A Time Point Proteomic Analysis Reveals Protein Dynamics of Plasmodium Oocysts.

The oocyst is a sporogonic stage of Plasmodium development that takes place in the mosquito midgut in about 2 weeks. The cyst is protected by a capsule of unknown composition, and little is known about oocyst biology. We carried out a proteomic analysis of oocyst samples isolated at early, mid, and late time points of development. Four biological replicates for each time point were analyzed, and almost 600 oocyst-specific candidates were identified. The analysis revealed that, in young oocysts, there is a strong activity of protein and DNA synthesis, whereas in mature oocysts, proteins involved in oocyst and sporozoite development, gliding motility, and invasion are mostly abundant. Among the proteins identified at early stages, 17 candidates are specific to young oocysts. Thirty-four candidates are common to oocyst and the merosome stages (sporozoite proteins excluded), sharing common features as replication and egress. Western blot and immunofluorescence analyses of selected candidates confirm the expression profile obtained by proteomic analysis.

Intracellular Plasmodium aquaporin 2 is important for sporozoite production in the mosquito vector and malaria transmission.

Malaria remains a devastating disease and, with current measures failing to control its transmission, there is a need for novel interventions. A family of proteins that have long been pursued as potential intervention targets are aquaporins, which are channels facilitating the movement of water and other solutes across membranes. We identify an aquaporin in malaria parasites and demonstrate that it is important for completion of Plasmodium development in the mosquito vector. Disruption of AQP2 in the human parasite Plasmodium falciparum and the rodent parasite Plasmodium berghei blocks sporozoite production inside oocysts established on mosquito midguts, greatly limiting parasite infection of salivary glands and transmission to a new host. In vivo epitope tagging of AQP2 in P. berghei, combined with immunofluorescence assays, reveals that the protein is localized in vesicle-like organelles found in the cytoplasm of gametocytes, ookinetes, and sporozoites. The number of these organelles varies between individual parasites and lifecycle stages suggesting that they are likely part of a dynamic endomembrane system. Phylogenetic analysis confirms that AQP2 is unique to malaria and closely related parasites and most closely resembles intracellular aquaporins. Structure prediction analyses identify several unusual features, including a large accessory extracellular loop and an arginine-to-phenylalanine substitution in the selectivity filter principally determining pore function, a unique feature among known aquaporins. This in conjunction with the importance of AQP2 for malaria transmission suggests that AQP2 may be a fruitful target of antimalarial interventions.

Overcoming the egress block of Plasmodium sporozoites expressing fluorescently tagged circumsporozoite protein.

Plasmodium sporozoites are the highly motile and invasive forms of the malaria parasite transmitted by mosquitoes. Sporozoites form within oocysts at the midgut wall of the mosquito, egress from oocysts and enter salivary glands prior to transmission. The GPI-anchored major surface protein, the circumsporozoite protein (CSP) is important for Plasmodium sporozoite formation, egress, migration and invasion. To visualize CSP, we previously generated full-length versions of CSP internally tagged with the green fluorescent protein, GFP. However, while these allowed for imaging of sporogony in oocysts, sporozoites failed to egress. Here, we explore different strategies to overcome this block in egress and obtain salivary gland resident sporozoites that express CSP-GFP. Replacing the N-terminal and repeat region with GFP did not allow sporozoite formation. Lowering expression of CSP-GFP at the endogenous locus allowed sporozoite formation but did not overcome egress block. Crossing of CSP-GFP expressing parasites that are blocked in egress with wild-type parasites yielded a small fraction of parasites that entered salivary glands and expressed various levels of CSP-GFP. Expressing CSP-GFP constructs from a silent chromosome region from promoters that are active only post salivary gland invasion yielded normal numbers of fluorescent salivary gland sporozoites, albeit with low levels of fluorescence. We also show that lowering CSP expression by 50% allowed egress from oocysts but not salivary gland entry. In conclusion, Plasmodium berghei parasites with normal CSP expression tolerate a certain level of CSP-GFP without disruption of oocyst egress and salivary gland invasion.

