Phosphodiesterase beta is the master regulator of cAMP signalling during malaria parasite invasion.

Cyclic nucleotide signalling is a major regulator of malaria parasite differentiation. Phosphodiesterase (PDE) enzymes are known to control cyclic GMP (cGMP) levels in the parasite, but the mechanisms by which cyclic AMP (cAMP) is regulated remain enigmatic. Here, we demonstrate that Plasmodium falciparum phosphodiesterase ß (PDEß) hydrolyses both cAMP and cGMP and is essential for blood stage viability. Conditional gene disruption causes a profound reduction in invasion of erythrocytes and rapid death of those merozoites that invade. We show that this dual phenotype results from elevated cAMP levels and hyperactivation of the cAMP-dependent protein kinase (PKA). Phosphoproteomic analysis of PDEß-null parasites reveals a >2-fold increase in phosphorylation at over 200 phosphosites, more than half of which conform to a PKA substrate consensus sequence. We conclude that PDEß plays a critical role in governing correct temporal activation of PKA required for erythrocyte invasion, whilst suppressing untimely PKA activation during early intra-erythrocytic development.

Safety, Pharmacokinetics, and Activity of High-Dose Ivermectin and Chloroquine against the Liver Stage of Plasmodium cynomolgi Infection in Rhesus Macaques.

Previously, ivermectin (1 to 10 mg/kg of body weight) was shown to inhibit the liver-stage development of Plasmodium berghei in orally dosed mice. Here, ivermectin showed inhibition of the in vitro development of Plasmodium cynomolgi schizonts (50% inhibitory concentration [IC50], 10.42 µM) and hypnozoites (IC50, 29.24 µM) in primary macaque hepatocytes when administered as a high dose prophylactically but not when administered in radical cure mode. The safety, pharmacokinetics, and efficacy of oral ivermectin (0.3, 0.6, and 1.2 mg/kg) with and without chloroquine (10 mg/kg) administered for 7 consecutive days were evaluated for prophylaxis or radical cure of P. cynomolgi liver stages in rhesus macaques. No inhibition or delay to blood-stage P. cynomolgi parasitemia was observed at any ivermectin dose (0.3, 0.6, and 1.2 mg/kg). Ivermectin (0.6 and 1.2 mg/kg) and chloroquine (10 mg/kg) in combination were well-tolerated with no adverse events and no significant pharmacokinetic drug-drug interactions observed. Repeated daily ivermectin administration for 7 days did not inhibit ivermectin bioavailability. It was recently demonstrated that both ivermectin and chloroquine inhibit replication of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro Further ivermectin and chloroquine trials in humans are warranted to evaluate their role in Plasmodium vivax control and as adjunctive therapies against COVID-19 infections.

Efficient synchronization of Plasmodium knowlesi in vitro cultures using guanidine hydrochloride.

BACKGROUND: Long-term in vitro culture of blood stage Plasmodium parasites invariably leads to asynchronous parasite development. The most often used technique to synchronize Plasmodium falciparum culture is sorbitol treatment, which differentially induces osmotic lysis of trophozoite- and schizont-infected red blood cells due to presence of the new permeation pathways in the membranes of these cells. However, sorbitol treatment does not work well when used to synchronize the culture-adapted Plasmodium knowlesi A1-H.1 line. METHODS: A number of common solutes were tested in lieu of sorbitol for synchronization of P. knowlesi A1-H.1 ring stage. RESULTS: Guanidine hydrochloride was found to selectively lyse trophozoite- and schizont-infected red blood cells, yielding highly synchronous and viable rings. CONCLUSIONS: A method for synchronization of P. knowlesi in human red blood cells was developed. Requiring only common laboratory reagents, this method is simple and should be applicable to most laboratory settings.

Preliminary studies on in vitro methods for the evaluation of anticoccidial efficacy/resistance in ruminants.

