Oocyst Sporozoites Research Articles

The Anticoccidial In Vitro Effects and Antioxidant Properties of Several Plants Traditionally Used for Coccidiosis in Togo.

Coccidiosis is a parasitic disease that often affects livestock. Identifying plants with inhibitory effects on the development of the parasite could help in finding new natural treatments. This study aimed to evaluate the anticoccidial potentials of extracts from Azadirachta indica leaves (AILs), Combretum micranthum leaves (CMLs), Carica papaya seeds (CPSs), Sarcocephalus latifolius roots (SLRs), and Vernonia amygdalina leaves (VALs). The in vitro anticoccidial efficacy of the extracts was evaluated through oocyst sporulation inhibition and sporozoite viability inhibition assays of Eimeria oocysts. The setup was examined for 72 h (every 24 h) of incubation. The DPPH radical scavenging activity and ferric reducing antioxidant power were used to evaluate the antioxidant potential of the extracts. Among the tested extracts, the SLR, CPS, and AIL extracts exhibited the maximum oocyst sporulation inhibition (75.85 ± 1.21%, 74.53 ± 1.65%, and 71.58 ± 0.24%, respectively) at a concentration of 75 mg/mL of plant extracts against the Eimeria species. The Sarcocephalus latifolius root extract showed the highest radical scavenging capacity (76.25 ± 0.53) and reducing power (86.21 ± 4.28). The biochemical screening of the selected plant extracts revealed the presence of antioxidant compounds such as phenols, flavonoids, alkaloids, saponins, and carbohydrates. The SLR extract contained the highest amounts of phenols (56.11 ± 0.33 µg/mL) and flavonoids (36.65 ± 1.85 µg/mL). In conclusion, the selected hydro-ethanolic extracts from these plants possess excellent anticoccidial and antioxidant activities, which can be attributed to the presence of medicinally important phytochemicals. Further research is needed to identify and isolate the active anticoccidial compounds from these plants, which could be utilized in the development of drugs against coccidiosis.

Revisiting the Plasmodium sporozoite inoculum and elucidating the efficiency with which malaria parasites progress through the mosquito

Malaria is initiated when infected anopheline mosquitoes inoculate sporozoites as they probe for blood. It is thought that all infected mosquitoes are equivalent in terms of their infectious potential, with parasite burden having no role in transmission success. In this study, using mosquitoes harboring the entire range of salivary gland sporozoite loads observed in the field, we demonstrate a strong and highly significant correlation between mosquito parasite burden and inoculum size. We then link the inoculum data to oocyst counts, the most commonly-used metric to assess mosquito infection in the field, and determine the efficiency with which oocyst sporozoites enter mosquito salivary glands. Taken together our data support the conclusion that mosquitoes with higher parasite burdens are more likely to initiate infection and contribute to onward transmission. Overall these data may account for some of the unexplained heterogeneity in transmission and enable more precise benchmarks for transmission-blocking interventions.

In vitro: Anti-coccidia activity ofCalotropis proceraleaf extract onEimeria papillataoocysts sporulation and sporozoite

AbstractNatural products play an important role as environmentally friendly agents that can be used against parasitic diseases. ManyEimeriaspecies cause eimeriosis in poultry. The negative effects of synthetic anti-coccidiosis medications necessitate the quest for alternative treatments derived from medicinal plants in the treatment of eimeriosis. The study was conducted to evaluate the effects ofCalotropis proceraleaf extract (CPLE) (Madar) on the sporulation ofEimeriaoocysts and sporozoites that affect mammalian jejunum and to obtain the best concentration for sporulation inhibition and infection prevention. Extracts were testedin vitroto prevent oocyst sporulation, wall deformity, and anti-sporozoite activity withEimeria papillata. The plant-chemical compounds analysis of CPLE some active compounds were shown as well as CPLEin vitroeffects at various concentrations (200, 100, 50, 25,12.5, and 6.25 mg/mL), while potassium dichromate solution 2.5% and Toltrazuril 25 mg/mL were administered as the control groups.C. proceraleaf extract showed the highest inhibitory percentage onE. papillataoocyst at 200 mg/mL of extract, approximately 91%. In addition, CPLE showed the sporozoite highest viability inhibitory percentage onE. papillataat 200 mg/mL of extract, approximately 88%, and the lowest efficacy was 5% at 6.25 mg/mL. Also, we noticed the deformation and destruction of the oocyst wall based on the concentration rate. Sporulation inhibition rate is significantly affected by incubation time and treatment concentration ratio. The results showed that Madar has an effective, inhibitory potential, and protective effect on coccidian oocyst sporulation and sporozoites ofE. papillata.

