Mature Schizonts Research Articles

Plasmepsin X activates the PCRCR complex of Plasmodium falciparum by processing PfRh5 for erythrocyte invasion

Plasmodium falciparum causes the most severe form of malaria in humans. The protozoan parasite develops within erythrocytes to mature schizonts, that contain more than 16 merozoites, which egress and invade fresh erythrocytes. The aspartic protease plasmepsin X (PMX), processes proteins and proteases essential for merozoite egress from the schizont and invasion of the host erythrocyte, including the leading vaccine candidate PfRh5. PfRh5 is anchored to the merozoite surface through a 5-membered complex (PCRCR), consisting of Plasmodium thrombospondin-related apical merozoite protein, cysteine-rich small secreted protein, Rh5-interacting protein and cysteine-rich protective antigen. Here, we show that PCRCR is processed by PMX in micronemes to remove the N-terminal prodomain of PhRh5 and this activates the function of the complex unmasking a form that can bind basigin on the erythrocyte membrane and mediate merozoite invasion. The ability to activate PCRCR at a specific time in merozoite invasion most likely masks potential deleterious effects of its function until they are required. These results provide an important understanding of the essential role of PMX and the fine regulation of PCRCR function in P. falciparum biology.

Dynamics of parasite growth in genetically diverse Plasmodium falciparum isolates

Multiple parasite lineages with different proliferation rates or fitness may coexist within a clinical malaria isolate, resulting in complex growth interactions and variations in phenotype. To elucidate the dynamics of parasite growth in multiclonal isolates, we measured growth rates (GRs) of three Plasmodium falciparum Cambodian isolates, including IPC_3445 (MRA-1236), IPC_5202 (MRA-1240), IPC_6403 (MRA-1285), and parasite lineages previously cloned from each of these isolates by limiting dilution. Following synchronization, in vitro cultures of each parasite line were maintained over four consecutive asexual cycles (192 h), with thin smears prepared at each 48-h cycle to estimate GR and fold change in parasitemia (FCP). Cell cycle time (CCT), the duration it takes for ring-stage parasites to develop into mature schizonts, was measured by monitoring the development of 0–3-h post-invasion rings for up to 52 h post-incubation. Laboratory lines 3D7 (MRA-102) and Dd2 (MRA-150) were used as controls. Significant differences in GR, FCP, and CCT were observed between parasite isolates and clonal lineages from each isolate. The parasite lines studied here have well-defined growth phenotypes and will facilitate basic malaria research and development of novel malaria interventions. These lines are available to malaria researchers through the MR4 collection of NIAID’s BEI Resources Program.

In vitro production of infectious Plasmodium falciparum sporozoites.

An effective vaccine is needed for the prevention and elimination of malaria. The only immunogens that have been shown to have a protective efficacy of more than 90% against human malaria are Plasmodium falciparum (Pf) sporozoites (PfSPZ) manufactured in mosquitoes (mPfSPZ)1-7. The ability to produce PfSPZ in vitro (iPfSPZ) without mosquitoes would substantially enhance the production of PfSPZ vaccines and mosquito-stage malaria research, but this ability is lacking. Here we reportthe production of hundreds of millions of iPfSPZ. iPfSPZ invaded human hepatocytes in culture and developed to mature liver-stage schizonts expressing P. falciparum merozoite surface protein 1 (PfMSP1) in numbers comparable to mPfSPZ. When injected into FRGhuHep mice containing humanized livers, iPfSPZ invaded the human hepatocytes and developed to PfMSP1-expressing late liver stage parasites at 45% the quantity ofcryopreserved mPfSPZ. Human blood from FRGhuHep mice infected with iPfSPZ produced asexual and sexual erythrocytic-stage parasites in culture, and gametocytes developed to PfSPZ when fed to mosquitoes, completing the P.falciparum life cycle from infectiousgametocyte to infectiousgametocyte without mosquitoes or primates.