Ovine Eimeria spp. infections cause increased mortality, reduced welfare and substantial economic losses, and anticocccidials are important for their control. Recent reports of anticoccidial resistance against ovine Eimeria spp. necessitate the development of in vitro methods for the detection of reduced anticoccidial efficacy, especially since the in vivo methods are both expensive, time consuming and requires the use of otherwise healthy animals. The aim of the present study was therefore to approach a preliminary standardization of in vitro assays for evaluation of the efficacy of the most commonly used anticoccidials in ruminants. For this purpose, apart from the evaluation of inhibition of oocyst sporulation, most effort was concentrated on assessment of the capacity of the different anticoccidials to inhibit both the invasion and further development (up to the first schizogony) of E. ninakohlyakimovae sporozoites in bovine colonic epithelial cells (BCEC). For this purpose, infected cultures were monitored 1, 8 and 15 days post infection to determine the infection rate, number of immature schizonts and number, size and appearance of mature schizonts, respectively. No clear inhibitory effect was found with any of the anticoccidial formulations tested, and we could not identify why there were no measurable effects from the different anticoccidials. Despite the lack of positive results, further investigations should be encouraged, as this could decrease the need for animal experiments and could be used in the initial assessment of anticoccidial efficacy of new drugs.

Metamorphosis of Ichthyophonus Schizonts Transiting the Gastrointestinal Tract of Experimentally Exposed Rainbow Trout.

Other than the initial infectious cell, schizonts are the only stage of the parasite Ichthyophonus sp. that has been identified in the tissues of a living host, and they are known to initiate new infections when ingested by a suitable host. However, after feeding Ichthyophonus-infected tissue to Rainbow Trout Oncorhynchus mykiss, we observed that once infection was initiated, some schizonts proceeded to develop into several other morphologic forms indistinguishable from those previously described from recently deceased hosts, decomposing infected corpses, and in vitro culture. It appeared that not all schizonts participated in the infection process; some initiated infection, as expected, while others passed into the intestines, where they morphed into multiple cell types (e.g., schizonts, some with partially digested or ruptured capsules, ameboid plasmodia, merozoites, hyphenated cells, and empty capsules). Some of these cells were viable when cultured, but none was infectious to naïve Rainbow Trout when administered by gavage. We posit that (1) not all tissue schizonts are programmed to perform the same function or (2) not all respond similarly to their environment. After consumption by a piscivore, those schizonts that do not initiate an infection do not die but rather metamorphose into different cell types as they transit the gastrointestinal tract and are ultimately released back into the aquatic environment through defecation. The fate of these cells after exiting the host is presently unknown, but they likely represent a segment of the Ichthyophonus life cycle.

Generation of transgenic rodent malaria parasites by transfection of cell culture-derived merozoites.

BACKGROUND: Malaria research is greatly dependent on and has drastically advanced with the possibility of genetically modifying Plasmodium parasites. The commonly used transfection protocol by Janse and colleagues utilizes blood stage-derived Plasmodium berghei schizonts that have been purified from a blood culture by density gradient centrifugation. Naturally, this transfection protocol depends on the availability of suitably infected mice, constituting a time-based variable. In this study, the potential of transfecting liver stage-derived merozoites was explored. In cell culture, upon merozoite development, infected cells detach from the neighbouring cells and can be easily harvested from the cell culture supernatant. This protocol offers robust experimental timing and temporal flexibility. METHODS: HeLa cells are infected with P. berghei sporozoites to obtain liver stage-derived merozoites, which are harvested from the cell culture supernatant and are transfected using the Amaxa Nucleofector® electroporation technology. RESULTS: Using this protocol, wild type P. berghei ANKA strain and marker-free PbmCherryHsp70-expressing P. berghei parasites were successfully transfected with DNA constructs designed for integration via single- or double-crossover homologous recombination. CONCLUSION: An alternative protocol for Plasmodium transfection is hereby provided, which uses liver stage-derived P. berghei merozoites for transfection. This protocol has the potential to substantially reduce the number of mice used per transfection, as well as to increase the temporal flexibility and robustness of performing transfections, if mosquitoes are routinely present in the laboratory. Transfection of liver stage-derived P. berghei parasites should enable generation of transgenic parasites within 8-18 days.

Ned-19 inhibition of parasite growth and multiplication suggests a role for NAADP mediated signalling in the asexual development of Plasmodium falciparum.