Effectiveness of Bacillus licheniformis-Fermented Products and Their Derived Antimicrobial Lipopeptides in Controlling Coccidiosis in Broilers.

Simple SummaryCoccidiosis is an important health problem in broilers, caused by infection with a highly contagious intestinal parasite of the genus Eimeria. Anti-coccidial drugs are widely used for the prevention of coccidiosis in broilers. However, multi-resistance coccidia is a potential threat to poultry production. In this study, we evaluated the potential of Bacillus licheniformis-fermented products (BLFP) and their derived antimicrobial lipopeptide, surfactin, on the prevention of coccidiosis in broilers. Results demonstrate that BLFPs and their derived antimicrobial lipopeptide, surfactin, exhibit anti-coccidial activity in vitro and in vivo.This study aimed to investigate the potential of Bacillus licheniformis-fermented products (BLFP) and their derived antimicrobial lipopeptide, surfactin, for the prevention of coccidiosis in broilers. Broilers were fed BLFP at 1.25 and 5 g/kg under Eimeria tenella challenge. At the end of experiment (35 days), the growth performance, survival rate, cecal morphology, cecal lesion scores, oocyst-count index, and anti-coccidial index were analyzed. The effects of the BLFP-derived surfactin on oocyst sporulation and sporozoite morphology in Eimeria species were also investigated in vitro. Results showed that BLFP supplementation at 1.25 and 5 g/kg improved cecal morphology and increased the survival rate of broilers under E. tenella challenge. Supplementation with 1.25 g/kg of BLFP reduced the lesion scores in the cecum of E. tenella-challenged broilers, while the oocyst-count index was reduced in broilers given 5 g/kg of BLFP. The anti-coccidial index of the 1.25 g/kg of BLFP-treated group was greater than 160, compared with the E. tenella-challenge-only group. Furthermore, surfactin inhibited Eimeria oocyst sporulation and disrupted sporozoite morphology. These results demonstrate that BLFPs and their derived antimicrobial lipopeptide, surfactin, exhibit anti-coccidial activity in vitro and in vivo. BLFP may be used as a natural feed additive for the prevention of coccidiosis in broilers, and 1.25 g/kg can be considered the optimum dosage.

Functional and Mechanistic Studies of Two Anti-coccidial Herbs, Bidens pilosa and Artemisia indica.

Currently, antibiotics are commonly used to treat coccidiosis, a severe protozoal disease in chickens. However, due to growing concerns about the antibiotic residue in meat and eggs, phytogenic formulations are becoming an attractive approach to manage this disease. In this study, we investigated the anti-coccidial function and mechanism of phytogenic formulations composed of Bidens pilosa, Artemisia indica, and both used in combination. We found that these formulations increased the survival rate and reduced body weight loss, the feed conversion ratio, oocyst excretion, bloody stools, and gut lesions of chickens. Mechanistic studies showed that A.indica, but not B. pilosa, reduced the survival of Eimeria oocysts. Accordingly, they both inhibited oocyst sporulation and sporozoite invasion into Madin-Darby bovine kidney (MDBK) cells. Overall, we demonstrate that these formulations protect chickens against coccidiosis. Moreover, a combination of B. pilosa and A.indica has an additive effect on coccidiosis control and growth performance in chickens compared to either one used alone.