Characterization of the dynamics of Plasmodium falciparum deoxynucleotide-triphosphate pool in a stage-specific manner

Understanding and characterizing the molecular background of the maintenance of genomic integrity might be a major factor in comprehending the exceptional ability of the malaria parasite, Plasmodium falciparum to adapt at a fast pace to antimalarials. A balanced nucleotide pool is an essential factor for high-fidelity replication. The lack of detailed studies on deoxynucleotide-triphosphate (dNTP) pools in various intraerythrocytic stages of Plasmodium falciparum motivated our present study. Here, we focused on the building blocks of DNA and utilized an EvaGreen-based dNTP incorporation assay to successfully measure the temporal dynamics of dNTPs in every intraerythrocytic stage and in drug-treated trophozoites. Our findings show that the ratio of dNTPs in the ring-stage parasites significantly differs from the more mature trophozoite and schizont stages. We were also able to detect dGTP levels that have never been shown before and found it to be the least abundant dNTP in all stages. Treatment with WR99210, a TS-DHFR inhibitor drug, affected not only dTTP, but also dGTP levels, despite its presumed selective action on pyrimidine biosynthesis. Results from our studies might assist in a better understanding of genome integrity mechanisms and may potentially lead to novel drug related aspects involving purine and pyrimidine metabolic targets.

Inhibitor of Cysteine Protease of Plasmodium malariae Regulates Malapains, Endogenous Cysteine Proteases of the Parasite.

Cysteine proteases of malaria parasites have been recognized as potential targets in antimalarial drug development as they play pivotal roles in the biology of these parasites. However, strict regulation of their activities is also necessary to minimize or prevent deleterious damage to the parasite and the host. Previously, we have characterized falcipain family cysteine proteases of Plasmodium malariae, named as malapains (MPs). MPs are active hemoglobinases. They also may participate in the release of merozoites from mature schizonts by facilitating remodeling of erythrocyte skeleton proteins. In this study, we identified and characterized an endogenous inhibitor of cysteine protease of P. malariae (PmICP). PmICP shared similar structural and biochemical properties with ICPs from other Plasmodium species. Recombinant PmICP showed a broad range of inhibitory activities against diverse cysteine proteases such as falcipain family enzymes (MP-2, MP-4, VX-3, VX-4, and FP-3), papain, and human cathepsins B and L, with stronger inhibitory activities against falcipain family enzymes. The inhibitory activity of PmICP was not affected by pH. PmICP was thermo-labile, resulting in rapid loss of its inhibitory activity at a high temperature. PmICP effectively inhibited hemoglobin hydrolysis by MPs and regulated maturation of MPs, suggesting its role as a functional regulator of MPs.

The Putative Bromodomain Protein PfBDP7 of the Human Malaria Parasite Plasmodium Falciparum Cooperates With PfBDP1 in the Silencing of Variant Surface Antigen Expression.

Epigenetic regulation is a critical mechanism in controlling virulence, differentiation, and survival of the human malaria parasite Plasmodium (P.) falciparum. Bromodomain proteins contribute to this process by binding to acetylated lysine residues of histones and thereby targeting the gene regulatory machinery to gene promoters. A protein complex containing the P. falciparum bromodomain proteins (PfBDP) 1 and PfBDP2 (BDP1/BDP2 core complex) was previously shown to play an essential role for the correct transcription of invasion related genes. Here, we performed a functional characterization of a third component of this complex, which we dubbed PfBDP7, because structural modelling predicted a typical bromodomain fold. We confirmed that PfBDP7 is a nuclear protein that interacts with PfBDP1 at invasion gene promoters in mature schizont stage parasites and contributes to their transcription. Although partial depletion of PfBDP7 showed no significant effect on parasite viability, conditional knock down of either PfBDP7 or PfBDP1 resulted in the de-repression of variant surface antigens (VSA), which are important pathogenicity factors. This de-repression was evident both on mRNA and protein level. To understand the underlying mechanism, we mapped the genome wide binding sites of PfBDP7 by ChIPseq and showed that in early schizonts, PfBDP7 and PfBDP1 are commonly enriched in heterochromatic regions across the gene body of all VSA families, including genes coding for PfEMP1, RIFIN, STEVOR, and PfMC-2TM. This suggests that PfBDP7 and PfBDP1 contribute to the silencing of VSAs by associating with heterochromatin. In conclusion, we identified PfBDP7 as a chromatin binding protein that is a constitutive part of the P. falciparum BDP1/BDP2 core complex and established PfBDP1 and PfBDP7 as novel players in the silencing of heterochromatin regulated virulence gene families of the malaria parasite P. falciparum.