BACKGROUND: Although malaria is a preventable and curable human disease, millions of people risk to be infected by the Plasmodium parasites and to develop this illness. Therefore, there is an urgent need to identify new anti-malarial drugs. Ca2+ signalling regulates different processes in the life cycle of Plasmodium falciparum, representing a suitable target for the development of new drugs. RESULTS: This study investigated for the first time the effect of a highly specific inhibitor of nicotinic acid adenine dinucleotide phosphate (NAADP)-induced Ca2+ release (Ned-19) on P. falciparum, revealing the inhibitory effect of this compound on the blood stage development of this parasite. Ned-19 inhibits both the transition of the parasite from the early to the late trophozoite stage and the ability of the late trophozoite to develop to the multinucleated schizont stage. In addition, Ned-19 affects spontaneous intracellular Ca2+ oscillations in ring and trophozoite stage parasites, suggesting that the observed inhibitory effects may be associated to regulation of intracellular Ca2+ levels. CONCLUSIONS: This study highlights the inhibitory effect of Ned-19 on progression of the asexual life cycle of P. falciparum. The observation that Ned-19 inhibits spontaneous Ca2+ oscillations suggests a potential role of NAADP in regulating Ca2+ signalling of P. falciparum.

Measuring ex vivo drug susceptibility in Plasmodium vivax isolates from Cambodia.

BACKGROUND: While intensive Plasmodium falciparum multidrug resistance surveillance continues in Cambodia, relatively little is known about Plasmodium vivax drug resistance in Cambodia or elsewhere. To investigate P. vivax anti-malarial susceptibility in Cambodia, 76 fresh P. vivax isolates collected from Oddar Meanchey (northern Cambodia) in 2013-2015 were assessed for ex vivo drug susceptibility using the microscopy-based schizont maturation test (SMT) and a Plasmodium pan-species lactate dehydrogenase (pLDH) ELISA. P. vivax multidrug resistance gene 1 (pvmdr1) mutations, and copy number were analysed in a subset of isolates. RESULTS: Ex vivo testing was interpretable in 80% of isolates using the pLDH-ELISA, but only 25% with the SMT. Plasmodium vivax drug susceptibility by pLDH-ELISA was directly compared with 58 P. falciparum isolates collected from the same locations in 2013-4, tested by histidine-rich protein-2 ELISA. Median pLDH-ELISA IC50 of P. vivax isolates was significantly lower for dihydroartemisinin (3.4 vs 6.3 nM), artesunate (3.2 vs 5.7 nM), and chloroquine (22.1 vs 103.8 nM) than P. falciparum but higher for mefloquine (92 vs 66 nM). There were not significant differences for lumefantrine or doxycycline. Both P. vivax and P. falciparum had comparable median piperaquine IC50 (106.5 vs 123.8 nM), but some P. falciparum isolates were able to grow in much higher concentrations above the normal standard range used, attaining up to 100-fold greater IC50s than P. vivax. A high percentage of P. vivax isolates had pvmdr1 Y976F (78%) and F1076L (83%) mutations but none had pvmdr1 amplification. CONCLUSION: The findings of high P. vivax IC50 to mefloquine and piperaquine, but not chloroquine, suggest significant drug pressure from drugs used to treat multidrug resistant P. falciparum in Cambodia. Plasmodium vivax isolates are frequently exposed to mefloquine and piperaquine due to mixed infections and the long elimination half-life of these drugs. Difficulty distinguishing infection due to relapsing hypnozoites versus blood-stage recrudescence complicates clinical detection of P. vivax resistance, while well-validated molecular markers of chloroquine resistance remain elusive. The pLDH assay may be a useful adjunctive tool for monitoring for emerging drug resistance, though more thorough validation is needed. Given high grade clinical chloroquine resistance observed recently in neighbouring countries, low chloroquine IC50 values seen here should not be interpreted as susceptibility in the absence of clinical data. Incorporating pLDH monitoring with therapeutic efficacy studies for individuals with P. vivax will help to further validate this field-expedient method.

Extensive differential protein phosphorylation as intraerythrocytic Plasmodium falciparum schizonts develop into extracellular invasive merozoites.