In silico identification of novel open reading frames in Plasmodium falciparum oocyte and salivary gland sporozoites using proteogenomics framework

BackgroundPlasmodium falciparum causes the deadliest form of malaria, which remains one of the most prevalent infectious diseases. Unfortunately, the only licensed vaccine showed limited protection and resistance to anti-malarial drug is increasing, which can be largely attributed to the biological complexity of the parasite’s life cycle. The progression from one developmental stage to another in P. falciparum involves drastic changes in gene expressions, where its infectivity to human hosts varies greatly depending on the stage. Approaches to identify candidate genes that are responsible for the development of infectivity to human hosts typically involve differential gene expression analysis between stages. However, the detection may be limited to annotated proteins and open reading frames (ORFs) predicted using restrictive criteria.MethodsThe above problem is particularly relevant for P. falciparum; whose genome annotation is relatively incomplete given its clinical significance. In this work, systems proteogenomics approach was used to address this challenge, as it allows computational detection of unannotated, novel Open Reading Frames (nORFs), which are neglected by conventional analyses. Two pairs of transcriptome/proteome were obtained from a previous study where one was collected in the mosquito-infectious oocyst sporozoite stage, and the other in the salivary gland sporozoite stage with human infectivity. They were then re-analysed using the proteogenomics framework to identify nORFs in each stage.ResultsTranslational products of nORFs that map to antisense, intergenic, intronic, 3′ UTR and 5′ UTR regions, as well as alternative reading frames of canonical proteins were detected. Some of these nORFs also showed differential expression between the two life cycle stages studied. Their regulatory roles were explored through further bioinformatics analyses including the expression regulation on the parent reference genes, in silico structure prediction, and gene ontology term enrichment analysis.ConclusionThe identification of nORFs in P. falciparum sporozoites highlights the biological complexity of the parasite. Although the analyses are solely computational, these results provide a starting point for further experimental validation of the existence and functional roles of these nORFs,

Transcriptomics and proteomics reveal two waves of translational repression during the maturation of malaria parasite sporozoites

Plasmodium sporozoites are transmitted from infected mosquitoes to mammals, and must navigate the host skin and vasculature to infect the liver. This journey requires distinct proteomes. Here, we report the dynamic transcriptomes and proteomes of both oocyst sporozoites and salivary gland sporozoites in both rodent-infectious Plasmodium yoelii parasites and human-infectious Plasmodium falciparum parasites. The data robustly define mRNAs and proteins that are upregulated in oocyst sporozoites (UOS) or upregulated in infectious sporozoites (UIS) within the salivary glands, including many that are essential for sporozoite functions in the vector and host. Moreover, we find that malaria parasites use two overlapping, extensive, and independent programs of translational repression across sporozoite maturation to temporally regulate protein expression. Together with gene-specific validation experiments, these data indicate that two waves of translational repression are implemented and relieved at different times during sporozoite maturation, migration and infection, thus promoting their successful development and vector-to-host transition.

Plasmodium berghei sporozoite specific genes- PbS10 and PbS23/SSP3 are required for the development of exo-erythrocytic forms

Plasmodium sporozoites are infective forms of the parasite to mammalian hepatocytes. Sporozoite surface or secreted proteins likely play an important role in recognition, invasion and successful establishment of hepatocyte infection. By approaches of reverse genetics, we report the functional analysis of two Plasmodium berghei (Pb) sporozoite specific genes- PbS10 and PbS23/SSP3 that encode for proteins with a putative signal peptide. The expression of both genes was high in oocyst and salivary gland sporozoite stages as compared to other life cycle stages and PbS23/SSP3 protein was detected in salivary gland sporozoites. Both mutants were indistinguishable to wild-type parasites with regard to asexual growth in RBC, ability to complete sexual reproduction and form sporozoites in vector host. While the sporozoite stage of both mutants were able to glide and invade hepatocytes normally in vitro and in vivo, PbS10 mutants suffered growth attenuation at an early stage while PbS23/SSP3 mutants manifested defect during late exo-erythrocytic form maturation. Interestingly, both mutants gave rare breakthrough infections, suggesting that while both were critical for liver stage development, their depletion did not completely abrogate blood stage infection. These findings have important implications for weakening sporozoites by multiple gene attenuation towards the generation of a safe whole organism vaccine.

Molecular characterization and expression profile of an alternate proliferating cell nuclear antigen homolog PbPCNA2 in Plasmodium berghei.