Putative prefoldin complex subunit 5 of Plasmodium berghei is crucial for microtubule formation and parasite development in the mosquito.

Plasmodium sporozoite development in and egress from oocysts in the Anopheles mosquito remains largely enigmatic. In a previously performed high-throughput knockout screen, the putative subunit 5 of the prefoldin complex (PbPCS5, PBANKA_0920100) was identified as essential for parasite development during mosquito and liver stage development. Here we generated and analyzed a PbPCS5 knockout parasite line during its development in the mosquito. Interestingly, PbPCS5 deletion does not significantly affect oocyst formation but leads to a growth defect resulting in aberrantly shaped sporozoites. Sporozoites produced in the absence of PbPCS5 were thinner, markedly elongated, and did, in most cases, not contain a nucleus. Sporozoites contained fewer subpellicular microtubules, which reached deep into the sporoblast during sporogony where they contacted and indented nuclei. These aberrantly shaped sporozoites did not reach the salivary glands, and we, therefore, conclude that PbPCS5 is essential for sporogony and the life cycle progression of the parasite during its mosquito stage.

In-vitro susceptibility and ex-vivo evaluation of macrocyclic lactone endectocides sub-lethal concentrations against Plasmodium vivax oocyst development in Anopheles arabiensis.

BACKGROUND: Asymptomatic malaria transmission has become a public health concern across malaria-endemic Africa including Ethiopia. Specifically, Plasmodium vivax is more efficient at transmitting earlier in the infection and at lower densities than Plasmodium falciparum. Consequently, a greater proportion of individuals infected with P. vivax can transmit without detectable gametocytaemia. Mass treatment of livestock with macrocyclic lactones (MLs), e.g., ivermectin and doramectin, was suggested as a complementary malaria vector tool because of their insecticidal effects. However, the effects of MLs on P. vivax in Anopheles arabiensis has not yet been fully explored. Hence, comparative in-vitro susceptibility and ex-vivo studies were conducted to evaluate the effects of ivermectin, doramectin and moxidectin sub-lethal concentrations on P. vivax oocyst development in An. arabiensis. METHODS: The 7-day sub-lethal concentrations of 25% (LC25) and 5% (LC5) were determined from in-vitro susceptibility tests on female An. arabiensis in Hemotek® membrane feeding assay. Next, an ex-vivo study was conducted using P. vivax gametocytes infected patient's blood spiked with the LC25 and LC5 of the MLs. At 7-days post-feeding, each mosquito was dissected under a dissection stereo microscope, stained with 0.5% (w/v) mercurochrome solution, and examined for the presence of P. vivax oocysts. Statistical analysis was based on a generalized mixed model with binomially distributed error terms. RESULTS: A 7-day lethal concentration of 25% (LC25, in ng/mL) of 7.1 (95% CI: [6.3;8.0]), 20.0 (95%CI:[17.8;22.5]) and 794.3 (95%CI:[716.4;1516.3]) were obtained for ivermectin, doramectin and moxidectin, respectively. Similarly, a lethal concentration of 5% (LC5, in ng/mL) of 0.6 (95% CI: [0.5;0.7]), 1.8 (95% CI:[1.6;2.0]) and 53.7 (95% CI:[ 48.4;102.5]) were obtained respectively for ivermectin, doramectin and moxidectin. The oocyst prevalence in treatment and control groups did not differ significantly (p > 0.05) from each other. Therefore, no direct effect of ML endectocides on P. vivax infection in An. arabiensis mosquitoes was observed at the sub-lethal concentration (LC25 and LC5). CONCLUSIONS: The effects of ivermectin and doramectin on malaria parasite is more likely via indirect effects, particularly by reducing the vectors lifespan and causing mortality before completing the parasite's sporogony cycle or reducing their vector capacity as it affects the locomotor activity of the mosquito.