Pathology of the most lethal form of malaria is caused by Plasmodium falciparum asexual blood stages and initiated by merozoite invasion of erythrocytes. We present a phosphoproteome analysis of extracellular merozoites revealing 1765 unique phosphorylation sites including 785 sites not previously detected in schizonts. All MS data have been deposited in the ProteomeXchange with identifier PXD001684 (http://proteomecentral.proteomexchange.org/dataset/PXD001684). The observed differential phosphorylation between extra and intraerythrocytic life-cycle stages was confirmed using both phospho-site and phospho-motif specific antibodies and is consistent with the core motif [K/R]xx[pS/pT] being highly represented in merozoite phosphoproteins. Comparative bioinformatic analyses highlighted protein sets and pathways with established roles in invasion. Within the merozoite phosphoprotein interaction network a subnetwork of 119 proteins with potential roles in cellular movement and invasion was identified and suggested that it is coregulated by a further small subnetwork of protein kinase A (PKA), two calcium-dependent protein kinases (CDPKs), a phosphatidyl inositol kinase (PI3K), and a GCN2-like elF2-kinase with a predicted role in translational arrest and associated changes in the ubquitinome. To test this notion experimentally, we examined the overall ubiquitination level in intracellular schizonts versus extracellular merozoites and found it highly upregulated in merozoites. We propose that alterations in the phosphoproteome and ubiquitinome reflect a starvation-induced translational arrest as intracellular schizonts transform into extracellular merozoites.

The malarial serine protease SUB1 plays an essential role in parasite liver stage development.

Transmission of the malaria parasite to its vertebrate host involves an obligatory exoerythrocytic stage in which extensive asexual replication of the parasite takes place in infected hepatocytes. The resulting liver schizont undergoes segmentation to produce thousands of daughter merozoites. These are released to initiate the blood stage life cycle, which causes all the pathology associated with the disease. Whilst elements of liver stage merozoite biology are similar to those in the much better-studied blood stage merozoites, little is known of the molecular players involved in liver stage merozoite production. To facilitate the study of liver stage biology we developed a strategy for the rapid production of complex conditional alleles by recombinase mediated engineering in Escherichia coli, which we used in combination with existing Plasmodium berghei deleter lines expressing Flp recombinase to study subtilisin-like protease 1 (SUB1), a conserved Plasmodium serine protease previously implicated in blood stage merozoite maturation and egress. We demonstrate that SUB1 is not required for the early stages of intrahepatic growth, but is essential for complete development of the liver stage schizont and for production of hepatic merozoites. Our results indicate that inhibitors of SUB1 could be used in prophylactic approaches to control or block the clinically silent pre-erythrocytic stage of the malaria parasite life cycle.

The Plasmodium vivax rhoptry neck protein 5 is expressed in the apical pole of Plasmodium vivax VCG-1 strain schizonts and binds to human reticulocytes.

BACKGROUND: Different proteins derived from the membrane or the apical organelles become involved in malarial parasite invasion of host cells. Among these, the rhoptry neck proteins (RONs) interact with a protein component of the micronemes to enable the formation of a strong bond which is crucial for the parasite's successful invasion. The present study was aimed at identifying and characterizing the RON5 protein in Plasmodium vivax and evaluating its ability to bind to reticulocytes. METHODS: Taking the Plasmodium falciparum and Plasmodium knowlesi RON5 amino acid sequences as template, an in-silico search was made in the P. vivax genome for identifying the orthologous gene. Different molecular tools were used for experimentally ascertaining pvron5 gene presence and transcription in P. vivax VCG-1 strain schizonts. Polyclonal antibodies against PvRON5 peptides were used for evaluating protein expression (by Western blot) and sub-cellular localization (by immunofluorescence). A 33 kDa PvRON5 fragment was expressed in Escherichia coli and used for evaluating the reactivity of sera from patients infected by P. vivax. Two assays were made for determining the RON5 recombinant fragment's ability to bind to reticulocyte-enriched human umbilical cord samples. RESULTS: The pvron5 gene (3,477 bp) was transcribed in VCG-1 strain schizonts and encoded a ~133 kDa protein which was expressed in the rhoptry neck of VCG-1 strain late schizonts, together with PvRON2 and PvRON4. Polyclonal sera against PvRON5 peptides specifically detected ~85 and ~30 kDa fragments in parasite lysate, thereby suggesting proteolytic processing in this protein. Comparative analysis of VCG-1 strain PvRON5 with other P. vivax strains having different geographic localizations suggested its low polymorphism regarding other malarial antigens. A recombinant fragment of the PvRON5 protein (rPvRON5) was recognized by sera from P. vivax-infected patients and bound to red blood cells, having a marked preference for human reticulocytes. CONCLUSIONS: The pvron5 gene is transcribed in the VCG-1 strain, the encoded protein is expressed at the parasite's apical pole and might be participating in merozoite invasion of host cells, taking into account its marked binding preference for human reticulocytes.