Synchronisation of Plasmodium falciparum and P. knowlesi In Vitro Cultures Using a Highly Specific Protein Kinase Inhibitor.

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

A Fast-Track Phenotypic Characterization of Plasmodium falciparum Vaccine Antigens through Lyse-Reseal Erythrocytes Mediated Delivery (LyRED) of RNA Interference for Targeted Translational Repression.

The minimal success of the malaria vaccine with available antigens indicates the need for intensive and accelerated research to identify and characterize new antigens that confer protection against infection, clinical manifestation, and even malaria transmission. Further, the genetic manipulation tools to characterize such antigens are very time-consuming and laborious due to the very low efficiency of transfection in the malaria parasite. Here, we report a human miRNA-mediated translational repression of antigens in Plasmodium falciparum as a fast-track method for understanding and validating their function. In this method, candidate miRNAs are designed based on favorable hybridization energy against a parasite gene, and miRNA mimics are delivered to the parasite by loading them as cargo in the erythrocytes by simple lyse-reseal method. Incubation of the miRNA loaded erythrocytes with purified mature trophozoites or schizonts results in the loaded erythrocytes' infection. The miRNA mimics are translocated to parasites, and the effect of miRNA-mediated translation repression can be monitored within 48-72h post-invasion. Unlike other transfection based methods, this method is fast, reproducible, and robust. We call this method as lyse-reseal erythrocytes for delivery (LyRED) of miRNA, which is a rapid and straight-forward method providing an efficient alternative to the existing genetic tools for P. falciparum to characterize the function of antigens or genes. The identification of crucial antigens from the different stages of the Plasmodium falciparum life cycle by the miRNA targeting approach can fuel the development of efficacious subunit vaccines against malaria.

Imported Malaria: A <i>Plasmodium falciparum</i> Malaria Case in Turkey with Rarely Seen Blood Stages of Infection

Malaria is an important public health problem, with about 219 million cases, which account for 435 thousand deaths of children in Africa in 2017. The infection is transmitted by female anopheles mosquito, including Plasmodium species pathogen for human. In Turkey, no domestic cases have been reported; however, according to Annual Health Statistics, 214 imported cases were reported in 2017. Although P. vivax exactly was reported in previous years, imported Plasmodium falciparum (P. falciparum) cases have increased in the last few years. Herein, we report a case of a 41-year-old male patient who returned two weeks ago from a one-week African journey. A limited number of different blood stages of the parasite (mature trophozoite, young and mature schizont) have been seen in the examination of the peripheral smear; however, the gametocyte stage was not seen. In the present case, detecting mature stages beside the early forms at the peripheral smear could not eliminate the probability of P. falciparum. To confirm the diagnosis and regulate the treatment protocol, molecular methods were employed to differentiate the potential mixture of infection. In this case study, we propose how to approach an uncertain case of severe falciparum malaria or mixture of malaria infection combined with another Plasmodium species, as a result of limited number of different blood stages of the infection at the peripheral smear.

Genome-Wide Expression Patterns of Rhoptry Kinases during the Eimeria tenella Life-Cycle.

Kinome from apicomplexan parasites is composed of eukaryotic protein kinases and Apicomplexa specific kinases, such as rhoptry kinases (ROPK). Ropk is a gene family that is known to play important roles in host–pathogen interaction in Toxoplasma gondii but is still poorly described in Eimeria tenella, the parasite responsible for avian coccidiosis worldwide. In the E. tenella genome, 28 ropk genes are predicted and could be classified as active (n = 7), inactive (incomplete catalytic triad, n = 12), and non-canonical kinases (active kinase with a modified catalytic triad, n = 9). We characterized the ropk gene expression patterns by real-time quantitative RT-PCR, normalized by parasite housekeeping genes, during the E. tenella life-cycle. Analyzed stages were: non-sporulated oocysts, sporulated oocysts, extracellular and intracellular sporozoites, immature and mature schizonts I, first- and second-generation merozoites, and gametes. Transcription of all those predicted ropk was confirmed. The mean intensity of transcription was higher in extracellular stages and 7–9 ropk were specifically transcribed in merozoites in comparison with sporozoites. Transcriptional profiles of intracellular stages were closely related to each other, suggesting a probable common role of ROPKs in hijacking signaling pathways and immune responses in infected cells. These results provide a solid basis for future functional analysis of ROPK from E. tenella.