Morphological and molecular characterization of Eimeria magna infecting local rabbit (Oryctolagus cuniculus) in Alkarg City, Saudi Arabia.

Coccidiosis is one of the most prevalent diseases found in local rabbits (Oryctolagus cuniculus), which is caused by the Eimeria. The study aimed to more reliably identify Eimeria species (Eimeria magna) infecting Local Rabbits in Alkarg City, Saudi Arabia, based the method on the molecular properties and morphological and molecular biological techniques. Sub-spheroidal oocysts measuring 21-27 × 12-16 (24 × 14.4) µm (20 n) and with a length/width (L/W) ratio of 0.9-1.1 (1.0) were identified by microscopic analysis of a fecal sample. Oocysts feature a bi-layered wall that is 1.0-1.2 (1.1) µm thick. About two-thirds of the wall's thickness is made up of a smooth outer layer. A polar granule is present, but neither a micropyle nor an oocyst residuum is present. The ovoidal sporozoites measure 15-18 × 8-11 (16.5 × 9.5) µm, have an L/W ratio of 1.6-1.8 (1.7), and take up around 21% of the oocyst's total surface. The mean size of the sub-Stieda body is 1.4 × 2.3 µm, while the average size of the Stieda body is 0.9 × 1.8 µm. The para-Stieda body is lacking. Sporocyst residuum appears membrane-bound and has an uneven form made up of several granules. With two refractile bodies below the striations and pronounced striations at the more pointed end, sporozoites are vermiform, measuring an average of 11.6 × 4.0 µm. The results of the sequencing for the 18S rDNA gene confirmed the species of Eimeria parasites found in the host (rabbits). The current parasite species is closely related to the previously described and deposited E. magna and deeply embedded in the genus Eimeria (family Eimeriidae). According to the findings, single oocyst molecular identification of Eimeria may be accomplished through consistent use of the morphological and molecular results. It is possible to draw the conclusion that the current research supplies relevant facts that help assess the potential infection and future control measures against rabbit coccidiosis to reduce the financial losses that can be incurred by the rabbit industry in Saudi Arabia.

Effects of monensin sodium and live attenuated oocyst vaccine as coccidiosis management programs on productive performance, bone quality and mineral utilisation in broiler chickens.

1. The following study was conducted to evaluate the influence of coccidiosis vaccine-induced metabolic stress on the utilisation of minerals in broilers. The starter, grower and finisher phase diets, including macro- and micro minerals at the recommended levels for the breed standards, were fed to chickens between 1 and 39 d of age.2. A total of 486, one-d-old male broilers were randomly distributed into three coccidiosis management programs (CMP) with six replications each. The CMP comprised: monensin sodium (MON), coccidiosis vaccine (VAC), not treated with MON or VAC (CNT).3. No significant differences between CMP were observed for body weight and weight gain among treatments. When compared to the CNT, the VAC program increased feed intake (P < 0.05) between d 1 to 13 and 14 to 26, while FCR worsened in the latter (P < 0.05) and the former (P = 0.05) periods.4. For birds in the MON and VAC programs, tibia bone length at d 13 and bone diameter at d 39 were both enhanced (P < 0.05). Meat yield characteristics were comparable among the CMP.5. Faeces of VAC birds had a lower (P < 0.05) dry matter and ash content than those in CNT program. CMP had no effect on serum or bone mineral concentrations at any point in time. For minerals, Mg, Na, and K faecal excretion was reduced (P < 0.01) as a result of the VAC program at d 13 with a trend at d 26.6. Compared to the CNT, the VAC program decreased the percentage ratio of drip loss (P = 0.08), water holding capacity (P < 0.01) and cooking loss (P < 0.01) in breast meat.7. Overall, the results showed that current broiler industry practices are capable of meeting the mineral needs of broilers vaccinated against coccidiosis.