The host endocytic pathway is essential for Plasmodium berghei late liver stage development.

The obligate intracellular liver stage of the Plasmodium parasite represents a bottleneck in the parasite life cycle and remains a promising target for therapeutic intervention. During this stage, parasites undergo dramatic morphological changes and achieve one of the fastest replication rates among eukaryotic species. Nevertheless, relatively little is known about the parasite interactions with the host hepatocyte. Using immunofluorescence, live cell imaging and electron microscopy, we show that Plasmodium berghei parasites are surrounded by vesicles from the host late endocytic pathway. We found that these vesicles are acidic and contain the membrane markers Rab7a, CD63 and LAMP1. When host cell vesicle acidification was disrupted using ammonium chloride or Concanamycin A during the late liver stage of infection, parasite survival was not affected, but schizont size was significantly decreased. Furthermore, when the host cell endocytic pathway was loaded with BSA-gold, gold particles were found within the parasite cytoplasm, showing the transport of material from the host endocytic pathway toward the parasite interior. These observations reveal a novel Plasmodium-host interaction and suggest that vesicles from the host endolysosomal pathway could represent an important source of nutrients exploited by the fast-growing late liver stage parasites.

Radicicol confers mid-schizont arrest by inhibiting mitochondrial replication in Plasmodium falciparum.

Radicicol, an antifungal antibiotic, was previously identified as a compound having antimalarial activity. However, its mechanism of action in Plasmodium falciparum was not elucidated. While characterizing its antimalarial function, we observed that radicicol manifested two distinct developmental defects in cultured P. falciparum in a concentration-dependent manner. At a low concentration of radicicol, a significant percentage of drug-treated parasites were arrested at the schizont stage, while at a higher concentration, the parasites were unable to multiply from schizont to ring. Also, the newly formed rings and trophozoites were extremely delayed in development, eventually leading to cell death. We intended to characterize the potential molecular target of radicicol at its sublethal doses. Our results demonstrated that radicicol specifically impaired mitochondrial replication. This decrement was associated with a severalfold increment of the topoisomerase VIB transcript as well as protein in treated cells over that of untreated parasites. Topoisomerase VIB was found to be localized in the organelle fraction. Our docking study revealed that radicicol fits into the Bergerat fold of Pf topoisomerase VIB present in its ATPase domain. Altogether, these data allow us to conclude that P. falciparum topoisomerase VIB might be one of the targets of radicicol causing inhibition of mitochondrial replication. Hence, radicicol can be suitably employed to explore the mitochondrial physiology of malaria parasites.

Magnetic isolation of Plasmodium falciparum schizonts iRBCs to generate a high parasitaemia and synchronized in vitro culture.

BACKGROUND: The establishment of methods for an in vitro continuous culture of Plasmodium falciparum is essential for gaining knowledge into its biology and for the development of new treatments. Previously, several techniques have been used to synchronize, enrich and concentrate P. falciparum, although obtaining cultures with high parasitaemia continues being a challenging process. Current methods produce high parasitaemia levels of synchronized P. falciparum cultures by frequent changes of culture medium or reducing the haematocrit. However, these methods are time consuming and sometimes lead to the loss of synchrony. METHODS: A procedure that combines Percoll and sorbitol treatments, the use of magnetic columns, and the optimization of the in vitro culture conditions to reach high parasitaemia levels for synchronized Plasmodium falciparum cultures is described. RESULTS: A new procedure has been established using P. falciparum 3D7, combining previous reported methodologies to achieve in vitro parasite cultures that reach parasitaemia up to 40% at any intra-erythrocytic stage. High parasitaemia levels are obtained only one day after magnetic column purification without compromising the parasite viability and synchrony. CONCLUSIONS: The described procedure allows obtaining a large scale synchronized parasite culture at a high parasitaemia with less manipulations than other methods previously described.

