increased by typical schizogony at higher density in PBM. Based on these observations, we determined an optimal cell density for continuous culture of P. beihaiensis isolates, confirmed their ability to proliferate and survive after cryopreservation, and describe an experimental protocol to culture this most recently described species in the genus Perkinsus.

Synchronisation of Plasmodium cultures is essential to investigate the complexities of time-dependent events associated with the asexual blood stage of the malaria parasite life cycle. Here we describe a procedure using ML10, a highly specific inhibitor of the parasite cyclic GMP-dependent protein kinase (PKG), to attain high synchronicity of Plasmodium falciparum and P. knowlesi asexual blood-stage cultures and to obtain high levels of arrested mature schizonts as well as viable released merozoites. Additionally, we describe how to use ML10 to improve the transfection efficiency of P. falciparum parasites and also how to derive the half maximal effective concentration (EC50) of ML10 in other P. falciparum laboratory lines and clinical isolates.

Malaria is a major global health problem which predominantly afflicts developing countries. Although many antimalarial therapies are currently available, the protozoan parasite causing this disease, Plasmodium spp., continues to evade eradication efforts. One biological phenomenon hampering eradication efforts is the parasite\'s ability to arrest development, transform into a drug-insensitive form, and then resume growth post-therapy. Currently, the mechanisms by which the parasite enters arrested development, or dormancy, and later recrudesces or reactivates to continue development, are unknown and the malaria field lacks techniques to study these elusive mechanisms. Since Plasmodium spp. salvage purines for DNA synthesis, we hypothesised that alkyne-containing purine nucleosides could be used to develop a DNA synthesis marker which could be used to investigate mechanisms behind dormancy. Using copper-catalysed click chemistry methods, we observe incorporation of alkyne modified adenosine, inosine, and hypoxanthine in actively replicating asexual blood stages of Plasmodium falciparum and incorporation of modified adenosine in actively replicating liver stage schizonts of Plasmodium vivax. Notably, these modified purines were not incorporated in dormant liver stage hypnozoites, suggesting this marker could be used as a tool to differentiate replicating and non-replicating liver forms and, more broadly, as a tool for advancing our understanding of Plasmodium dormancy mechanisms.

During co-evolution Plasmodium parasites and vertebrates went through a process of selection resulting in defined and preferred parasite-host combinations. As such, Plasmodium falciparum (Pf) sporozoites can infect human hepatocytes while seemingly incompatible with host cellular machinery of other species. The compatibility between parasite invasion ligands and their respective human hepatocyte receptors plays a key role in Pf host selectivity. However, it is unclear whether the ability of Pf sporozoites to mature in cross-species infection also plays a role in host tropism. Here we used fresh hepatocytes isolated from porcine livers to study permissiveness to Pf sporozoite invasion and development. We monitored intra-hepatic development via immunofluorescence using anti-HSP70, MSP1, EXP1, and EXP2 antibodies. Our data shows that Pf sporozoites can invade non-human hepatocytes and undergo partial maturation with a significant decrease in schizont numbers between day three and day five. A possible explanation is that Pf sporozoites fail to form a parasitophorous vacuolar membrane (PVM) during invasion. Indeed, the observed aberrant EXP1 and EXP2 staining supports the presence of an atypical PVM. Functions of the PVM include the transport of nutrients, export of waste, and offering a protective barrier against intracellular host effectors. Therefore, an atypical PVM likely results in deficiencies that may detrimentally impact parasite development at multiple levels. In summary, despite successful invasion of porcine hepatocytes, Pf development arrests at mid-stage, possibly due to an inability to mobilize critical nutrients across the PVM. These findings underscore the potential of a porcine liver model for understanding the importance of host factors required for Pf mid-liver stage development.