Attractive targeted sugar bait: the pyrrole insecticide chlorfenapyr and the anti-malarial pharmaceutical artemether-lumefantrine arrest Plasmodium falciparum development inside wild pyrethroid-resistant Anopheles gambiae s.s. mosquitoes.

BACKGROUND: Attractive targeted sugar bait (ATSB) is a novel approach to vector control, offering an alternative mode of insecticide delivery via the insect alimentary canal, with potential to deliver a variety of compounds new to medical entomology and malaria control. Its potential to control mosquitoes was recently demonstrated in major field trials in Africa. The pyrrole chlorfenapyr is an insecticide new to malaria vector control, and through its unique mode of action-disruption of ATP mediated energy transfer in mitochondria-it may have direct action on energy transfer in the flight muscle cells of mosquitoes. It may also have potential to disrupt mitochondrial function in malarial parasites co-existing within the infected mosquito. However, little is known about the impact of such compounds on vector competence in mosquitoes responsible for malaria transmission. METHODS: In this study, ATSBs containing chlorfenapyr insecticide and, as a positive control, the anti-malarial drugs artemether/lumefantrine (A/L) were compared for their effect on Plasmodium falciparum development in wild pyrethroid-resistant Anopheles gambiae sensu stricto (s.s.) and for their capacity to reduce vector competence. Female mosquitoes were exposed to ATSB containing either sublethal dose of chlorfenapyr (CFP: 0.025%) or concentrations of A/L ranging from 0.4/2.4 mg/ml to 2.4/14.4 mg/ml, either shortly before or after taking infective blood meals. The impact of their component compounds on the prevalence and intensity of P. falciparum infection were compared between treatments. RESULTS: Both the prevalence and intensity of infection were significantly reduced in mosquitoes exposed to either A/L or chlorfenapyr, compared to unexposed negative control mosquitoes. The A/L dose (2.4/14.4 mg/ml) totally erased P. falciparum parasites: 0% prevalence of infection in female mosquitoes exposed compared to 62% of infection in negative controls (df = 1, χ2 = 31.23 p < 0.001). The dose of chlorfenapyr (0.025%) that killed < 20% females in ATSB showed a reduction in oocyte density of 95% per midgut (0.18/3.43 per midgut). CONCLUSION: These results are evidence that chlorfenapyr, in addition to its direct killing effect on the vector, has the capacity to block Plasmodium transmission by interfering with oocyte development inside pyrethroid-resistant mosquitoes, and through this dual action may potentiate its impact under field conditions.

Transmission efficiency of Plasmodium vivax at low parasitaemia.

BACKGROUND: Plasmodium vivax is responsible for much of malaria outside Africa. Although most P. vivax infections in endemic areas are asymptomatic and have low parasite densities, they are considered a potentially important source of transmission. Several studies have demonstrated that asymptomatic P. vivax carriers can transmit the parasite to mosquitoes, but the efficiency has not been well quantified. The aim of this study is to determine the relationship between parasite density and mosquito infectivity, particularly at low parasitaemia. METHODS: Membrane feeding assays were performed using serial dilutions of P. vivax-infected blood to define the relationship between parasitaemia and mosquito infectivity. RESULTS: The infection rate (oocyst prevalence) and intensity (oocyst load) were positively correlated with the parasite density in the blood. There was a broad case-to-case variation in parasite infectivity. The geometric mean parasite density yielding a 10% mosquito infection rate was 33 (CI 95 9-120) parasites/µl or 4 (CI 95 1-17) gametocytes/µl. The geometric mean parasite density yielding a 50% mosquito infection rate was 146 (CI 95 36-586) parasites/µl or 13 (CI 95 3-49) gametocytes/µl. CONCLUSION: This study quantified the ability of P. vivax to infect Anopheles dirus at over a broad range of parasite densities. It provides important information about parasite infectivity at low parasitaemia common among asymptomatic P. vivax carriers.

DNA Aptamer-Based Staining and Fluorescence Microscopy for Rapid Detection of Cyclospora Cayetanensis Oocysts.