IL-17A regulates Eimeria tenella schizont maturation and migration in avian coccidiosis.

Although IL17A is associated with the immunological control of various infectious diseases, its role in host response to Eimeria infections is not well understood. In an effort to better dissect the role of IL17A in host-pathogen interactions in avian coccidiosis, a neutralizing antibody (Ab) to chicken IL17A was used to counteract IL17A bioactivity in vivo. Chickens infected with Eimeria tenella and treated intravenously with IL17A Ab, exhibited reduced intracellular schizont and merozoite development, diminished lesion score, compared with untreated controls. Immunohistological evaluation of cecal lesions in the parasitized tissues indicated reduced migration and maturation of second-generation schizonts and reduced lesions in lamina propria and submucosa. In contrast, untreated and infected chickens had epithelial cells harboring second-generation schizonts, which extend into the submucosa through muscularis mucosa disruptions, maturing into second generation merozoites. Furthermore, IL17A Ab treatment was associated with increased parameters of Th1 immunity (IL2- and IFNγ- producing cells), reduced levels of reactive oxygen species (ROS), and diminished levels of serum matrix metalloproteinase-9 (MMP-9). Finally, schizonts from untreated and infected chickens expressed S100, Wiskott-Aldrich syndrome protein family member 3 (WASF3), and heat shock protein-70 (HSP70) proteins as merozoites matured, whereas the expression of these proteins was absent in IL17A Ab-treated chickens. These results provide the first evidence that the administration of an IL17A neutralizing Ab to E. tenella-infected chickens inhibits the migration of parasitized epithelial cells, markedly reduces the production of ROS and MMP-9, and decreases cecal lesions, suggesting that IL17A might be a potential therapeutic target for coccidiosis control.

Isoprenoid biosynthesis inhibition disrupts Rab5 localization and food vacuolar integrity in Plasmodium falciparum.

The antimalarial agent fosmidomycin is a validated inhibitor of the nonmevalonate isoprenoid biosynthesis (methylerythritol 4-phosphate [MEP]) pathway in the malaria parasite, Plasmodium falciparum. Since multiple classes of prenyltransferase inhibitors kill P. falciparum, we hypothesized that protein prenylation was one of the essential functions of this pathway. We found that MEP pathway inhibition with fosmidomycin reduces protein prenylation, confirming that de novo isoprenoid biosynthesis produces the isoprenyl substrates for protein prenylation. One important group of prenylated proteins is small GTPases, such as Rab family members, which mediate cellular vesicular trafficking. We have found that Rab5 proteins dramatically mislocalize upon fosmidomycin treatment, consistent with a loss of protein prenylation. Fosmidomycin treatment caused marked defects in food vacuolar morphology and integrity, consistent with a defect in Rab-mediated vesicular trafficking. These results provide insights to the biological functions of isoprenoids in malaria parasites and may assist the rational selection of secondary agents that will be useful in combination therapy with new isoprenoid biosynthesis inhibitors.

Inhibition of Plasmodium falciparum CDPK1 by conditional expression of its J-domain demonstrates a key role in schizont development.