Identification and Assessment of Plasmodium berghei Merozoites and Cell Cycle by Flow Cytometry.

The asexual blood stages of the Plasmodium berghei life cycle including merozoites are attractive targets for transmission blocking vaccines and drugs. Improved understanding of P. berghei life cycle stage growth and development would provide new opportunities to evaluate antimalarial vaccines and drugs. Blood stage samples from C57BL/6 albino mice infected with P. berghei sporozoites were singly stained with a high binding affinity deoxyribonucleic acid dye, YOYO-1, and measured by flow cytometry (FCM). Duplicate slides were made from samples and stained with diluted Giemsa's and YOYO-1, respectively. Correlated results were compared by FCM, light microscopy, and fluorescent microscopy. Complete life cycle stage determination and analysis by FCM is reported to include merozoites, ring forms, trophozoites, immature, and mature schizonts. FCM demonstrated a clear separation between each stage using their unique fluorescence distribution. When compared to light microscopy, a strong correlation (r 2 = 0.925 to 0.974) was observed in determining the ring forms, trophozoites, and schizonts phases, but only a moderate correlation (r 2 = 0.684 to 0.778) was observed for merozoites. The identification and measurement of merozoites suggest that FCM is a useful technique to monitor the entire life stage of the parasite. Initial stage-specific data demonstrated that mefloquine has a mode of action on mature parasite forms, and artesunic acid was rapidly effective against merozoites and other immature and mature parasite forms with higher killing. Blood stage parasites in each individual life stage, including merozoites, are reliably identified and quantified quickly by FCM, making this technique an ideal alternative to microscopy. This integrated whole life stage model, particularly with confirmed determination of merozoite population, could widely be used for drug and vaccine research in malaria therapy and prophylaxis.

Eimeria tenella Translation Initiation Factor eIF-5A That Interacts With Calcium-Dependent Protein Kinase 4 Is Involved in Host Cell Invasion.

Eimeria tenella is an apicomplexan, parasitic protozoan known to infect poultry worldwide. An important calcium-dependent protein kinase (CDPK) has been identified in plants, green algae, ciliates and apicomplexan, such as E. tenella. CDPKs are effector molecules involved in calcium signaling pathways, which control important physiological processes such as gliding motility, reproduction, and host cell invasion. Given that CDPKs are not found in the host, studying the functions of CDPKs in E. tenella may serve as a basis for developing new therapeutic drugs and vaccines. To assess the function of CDPK4 in E. tenella (EtCDPK4), a putative interactor, translation initiation factor eIF-5A (EteIF-5A), was screened by both co-immunoprecipitation (co-IP) and His pull-down assays followed by mass spectrometry. The interaction between EteIF-5A and EtCDPK4 was determined by bimolecular fluorescence complementation (BiFC), GST pull-down, and co-IP. The molecular characteristics of EteIF-5A were then analyzed. Quantitative real-time polymerase chain reaction and western blotting were used to determine the transcription and protein levels of EteIF-5A in the different developmental stages of E. tenella. The results showed that the transcription level of EteIF-5A mRNA was highest in second-generation merozoites, and the protein expression level was highest in unsporulated oocysts. Indirect immunofluorescence showed that the EteIF-5A protein was found throughout the cytoplasm of sporozoites, but not in the refractile body. As the invasion of DF-1 cells progressed, EteIF-5A fluorescence intensity increased in trophozoites, decreased in immature schizonts, and increased in mature schizonts. The secretion assay results, analyzed by western blotting, indicated that EteIF-5A was a secreted protein but not from micronemes. The results of invasion inhibition assays showed that rabbit anti-rEteIF-5A polyclonal antibodies effectively inhibited cell invasion by sporozoites, with an inhibition rate of 48%.