Proliferative cell nuclear antigen (PCNA) is the processivity factor for various DNA polymerases and it functions in response to DNA damage in eukaryotic system. Plasmodium falciparum contains two PCNAs, while PCNA1 has been attributed to DNA replication, the role of PCNA2 has been assigned to DNA damage response in erythrocytic developmental stages. Although a recent transposon mediated knockout strategy qualified pcna2 as a nonessential gene in Plasmodium berghei, a conventional homologous recombination-based knockout strategy has not been employed for this gene yet. Moreover, the cellular dynamics of PCNA2 in extraerythrocytic stages still remain elusive in Plasmodium. We attempted multiple times to knock out PbPCNA2 from the parasite genome using homologous recombination strategy without much success. However, we were able to generate PbPCNA2-GFP tagged transgenic parasites confirming that the pcna2 locus is amenable to genetic manipulation. The GFP-tagged parasites showed similar growth phenotype, compared to wild-type parasites, in both erythrocytic and sporogonic cycle, suggesting that tagging had no effect on parasite physiology. PbPCNA2 expression was also observed during the sporogonic cycle in midgut oocyst and salivary gland sporozoites. The PbPCNA2 expression was upregulated in the presence of DNA damaging agents like hydroxyurea and methyl methanesulphonate. Our inability to knock out PCNA2 suggested its essentiality in the parasite development and elevated expression during DNA damaging condition hint at a critical role of the protein in parasite physiology. © 2019 IUBMB Life, 71(9):1293-1301, 2019.

Anti-coccidial properties and mechanisms of an edible herb, Bidens pilosa, and its active compounds for coccidiosis

Avian coccidiosis is an economically important disease in the poultry industry. In view of the disadvantages of anti-coccidial drugs in chickens, edible plants and their compounds are re-emerging as an alternative strategy to combat this disease. A previous publication reported that the edible plant B. pilosa showed promise for use against coccidiosis. Here, we first investigated into the anti-coccidial effects of B. pilosa. We found that B. pilosa at 100 ppm or more significantly suppressed E. tenella as evidenced by reduction in mortality rate, oocyst excretion and gut pathological severity in chickens and its minimum prophylactic duration was 3 days. Next, we explored the mode of action of anti-coccidial mechanism of B. pilosa. The E. tenella oocysts were not directly killed by B. pilosa; however, administration of the plant suppressed oocyst sporulation, sporozoite invasion, and schizonts in the life cycle of E. tenella. Besides, B. pilosa boosted T cell-mediated immunity. Finally, we characterized the related anti-coccidial phytochemicals and their mode of action. One of three potent polyynes present in B. pilsoa, Compound 1 (cytopiloyne), acted against coccidiosis in chickens in a similar manner to B. pilosa. These data illustrate the anti-coccidial potency and mechanism of B. pilosa and one of its active compounds, and provide a cornerstone for development of novel herbal remedies for avian coccidiosis.

T Lymphocytes Response to Caecal Coccidiosis in Broilers Infected with Exo and Endogenous Stages of Eimeria Tenella

The present study reveals the proliferation of cytokines in treated and non- treated broilers consisting of IFN- γ, IL-1, IL-2, IL-4, IL-6, TNF and TGF. The CD4 count in the treated and non- treated broilers orally administered with various developmental stages of the parasite reached a peak on day 10 at primary; secondary and day 24 at tertiary infections. There was significant difference in the CD4 cell count between groups of the infected broiler chickens (p < 0.05). The current study observed a relationship between the different developmental stages of the parasite and lymphocytes response. Broiler chickens infected with sporulated oocyst (sporozoites) and merozoites treated and non-treated gave high CD4 T- lymphocyte numbers than the other groups throughout the experimental periods.

Immune Regulation of Plasmodium Is Anopheles Species Specific and Infection Intensity Dependent.

ABSTRACTMalaria parasite ookinetes must traverse the vector mosquito midgut epithelium to transform into sporozoite-producing oocysts. The Anopheles innate immune system is a key regulator of this process, thereby determining vector competence and disease transmission. The role of Anopheles innate immunity factors as agonists or antagonists of malaria parasite infection has been previously determined using specific single Anopheles-Plasmodium species combinations. Here we show that the two C-type lectins CTL4 and CTLMA2 exert differential agonistic and antagonistic regulation of parasite killing in African and South American Anopheles species. The C-type lectins regulate both parasite melanization and lysis through independent mechanisms, and their implication in parasite melanization is dependent on infection intensity rather than mosquito-parasite species combination. We show that the leucine-rich repeat protein LRIM1 acts as an antagonist on the development of Plasmodium ookinetes and as a regulator of oocyst size and sporozoite production in the South American mosquito Anopheles albimanus. Our findings explain the rare observation of human Plasmodium falciparum melanization and define a key factor mediating the poor vector competence of Anopheles albimanus for Plasmodium berghei and Plasmodium falciparum.