PfCDPK1 [Plasmodium falciparum CDPK1 (calcium-dependent protein kinase 1)] is highly expressed in parasite asexual blood and mosquito stages. Its role is still poorly understood, but unsuccessful gene knockout attempts suggest that it is essential for parasite replication and/or RBC (red blood cell) invasion. In the present study, by tagging endogenous CDPK1 with GFP (green fluorescent protein), we demonstrate that CDPK1 localizes to the parasite plasma membrane of replicating and invasive forms as well as very young intracellular parasites and does not appear to be exported into RBCs. Although a knockdown of endogenous CDPK1 was achieved using a destabilization domain, parasites tolerated reduced expression without displaying a phenotype. Because of this, the PfCDPK1 auto-inhibitory J (junction) domain was explored as a means of achieving inducible and specific inhibition. Under in vitro conditions, a fusion protein comprising a J-GFP fusion specifically bound to PfCDPK1 and inhibited its activity. This fusion protein was conditionally expressed in P. falciparum asexual blood stages under the regulation of a DD (destabilization domain) (J-GFP-DD). We demonstrate that J-GFP-DD binds to CDPK1 and that this results in the arrest of parasite development late in the cell cycle during early schizogony. These data point to an early schizont function for PfCDPK1 and demonstrate that conditionally expressing auto-inhibitory regions can be an effective way to address the function of Plasmodium enzymes.

Eimeria arloingi: further studies on the development of some endogenous stages.

The present study investigated the ultrastructural characteristics of gamogony and oocyst wall formation of Eimeria arloingi in experimentally infected kids. The 18 newborn animals allocated to 3 equal groups. Two of groups, A, B were inoculated with a single dose of 1×10(3) and 1×10(5) sporulated oocysts of E. arloingi, respectively. At 7, 14, 21, 28, 35, and 42days postinoculation (DPI), 1 kid from each group was necropsied for ultrastructural studies. Transmission electron microscopy was used to screen for the presence of developmental stages of the parasite. All stages of microgametocyte and macrogametocyte developments and also oocyst wall formation were observed from 7 to 42DPI. Different stages of schizigony accompanied by marked proliferation of endoplasmic reticulum, mitochondria and several granular dividing nuclei were diagnosed in the affected epithelial cells. Young microgamonts were recognizable by an electron lucent parasitophorous vacuole and several dividing nuclei with prominent nucleolar and peripheral chromatin in the cytoplasm. At a later stage, the nuclei began to elongate and a single mitochondrion and two basal bodies were observed in close proximity nucleus. These bodies eventually protruded from the surface of the gametocyte and formed two flagellar structures. Up to 80-120 microgametes were produced per microgamont. Macrogamonts were recognized by the presence of wall-forming bodies of types 1 and 2. Electron lucent WFB2 appeared earlier than the electron denser WFB1 during the process of macrogametogenesis. The outer layer of the oocyst wall was formed by the release of the contents of WFB1 at the surface to form an electron dense layer. The WFB2 appeared, subsequently, to give rise to the electron lucent inner layer. WFB1 plays a major role in oocyst wall formation.

Maturation of Plasmodium falciparum in multiply infected erythrocytes and the potential role in malaria pathogenesis.

Erythrocytes containing two or more parasites, referred to here as multiply infected erythrocytes (MIEs), are common in the blood of humans infected by Plasmodium falciparum. It is necessary to study these cells closely because the excess numbers of parasites they contain suggest that they could be overloaded with virulence factors. Here, microscopic examinations of blood smears from patients showed that up to seven merozoites can successfully invade an erythrocyte and mature to ring stage. However, in vitro culture showed that only up to three parasites can mature to late schizont stage. These observations were made by culturing the parasites in erythrocytes containing hemoglobin AA (HbAA), HbAS, and HbSS. Biochemical analysis of saponin-concentrated culture suggests that more hemozoin is produced in a MIE than in a singly infected erythrocyte (SIE). Studies have shown that ingestion of excessive hemozoin destroys monocytes and neutrophils, which could impair the immune system. Cultured parasites were also examined by transmission electron microscopy, and it was found that the quantity of knobs was dramatically increased on the membranes of erythrocytes containing multiple schizonts, compared to those containing only one schizont. Knobs contain, among other things, P. falciparum erythrocyte membrane protein 1 (PfEMP1) complex which mediates sequestration and promotes severe malaria. These findings suggest that P. falciparum increases its virulence by producing MIEs. On sexual life cycle of the parasite, microphotographs are presented in this report showing, for the first time, that two gametocytes can develop in one erythrocyte; they are referred to here as twin gametocytes. It is not known whether they can infect mosquitoes.

Temporal gene expression during asexual development of the apicomplexan Sarcocystis neurona.

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. IMPORTANCE: 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.