Comprehensive Analysis of Transcript and Protein Relative Abundance During Blood Stages of Plasmodium falciparum Infection.

Plasmodium falciparum is the main causative agent of human malaria. During the intraerythrocytic development cycle, the P. falciparum morphology changes dramatically from circulating young rings to sequestered mature trophozoites and schizonts. Sequestered forms contribute to the pathophysiology of severe malaria as the infected erythrocytes obstruct the microvascular flow in deep organs and induce local inflammation. However, the sequestration mechanism limits the access to the corresponding parasitic form in the clinical samples from patients infected with P. falciparum. To complement this deficiency, we aimed to evaluate the relevance of mRNA study as a proxy of protein expression in sequestered parasites. To do so, we conducted a proteotranscriptomic analysis using five independent P. falciparum laboratory strain samples. RNA sequencing was performed, and the mRNA expression level was assessed on circulating ring-stage parasites. The level of protein expression were measured by LC-MS/MS on the corresponding sequestered mature forms after 18-24 h of maturation. Overall, our results showed a strong transcriptome/transcriptome and a very strong proteome/proteome correlation between samples. Moreover, positive correlations of mRNA and protein expression levels were found between ring-stage transcriptomes and mature form proteomes. However, twice more transcripts were identified at the ring stage than proteins at the mature trophozoite stage. A high level of transcript expression did not guarantee the detection of the corresponding protein. Finally, we pointed out discrepancies at the individual gene level. Taken together, our results show that transcript and protein expressions are overall correlated. However, mRNA abundance is not a perfect proxy of protein expression at the individual level. Importantly, our study shows limitations of the "blind" use of RNA-seq and the importance of multiomics approaches for P. falciparum blood stage study in clinical samples.

A Chimeric Plasmodium vivax Merozoite Surface Protein Antibody Recognizes and Blocks Erythrocytic P. cynomolgi Berok Merozoites In Vitro.

Research on erythrocytic Plasmodium vivax merozoite antigens is critical for identifying potential vaccine candidates in reducing P. vivax disease. However, many P. vivax studies are constrained by its inability to undergo long-term culture in vitro Conserved across all Plasmodium spp., merozoite surface proteins are essential for invasion into erythrocytes and highly expressed on erythrocytic merozoites, thus making it an ideal vaccine candidate. In clinical trials, the P. vivax merozoite surface protein 1 (PvMSP1-19) vaccine candidate alone has shown to have limited immunogenicity in patients; hence, we incorporate the highly conserved and immunogenic C terminus of both P. vivax merozoite surface protein 8 (PvMSP8) and PvMSP1-19 to develop a multicomponent chimeric protein rPvMSP8+1 for immunization of mice. The resulted chimeric rPvMSP8+1 antibody was shown to recognize native protein MSP8 and MSP1-19 of mature P. vivax schizonts. In the immunized mice, an elevated antibody response was observed in the rPvMSP8+1-immunized group compared to that immunized with single-antigen components. In addition, we examined the growth inhibition of these antibodies against Plasmodium cynomolgi (Berok strain) parasites, which is phylogenetically close to P. vivax and sustains long-term culture in vitro Similarly, the chimeric anti-rPvMSP8+1 antibodies recognize P. cynomolgi MSP8 and MSP1-19 on mature schizonts and showed strong inhibition in vitro via growth inhibition assay. This study provides support for a new multiantigen-based paradigm rPvMSP8+1 to explore potential chimeric vaccine candidates against P. vivax malaria using sister species P. cynomolgi.