There are no commercial antibodies for detection of Cyclospora cayetanensis, only a relatively slow polymerase chain reaction (PCR) test developed by the U.S. Food and Drug Administration (FDA). However, DNA aptamers have recently been developed by our group against known proteins and whole oocysts of C. cayetanensis and shown to specifically detect the oocysts when attached on their 5' ends to red-emitting fluorophores and used as probes for fluorescence microscopy. Aptamers developed against recombinant wall protein 2 and TA4 antigen-like protein as well as whole oocysts specifically stained C. cayetanensis oocysts while exhibiting little, if any, staining of numerous other waterborne parasite species. Interestingly, the aptamers stained both exterior cell wall moieties and internal structures, suggesting that the aptamers penetrate the oocysts even without added detergents.

Development of duplex real-time PCR for quick detection of cryptosporidiosis in goats.

Cryptosporidium spp. is the most important foodborne and waterborne pathogens and a leading cause of mortality from foodborne and waterborne gastrointestinal diseases. In neonates of domestic animals, it is associated with consistent diarrhea and dehydration. Cryptosporidium infection begins with the ingestion of sporulated oocytes disseminated by carrier animals that consistently contaminate the environment. Many diagnostic tests are available including microscopy and antigen trap-ELISA, but none of the diagnostic tests available currently cannot differentiate between active and passive infection in the host. In the current study, to address this challenge an mRNA-based duplex TaqMan® probe PCR was developed to target the Cryptosporidium oocyst wall protein gene and 18SSU rRNA gene in a single tube that can detect metabolically active cryptosporidial oocysts. The mRNA transcripts are the direct indicator of any actively replicating cell and they will help decipher the active stages of its lifecycle in a host. This diagnostic assay was standardized by computing transcript copy number-based limit of detection (LOD). For COWP and 18SSU rRNA genes, the LOD was 7.08 × 1004 and 5.95 × 1005 , respectively. During active infections, the oocyst wall protein will be active and so its COWP gene transcripts will act as a marker for active infection. While transcripts for 18SSU rRNA are constitutively expressed in cryptosporidial life cycle. This current diagnostic assay will be a quantitative marker that will help assess the active stages of Cryptosporidium infection in neonates. The disease dynamics will help better understand to formulate the control strategies and contain infection among healthy animals.

Dispersal and invasive stages of Urospora eugregarines (Apicomplexa) from brown bodies of a polychaete host.

Urosporid eugregarines (Apicomplexa: Urosporidae) are unicellular eukaryotic parasites inhabiting the coelom or the intestine of marine invertebrates such as annelids, molluscs, nemerteans, and echinoderms. Despite the availability of published morphological and phylogenetical analyses of coelomic gregarines, their long-term survival in the host body cavity and dispersal routes into the marine environment remain unclear. Here, we focus on Urospora gametocysts and oocysts with sporozoites, which were found viable inside the so-called brown bodies floating in the body cavity of the polychaete Travisia forbesii. Brown bodies form as a result of host defence where coelomocytes encapsulate dead host cells and foreign objects including potential pathogens. We hypothesise the long-term persistence of Urospora eugregarines in brown bodies through evasion of the host immunity and outline possible pathways for their egress into the marine environment, applicable as dispersal routes for other parasites as well. Unique features revealed by detailed ultrastructural analysis of detected eugregarine stages include asynchronous sporogony, a massive sporozoite secretion apparatus, as well as the presence of free (possibly autoinfective) sporozoites within the gametocyst. The assignment to the genus Urospora and the complete identity with U. ovalis and U. travisiae were confirmed by analysing 18S rDNA sequences obtained from isolated gametocysts. The 18S rDNA phylogeny confirmed the affiliation of Urosporidae to Lecudinoidea and the grouping of all Urospora sequences with Difficilina from nemerteans and environmental sequences from the Artic region. We also enriched the Apicomplexa set by partial 28S rDNA sequences of two Urospora species enabling more complex phylogenetic analyses prospectively.