Fe-S] cluster assembly in the apicoplast and its indispensability in mosquito stages of the malaria parasite.

The relict plastid (apicoplast) of the malaria parasite is the site for important biochemical pathways and is essential for parasite survival. The sulfur mobilization (SUF) pathway of iron-sulfur [Fe-S] cluster assembly in the apicoplast of Plasmodium spp. is of interest due to its absence in the human host suggesting the possibility of antimalarial intervention through apicoplast [Fe-S] biogenesis. We report biochemical characterization of components of the Plasmodium falciparum apicoplast SUF pathway after the first step of SUF. In vitro interaction experiments and in vivo cross-linking showed that apicoplast-encoded PfSufB and apicoplast-targeted PfSufC and PfSufD formed a complex. The PfSufB-C2 -D complex could function as a scaffold to assemble [4Fe-4S] clusters in vitro and activity of the PfSufC ATPase was enhanced by PfSufD. Two carrier proteins, the NifU-like protein PfNfu and the A-type carrier PfSufA are homodimers, the former mediating transfer of [4Fe-4S] from the scaffold to a model [4Fe-4S] target protein with higher efficiency. Conditional knockout of SufS, the enzyme catalyzing the first step of SUF, by selective excision in the mosquito stages of Plasmodium berghei severely impaired development of sporozoites in oocysts establishing essentiality of the SUF machinery in the vector. Our results delineate steps of the complete apicoplast SUF pathway and demonstrate its critical role in the parasite life cycle.

Plasmodium AdoMetDC/ODC bifunctional enzyme is essential for male sexual stage development and mosquito transmission.

ABSTRACTPolyamines are positively-charged organic molecules that are important for cellular growth and division. Polyamines and their synthesizing enzymes are particularly abundant in rapidly proliferating eukaryotic cells such as parasitic protozoa and cancer cells. Polyamine biosynthesis inhibitors, such as Elfornithine, are now being considered for cancer prevention and have been used effectively against Trypanosoma brucei. Inhibitors of polyamine biosynthesis have caused growth arrest of Plasmodium falciparum blood stages in vitro, but in P. berghei only partial inhibition has been observed. While polyamine biosynthesis enzymes are characterized and conserved in Plasmodium spp., little is known on the biological roles of these enzymes inside malaria parasite hosts. The bifunctional polyamine biosynthesis enzyme S-adenosyl methionine decarboxylase/ornithine decarboxylase (AdoMetDC/ODC) was targeted for deletion in P. yoelii. Deletion of AdoMetDC/ODC significantly reduced blood stage parasitemia but Anopheles transmission was completely blocked. We showed that male gametocytogenesis and male gamete exflagellation were abolished and consequently no ookinetes or oocyst sporozoites could be generated from adometdc/odc(–) parasites. Supplementation of putrescine and spermidine did not rescue the defective phenotypes of male gametocytes and gametes of the knockout parasites. These results highlight the crucial role of polyamine homeostasis in the development and functions of Plasmodium erythrocytic stages in the blood and in the mosquito vector and validate polyamine biosynthesis pathway enzymes as drug targeting candidates for malaria parasite transmission blocking.

Vital and dispensable roles of Plasmodium multidrug resistance transporters during blood- and mosquito-stage development.

Multidrug resistance (MDR) proteins belong to the B subfamily of the ATP Binding Cassette (ABC) transporters, which export a wide range of compounds including pharmaceuticals. In this study, we used reverse genetics to study the role of all seven Plasmodium MDR proteins during the life cycle of malaria parasites. Four P. berghei genes (encoding MDR1, 4, 6 and 7) were refractory to deletion, indicating a vital role during blood stage multiplication and validating them as potential targets for antimalarial drugs. Mutants lacking expression of MDR2, MDR3 and MDR5 were generated in both P. berghei and P. falciparum, indicating a dispensable role for blood stage development. Whereas P. berghei mutants lacking MDR3 and MDR5 had a reduced blood stage multiplication in vivo, blood stage growth of P. falciparum mutants in vitro was not significantly different. Oocyst maturation and sporozoite formation in Plasmodium mutants lacking MDR2 or MDR5 was reduced. Sporozoites of these P. berghei mutants were capable of infecting mice and life cycle completion, indicating the absence of vital roles during liver stage development. Our results demonstrate vital and dispensable roles of MDR proteins during blood stages and an important function in sporogony for MDR2 and MDR5 in both Plasmodium species.