In-vitro anti-malarial activity of Chikadoma plant from the rainforest of Southern Nigeria

Background: Malaria remains a life-threatening tropical disease. Due to the development of resistance to the commonly available orthodox antimalarials which of course, poses a great challenge in malaria-controlling-program, alternative and complementary approach becomes imperative thereby making phytotherapy a research focus. Objectives: To investigate the effect of chikadoma plant using its methanol leaf extract against a plasmodium-mediated tropical disease, malaria. Materials and Methods: The culture samples of Plasmodium (P.) falciparum from 20 symptomatic adult outpatients were used in the antimalarial in-vitro test. For cultivation of P. falciparum, the culture medium employed was Roswell Park Memorial Institute (RPMI) 1640. Optical microscopy was used for parasite quantification in the performance of antiplasmodial in-vitro assays. The leaf extract of chikadoma dissolved in dimethylsulphoxide (DMSO) was the treatment, prepared into 7 different levels of concentration (3.125, 6.25, 12.5, 25, 50, 100, and 200 mg/mL) while culture medium with the malarial parasite alone served as negative control. Micromalarial culture preceded by culture synchronized with sorbitol 5%, were divided into “control” and “treated groups”, followed by incubation in CO2 candle jar at 370C for 72 h. The percentage of parasitemia was measured 8 h, showing the activity of the extract on P. falciparum stages of proliferation. Thin blood smear from the erythrocytes layer was made and stained with 10% Giemsa for 30 mins to estimate the parasitemia. The antimalarial activity of the extract was calculated using Probit analysis by counting the 50% growth inhibition (IC50). Results: The growth of P. falciparum was inhibited by the extract on mature schizont stage; and the IC50 of the extract after 40 h incubation was 3.0 mg/mL. Conclusion: The leaf extract of chikadoma significantly has antimalarial effect in-vitro against P. falciparum.
 Keywords: Chikadoma; Lupinus arboreus; antimalarial activity; tropical disease; Nigeria.

Plasmodium falciparum Merozoite Associated Armadillo Protein (PfMAAP) Is Apically Localized in Free Merozoites and Antibodies Are Associated With Reduced Risk of Malaria.

Understanding the functional role of proteins expressed by Plasmodium falciparum is an important step toward unlocking potential targets for the development of therapeutic or diagnostic interventions. The armadillo (ARM) repeat protein superfamily is associated with varied functions across the eukaryotes. Therefore, it is important to understand the role of members of this protein family in Plasmodium biology. The Plasmodium falciparum armadillo repeats only (PfARO; Pf3D7_0414900) and P. falciparum merozoite organizing proteins (PfMOP; Pf3D7_0917000) are armadillo-repeat containing proteins previously characterized in P. falciparum. Here, we describe the characterization of another ARM repeat-containing protein in P. falciparum, which we have named the P. falciparum Merozoites-Associated Armadillo repeats protein (PfMAAP). Antibodies raised to three different synthetic peptides of PfMAAP show apical staining of free merozoites and those within the mature infected schizont. We also demonstrate that the antibodies raised to the PfMAAP peptides inhibited invasion of erythrocytes by merozoites from different parasite isolates. In addition, naturally acquired human antibodies to the N- and C- termini of PfMAAP are associated with a reduced risk of malaria in a prospective cohort analysis.

Plasmodium Condensin Core Subunits SMC2/SMC4 Mediate Atypical Mitosis and Are Essential for Parasite Proliferation and Transmission

SummaryCondensin is a multi-subunit protein complex regulating chromosome condensation and segregation during cell division. In Plasmodium spp., the causative agent of malaria, cell division is atypical and the role of condensin is unclear. Here we examine the role of SMC2 and SMC4, the core subunits of condensin, during endomitosis in schizogony and endoreduplication in male gametogenesis. During early schizogony, SMC2/SMC4 localize to a distinct focus, identified as the centromeres by NDC80 fluorescence and chromatin immunoprecipitation sequencing (ChIP-seq) analyses, but do not form condensin I or II complexes. In mature schizonts and during male gametogenesis, there is a diffuse SMC2/SMC4 distribution on chromosomes and in the nucleus, and both condensin I and condensin II complexes form at these stages. Knockdown of smc2 and smc4 gene expression reveals essential roles in parasite proliferation and transmission. The condensin core subunits (SMC2/SMC4) form different complexes and may have distinct functions at various stages of the parasite life cycle.