Mattesia cf. geminata, an ant-pathogenic neogregarine (Apicomplexa: Lipotrophidae) in two Temnothorax species (Hymenoptera: Formicidae).

An ant-pathogenic neogregarine in Temnothorax affinis and T. parvulus (Hymenoptera: Formicidae) is described based on morphological and ultrastructural characteristics. The pathogen infects the hypodermis of the ants. The infection was mainly synchronous so that only gametocysts and oocysts could be observed simultaneously in the host body. Gametogamy resulted in the formation of two oocysts within a gametocyst. The lemon-shaped oocysts measured 11-13 µm in length and 8-10 µm in width. The surface of the oocysts is not smooth but contains many buds. A ring-shaped line containing rosary-arrayed buds line up in the equatorial plane of the oocyst. These specific characteristics were observed for the first time in neogregarine oocysts from ants. Polar plugs were recognizable clearly by light and electron microscopy. The oocyst wall was quite thick, measuring 775 to 1000 nm. Each oocyst contained eight sporozoites. The neogregarines in the two Temnothorax species show many similarities such as the size and shape of the oocysts, a relatively fragile gametocyst membrane, host affinity, and tissue preference. We identified these neogregarines as Mattesia cf. geminata, which is here recorded from natural ant populations in the Old World for the first time. All neogregarine pathogens infecting ants in nature so far have been recorded from the New World. We present the two ant species, Temnothorax affinis and T. parvulus, as new natural hosts for M. cf. geminata. Furthermore, the morphological and ultrastructural characteristics of the oocyst of M. cf. geminata are documented by scanning and transmission electron microscopy for the first time.

A pilot study for the isolation of Eimeria spp. oocysts from environmental straw samples in comparison with individual faecal examination of fattening calves.

The diagnosis of eimeriosis in calves mainly relies on the presence of diarrhoea and the excretion of Eimeria oocysts in the faeces. Restraining the animals to collect rectal samples for diagnostic purposes is stressful and time-consuming. The aim of this study was to evaluate a method for the quantification of oocysts in environmental barn straw samples. To investigate the recovery rate of the method, straw and Eimeria negative faeces were spiked with Eimeria oocysts in plastic bags and mixed with water and 0.05% Tween 20 (v/v); the liquids were filtered twice through sieves (mesh size 300 and 52 µm), centrifuged and the number of oocysts in the sediment determined using a McMaster counting chamber. A recovery rate of 52.4% (95% confidence interval: 48.2-56.5%) was obtained. In the following, field straw (n = 156) and individual faecal samples (n = 195, also analysed by McMaster counting chambers) were collected on four different farms. Eimeria oocysts were present on all farms in faecal (84/195, 43.1%) and straw samples (119/156, 76.3%). In 37 (23.7%) straw samples, sporulated oocysts were observed, with a sporulation rate ranging from 0 to 40%. Despite high variability between farms and examination days, mean numbers of oocysts in the straw positively correlated with mean numbers of oocysts excreted in the faeces (ρSpearman = 0.60). The examination of environmental straw samples may represent an easy-to-perform, non-invasive, inexpensive preliminary diagnostic approach for surveillance of eimeriosis at group level, having the potential to assess the infection pressure.

Proteomic Analysis of Fractionated Eimeria tenella Sporulated Oocysts Reveals Involvement in Oocyst Wall Formation.