Effects of Developmental Temperature on Gametocysts and Oocysts of Two Species of Gregarines Blabericola migrator and Blabericola cubensis (Apicomplexa: Eugregarinida: Blabericolidae) Parasitizing Blaberid Cockroaches (Dictyoptera: Blaberidae).

Abiotic environmental conditions, especially temperature and humidity, have profound effects on the growth and development of gregarines, but these effects remain largely undocumented. Quantifying the effects of environmental conditions on the growth and development of exogenous gregarine ontogenetic stages is an important first step in understanding the transmission, population dynamics, and environmental persistence of gregarine infection. In this study, we examined the effect of 6 environmental temperatures (10, 18, 22, 27, 35, and 40 C) at constant humidity (0 mmHg vapor pressure deficit) on gametocyst development and oocyst viability in 2 gregarine species: Blabericola migrator and Blabericola cubensis parasitizing the Tiger-striped Hissing Cockroach, Princisia vanwaerebecki, and the Discoid Cockroach, Blaberus discoidalis, respectively. Temperature has a significant effect on gametocyst development and oocyst viability for both gregarine species. Gametocyst development for both gregarine species displays a similar threshold response to environmental temperature: 10 and 40 C represent extremes outside their developmental range, but within these extremes, the relationship between gametocyst development and temperature is weakly direct. Dehiscence increased with temperature from 68% at 18 C to 93% at 22 C and remained at that level through 35 C. Developmental temperature also has a meaningful but inverse effect on oocyst viability of both B. migrator and B. cubensis. For both species, oocyst viability is highest at 18 and 22 C and is significantly reduced at 27 and 35 C. Thus oocyst production and sporozoite viability are linked but environmentally independent phenomena. Overall, there is an acceptable developmental temperature zone for B. migrator and B. cubensis that ranges from 18 to 27 C, but production of viable sporozoites is greatest in a relatively narrow zone around 22 C. Prior studies have postulated that mechanisms that concentrate oocysts and hosts, such as host behavior or host microhabitat preference, increase the host-oocyst encounter rate and thus transmission. This study indicates that abiotic influences on gametocyst development may also lead to heterogeneous oocyst distributions in the environment and increase the likelihood of host-oocyst encounters.

SSP3 is a novel Plasmodium yoelii sporozoite surface protein with a role in gliding motility.

Plasmodium sporozoites develop within oocysts in the mosquito midgut wall and then migrate to the salivary glands. After transmission, they embark on a complex journey to the mammalian liver, where they infect hepatocytes. Proteins on the sporozoite surface likely mediate multiple steps of this journey, yet only a few sporozoite surface proteins have been described. Here, we characterize a novel, conserved sporozoite surface protein (SSP3) in the rodent malaria parasite Plasmodium yoelii. SSP3 is a putative type I transmembrane protein unique to Plasmodium. By using epitope tagging and SSP3-specific antibodies in conjunction with immunofluorescence microscopy, we showed that SSP3 is expressed in mosquito midgut oocyst sporozoites, exhibiting an intracellular localization. In sporozoites derived from the mosquito salivary glands, however, SSP3 localized predominantly to the sporozoite surface as determined by immunoelectron microscopy. However, the ectodomain of SSP3 appeared to be inaccessible to antibodies in nonpermeabilized salivary gland sporozoites. Antibody-induced shedding of the major surface protein circumsporozoite protein (CSP) exposed the SSP3 ectodomain to antibodies in some sporozoites. Targeted deletion of SSP3 adversely affected in vitro sporozoite gliding motility, which, surprisingly, impacted neither their cell traversal capacity, host cell invasion in vitro, nor infectivity in vivo. Together, these data reveal a previously unappreciated complexity of the Plasmodium sporozoite surface proteome and the roles of surface proteins in distinct biological activities of sporozoites.