Antimalarial drug sensitivity test: A new approach toward management of severity of Plasmodium falciparum

Background: Malaria despite everything represents a danger to the strength of occupants and voyagers in tropical nations. The Plasmodium species that are known to infect humans worldwide are Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. Of these, P. falciparum causes high morbidity and mortality, and it is during the asexual erythrocytic stages that the majority of the indications of malaria are shown. Aim: The aim of this study was to standardize an in vitro assay for antimalarial drug sensitivity of P. falciparum isolates. Materials and Methods: This prospective study was carried out over a period of 1 year from July 2014 to June 2015. We effectively completed in vitro drug susceptibility testing on 34 isolates of P. falciparum, which were collected from the patient admitted in Mahatma Gandhi Mission’s Teaching Hospital, Navi Mumbai, Maharashtra, India. We obtained the P. falciparum 3D7 strain from Haffkine Institute for Training, Research and Testing, and Indian Institute of Technology (IIT) Bombay, Mumbai, Maharashtra, India. World Health Organization (WHO) standard in vitro micro-test (Mark III) method was used for the assessment of the response of P. falciparum to chloroquine, mefloquine, quinine, amodiaquine, sulfadoxine/pyrimethamine, and artemisinin. The method was partially modified by replacement of leukocytes with AB serum in Roswell Park Memorial Institute (RPMI)-1640 medium. Results: The development of P. falciparum 3D7 chloroquine-sensitive strain from the ring stage to schizonts (not fewer than six merozoites) in control wells containing chloroquine was 100% recorded. This level of development in the control wells empowered fast minute assurance (5min for isolate per drug) of the Minimum inhibitory concentrations (MICs) of chloroquine. The test well shows the development from the ring stage to mature schizont stages were 67.65% in well containing chloroquine, 17.65% in well containing amodiaquine, and 14.71% in well containing sulfadoxine/pyrimethamine, it means these drugs are resistant to P. falciparum, and however, there is no development of the P. falciparum from the ring stage to mature schizont (with at least six merozoites) stage in the well containing artesunate, mefloquine and quinine were equal to the control well, it means these antimalarial drugs are sensitive to the P. falciparum. Conclusion: Antimalarial drug resistance has developed as a result of prescribing this medication without having malaria. Early diagnosis of malaria and drug sensitivity testing may prevent the emergence of resistance to antimalarial drugs. Monotherapy ought to be kept up a key decent way from; the combined antimalarial drug therapy may reduce the chances of emergence of antimalarial drug resistance.

Molecular characterization and immune protection of an AN1-like zinc finger protein of Eimeria tenella.

Coccidiosis is caused by multiple species of the apicomplexan protozoa Eimeria. Among them, Eimeria tenella is frequently considered to be the most pathogenic. Zinc finger proteins (ZnFPs) are a type of protein containing zinc finger domains. In the present study, a putative Eimeria tenella AN1-like ZnFP (E. tenella AN1-like zinc finger domain-containing protein, putative partial mRNA, EtAN1-ZnFP) was cloned and characterized, and its immune protective effects were evaluated. The 798-bp ORF sequence of EtAN1-ZnFP that encoded a protein of approximately 27.0 kDa was obtained. The recombinant EtAN1-ZnFP protein (rEtAN1-ZnFP) was expressed in Escherichia coli. Western blot analysis showed that the recombinant protein was recognized by the anti-GST monoclonal antibody and anti-sporozoite protein rabbit serum. qPCR analysis revealed that EtAN1-ZnFP was highly expressed in unsporulated oocysts and sporozoites. Immunostaining with an anti-rEtAN1-ZnFP antibody indicated that EtAN1-ZnFP was uniformly distributed in the cytoplasm of sporozoites, except for the refractive body; furthermore, this protein was evenly distributed in the cytoplasm of immature schizonts but seldom distributed in mature schizonts. The results of the in vitro invasion inhibition assay indicated that the antibodies against rEtAN1-ZnFP efficiently reduced the ability of E. tenella sporozoites to invade host cells. Animal challenge experiments demonstrated that the chickens immunized with rEtAN1-ZnFP protein significantly decreased mean lesion scores and fecal oocyst output compared with challenged control group. The results suggest that EtAN1-ZnFP can induce partial immune protection against infection with E. tenella and could be an effective candidate for the development of new vaccines.

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.