Eimeria tenella is the most pathogenic intracellular protozoan parasite of the Eimeria species. Eimeria oocyst wall biogenesis appears to play a central role in oocyst transmission. Proteome profiling offers insights into the mechanisms governing the molecular basis of oocyst wall formation and identifies targets for blocking parasite transmission. Tandem mass tags (TMT)-labeled quantitative proteomics was used to analyze the oocyst wall and sporocysts of E. tenella. A combined total of 2865 E. tenella proteins were identified in the oocyst wall and sporocyst fractions; among these, 401 DEPs were identified, of which 211 were upregulated and 190 were downregulated. The 211 up-regulated DEPs were involved in various biological processes, including DNA replication, fatty acid metabolism and biosynthesis, glutathione metabolism, and propanoate metabolism. Among these proteins, several are of interest for their likely role in oocyst wall formation, including two tyrosine-rich gametocyte proteins (EtGAM56, EtSWP1) and two cysteine-rich proteins (EtOWP2, EtOWP6). Concurrently, 96 uncharacterized proteins may also participate in oocyst wall formation. The present study significantly expands our knowledge of the proteome of the oocyst wall of E. tenella, thereby providing a theoretical basis for further understanding of the biosynthesis and resilience of the E. tenella oocyst wall.

Fluorescent tagging of Plasmodium circumsporozoite protein allows imaging of sporozoite formation but blocks egress from oocysts.

The circumsporozoite protein, CSP, is the major surface protein of Plasmodium sporozoites, the form of malaria parasites transmitted by mosquitoes. CSP is involved in sporozoite formation within and egress from oocysts, entry into mosquito salivary glands and mammalian liver as well as migration in the skin. Yet, how CSP facilitates sporozoite formation, oocyst egress and hepatocyte specific invasion is still not fully understood. Here, we aimed at generating a series of parasites expressing full-length versions of CSP with internally inserted green fluorescent protein between known domains at the endogenous csp locus. This enabled the investigation of sporozoite formation in living oocysts. GFP insertion after the signal peptide leads to cleavage of GFP before the fusion protein reached the plasma membrane while insertion of GFP before or after the TSR domain prevented sporozoite egress and liver invasion. These data suggest different strategies for obtaining mature salivary gland sporozoites that express GFP-CSP fusions.

NAD(P) transhydrogenase has vital non-mitochondrial functions in malaria parasite transmission.

Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form (NADP) are vital for cell function in all organisms and form cofactors to a host of enzymes in catabolic and anabolic processes. NAD(P) transhydrogenases (NTHs) catalyse hydride ion transfer between NAD(H) and NADP(H). Membrane-bound NTH isoforms reside in the cytoplasmic membrane of bacteria, and the inner membrane of mitochondria in metazoans, where they generate NADPH. Here, we show that malaria parasites encode a single membrane-bound NTH that localises to the crystalloid, an organelle required for sporozoite transmission from mosquitos to vertebrates. We demonstrate that NTH has an essential structural role in crystalloid biogenesis, whilst its enzymatic activity is required for sporozoite development. This pinpoints an essential function in sporogony to the activity of a single crystalloid protein. Its additional presence in the apicoplast of sporozoites identifies NTH as a likely supplier of NADPH for this organelle during liver infection. Our findings reveal that Plasmodium species have co-opted NTH to a variety of non-mitochondrial organelles to provide a critical source of NADPH reducing power.

Morphometric analysis of aerobic Eimeria bovis sporogony using live cell 3D holotomographic microscopy imaging.