Plasmodium yoelii vitamin B5 pantothenate transporter candidate is essential for parasite transmission to the mosquito.

In nearly all non-photosynthetic cells, pantothenate (vitamin B5) transport and utilization are prerequisites for the synthesis of the universal essential cofactor Coenzyme A (CoA). Early studies showed that human malaria parasites rely on the uptake of pantothenate across the parasite plasma membrane for survival within erythrocytes. Recently, a P. falciparum candidate pantothenate transporter (PAT) was characterized by functional complementation in yeast. These studies revealed that PfPAT mediated survival of yeast cells in low pantothenate concentrations and restored sensitivity of yeast cells lacking pantothenate uptake to fenpropimorph. In addition, PfPAT was refractory to deletion in P. falciparum in vitro, but nothing is known about the in vivo functions of PAT in Plasmodium life cycle stages. Herein, we used gene-targeting techniques to delete PAT in Plasmodium yoelii. Parasites lacking PAT displayed normal asexual and sexual blood stage development compared to wild-type (WT) and WT-like p230p(-) parasites. However, progression from the ookinete to the oocyst stage and sporozoite formation were completely abolished in pat(-) parasites. These studies provide the first evidence for an essential role of a candidate pantothenate transport in malaria transmission to Anopheles mosquitoes. This will set the stage for the development of PAT inhibitors against multiple parasite life cycle stages.

Transstadial transmission of Hepatozoon canis from larvae to nymphs of Rhipicephalus sanguineus

Hepatozoon canis is an apicomplexan parasite of dogs, which is known to become infected by ingesting Rhipicephalus sanguineus adult ticks. To investigate the possibility of H. canis transovarial and transstadial transmission from larvae to nymphs, engorged adult female ticks were collected from a private animal shelter in southern Italy, where H. canis infection is highly prevalent. Female ticks (n=35) and egg batches were tested by PCR for H. canis. All eggs examined were PCR-negative whereas 88.6% of females from the environment tested positive. Additionally, fed larvae (n=120) from a dog naturally infected by H. canis were dissected at different time points post collection (i.e. 0, 10, 20 and 30 days). Molted nymphs dissected at 20 days post collection revealed immature oocysts displaying an amorphous central structure in 50% of the specimens, and oocysts containing sporocysts with sporozoites were found in 53.3% of the nymphs dissected at 30 days post collection. This study demonstrates that H. canis is not transmitted transovarially, but it is transmitted transstadially from larvae to nymphs of R. sanguineus and develops sporozoites in oocysts that may infect dogs.

Expression profile of the Plasmodium falciparum intra-erythrocytic stage protein, PF3D7_1363700

BackgroundEfforts to control malaria are demanding due to drug-resistant parasites, insecticide-resistant mosquitoes and poor health infrastructure in malaria-endemic countries. Therefore, the research and development of additional malaria control methods are crucial. For host-parasite interactions, surface antigens and secreted proteins are likely to be involved in infectivity and invasion of host tissues and therefore can be effective targets for control by vaccines, drug therapy, or novel mosquito control methods. In an effort to identify and characterize genes that may have a role in host-parasite interaction, this study describes the expression profile of Plasmodium falciparum PF3D7_1363700.MethodsA P. falciparum gene, PF3D7_1363700, was identified by a search of the annotated Plasmodium genome database. Protein alignments of PF3D7_1363700 orthologues from various Plasmodium species were performed to demonstrate protein similarity. Transcript expression profiles of PF3D7_1363700 were determined via reverse-transcriptase PCR and protein expression was investigated by immunofluorescence assays, western blot analysis and green fluorescent trafficking studies.ResultsThe PF3D7_1363700 protein demonstrates significant similarity with orthologues in other Plasmodium species and appears to be unique to Apicomplexans. The PF3D7_1363700 transcription profile demonstrated expression during the intra-erythrocytic, oocyst sporozoite, and salivary gland sporozoite stages while the PF3D7_1363700 protein was only detected during the intra-erythrocytic stages.ConclusionsThis research utilized an in silico approach to identify a well-conserved protein known as PF3D7_1363700. By molecular, biochemical and cellular analyses, PF3D7_1363700 was discovered to be an intra-erythrocytic-specific stage protein that is unique to Apicomplexans.