BACKGROUND: Cytauxzoon felis is a life-threatening protozoan disease of cats. Identification of schizont-laden macrophages is a point-of-care diagnostic test for acute cytauxzoonosis.
OBJECTIVE: The primary objective determined cytologic agreement between sample types to diagnose acute cytauxzoonosis. The secondary objective evaluated novices\' ability to identify cytauxzoon organisms in blood films and tissue aspirates.
METHODS: Thirty-eight cats with suspected acute cytauxzoonosis and 5 controls examined postmortem.
METHODS: Cases were prospectively submitted and collected. Blood film, lymph node, and splenic aspirates were blindly reviewed for sample quality, presence of schizont-laden macrophages, and agreement between sample types. A subset of cases and controls were evaluated by 12 blinded novice observers to determine sensitivity and specificity for identifying organisms in various sample types.
RESULTS: Acute cytauxzoonosis diagnosis was made on at least 1 sample type in 28/38 cats. Schizont-laden macrophages were seen on 33% (10/30) of blood films, 56% (19/34) lymph node aspirates, 77% (26/34) splenic aspirates. Schizont-laden macrophages were more likely seen on splenic than lymph node aspirates (McNemar\'s, P = .03) or blood film (McNemar\'s, P = <.001). Novice observers were more likely to agree with experts when identifying schizont-laden macrophages in splenic aspirates (sensitivity = 77.1%, specificity = 94.4%) versus lymph node aspirates (sensitivity = 52.8%, specificity = 96.4%) or blood films (sensitivity = 41.7%, specificity = 96.9%).
CONCLUSIONS: Schizont-laden macrophages are most frequently identified in spleen, even by novice observers. If the diagnosis of acute cytauxzoonosis cannot be confirmed via blood film, then splenic, followed by peripheral lymph node aspirates can be considered.

Theileria parasites commonly infect African wild artiodactyls. In rare roan (Hippotragus equinus) and sable (H. niger) antelopes, Theileria sp. (sable)-associated calf mortalities constrain breeding programs. The pathogenicity of most leukocyte-transforming Theileria spp. originates in their invasion of and multiplication in various mononuclear leukocytes, the transformation of both infected and uninfected leukocytes, and their infiltration of multiple organs. Understanding the pathogenesis of theileriosis can be improved by the use of immunohistochemistry (IHC) to identify the localization of the parasites in tissue sections. Our aim was to develop a reproducible IHC assay to detect leukocyte-associated Theileria parasites in formalin-fixed, paraffin-embedded roan and sable tissues. Polyclonal antibodies were purified from the sera of 5 roans from an area endemic for Theileria sp. (sable) and tested for IHC reactivity in 55 infected and 39 control roan and sable antelopes, and for antigen and species cross-reactivity in an additional 58 cases. The 3 strongest antibodies consistently detected intraleukocytic theilerial antigens in known positive cases in roan and sable antelopes, and also detected other Theileria spp. in non-hippotraginid wild artiodactyl tissues. The antibodies did not cross-react with other apicomplexan protozoa, with the exception of Cryptosporidium. Given that PCR on its own cannot determine the significance of theilerial infection in wild ruminants, IHC is a useful laboratory test with which to confirm the diagnosis in these species.

Toxoplasma gondii and Eimeria spp. are widely prevalent Coccidian parasites that undergo sexual reproduction during their life cycle. T. gondii can infect any warm-blooded animal in its asexual cycle; however, its sexual cycle is restricted to felines. Eimeria spp. are usually restricted to one host species, and their whole life cycle is completed within this same host. The literature reviewed in this article comprises the recent findings regarding the unique biology of the sexual development of T. gondii and Eimeria spp. The molecular basis of sex in these pathogens has been significantly unraveled by new findings in parasite differentiation along with transcriptional analysis of T. gondii and Eimeria spp. pre-sexual and sexual stages. Focusing on the metabolic networks, analysis of these transcriptome datasets shows enrichment for several different metabolic pathways. Transcripts for glycolysis enzymes are consistently more abundant in T. gondii cat infection stages than the asexual tachyzoite stage and Eimeria spp. merozoite and gamete stages compared to sporozoites. Recent breakthroughs in host-pathogen interaction and host restriction have significantly expanded the understating of the unique biology of these pathogens. This review aims to critically explore advances in the sexual cycle of Coccidia parasites with the ultimate goal of comparing and analyzing the sexual cycle of Eimeria spp. and T. gondii.