M onoxenous Eimeria species are widespread enteropathogenic apicomplexan protozoa with a high economic impact on livestock. In cattle, tenacious oocysts shed by E. bovis-infected animals are ubiquitously found and making infection of calves almost inevitable. To become infectious oocysts, exogenous oxygen-dependent E. bovis sporogony must occur leading to the formation of sporulated oocysts containing four sporocysts each harboring two sporozoites. Investigations on sporogony by live cell imaging techniques of ruminant Eimeria species are still absent in literature as commonly used fluorescent dyes do not penetrate resistant oocyst bi-layered wall. Sporogonial oocysts were daily analyzed by a 3D Cell Explorer Nanolive microscope to explore ongoing aerobic-dependent sporogony as close as possible to an in vivo situation. Subsequently, 3D holotomographic images of sporulating E. bovis oocysts were digitally stained based on refractive indices (RI) of oocyst bi-layered wall and sub-compartments of circumplasm using STEVE software (Nanolive), and the cellular morphometric parameters were obtained. Overall, three different E. bovis sporogony phases, each of them divided into two sub-phases, were documented: (i) sporoblast/sporont transformation into sporogonial stages, (ii) cytokinesis followed by nuclear division, and finally (iii) formation of four sporocysts with two fully developed sporozoites. Approximately 60% of sporulating E. bovis oocysts accomplished aerobic sporogony in a synchronized manner. E. bovis sporogony was delayed (i.e., 6 days) when compared to an in vivo situation where 2-3 days are required but under optimal environmental conditions. Live cell 3D holotomography analysis might facilitate the evaluation of either novel disinfectants- or anti-coccidial drug-derived effects on ruminant/avian Eimeria sporogony in vitro as discrimination of sporogony degrees based on compactness, and dry mass was here successfully achieved. Main changes were observed in the oocyst area, perimeter, compactness, extent, and granularity suggesting those parameters as an efficient tool for a fast evaluation of the sporulation degree.

Functional characterization of Clonorchis sinensis sodium-bile acid co-transporter (CsSBAT) as a steroid sulfate transporter.

Clonorchis sinensis (Cs) is a common trematode in Asian countries. Infection by Cs can result in many clinical symptoms. Here, a cDNA encoding a Cs apical sodium-dependent bile acid transporter (CsSBAT) was isolated from a Cs cDNA library, and functional characterization was performed using Xenopus laevis oocyte expression system. When expressed in Xenopus laevis oocytes, CsSBAT mediated the transport of radiolabeled estrone sulfate and dehydroepiandrosterone sulfate. No trans-uptake of carnitine, estradiol 17 ß-D glucuronide, prostaglandin E2, p-aminohippuric acid, α-ketoglutaric acid, and tetraethylammonium was observed. CsSBAT-mediated estrone sulfate uptake was in a time- and sodium-dependent manner. CsSBAT showed no exchange properties in efflux experiments. Concentration-dependent results showed saturable kinetics consistent with the Michaelis-Menten equation. Nonlinear regression analyses yielded a Km value of 0.3 ± 0.04 µM for [3H]estrone sulfate. CsSBAT-mediated estrone sulfate uptake was strongly inhibited by sulfate conjugates but not glucuronide conjugates. These findings contribute to our understanding of CsSBAT transport properties and the cascade of estrogen metabolite movement in Cs.

Development of Toxoplasma gondii Chinese I genotype Wh6 Strain in Cat Intestinal Epithelial Cells.

Felids are the unique definitive host of Toxoplasma gondii. The intestine of felid is the only site for initiating Toxoplasma gondii sexual reproduction. T. gondii excretes millions of infectious oocysts from the intestine, which are the primary source of infection. There are many difficulties in developing vaccines and drugs to control oocyst excretion due to the lack of an appropriate experimental model. Here, we established an in vitro feline intestinal epithelial cell (IEC) infection system and an efficient animal model of T. gondii Chinese 1 genotype, Wh6 strain (TgCtwh6). The Kunming mice brain tissues containing TgCtwh6 cysts were harvested 42-day post-infection. The bradyzoites were co-cultured with cat IECs in vitro at a ratio of 1:10. Five 3-month-old domestic cats were orally inoculated with 600 cysts each. The oocysts were detected by daily observation of cat feces by microscopy and polymerase chain reaction. We found that the parasite adhered and invaded cat IECs in vitro, transformed into tachyzoites, and then divided to form rose-like structures. These parasites eventually destroyed host cells, escaped, and finished the asexual reproduction process. Schizonts associated with sexual reproduction have not been observed during development in vitro cultured cells. However, schizonts were detected in all infected cat intestinal epithelial cells, and oocysts were presented in all cat feces. Our study provides a feasible cell model and an efficient infection system for the following studies of T. gondii sexual reproduction, and also lays a foundation to develop drugs and vaccines for blocking excretion and transmission of oocysts.