Anti-Eimeria tenella activity of Ethanamizuril in vitro and in vivo.

The prevalence of chicken coccidiosis in the poultry industry is a significant concern, further exacerbated by the emergence of drug-resistant coccidia resulting from the indiscriminate use of medications. Ethanamizuril, a novel triazine anti-coccidial compound, has been used to combat drug resistance. Currently, it is known that Ethanamizuril acts on the second-generation merozoites and early gametogenesis stages of Eimeria. Limited information exists regarding its impact on the early merozoites and exogenous stage of Eimeria. In the present study, the anti-coccidial properties of Ethanamizuril were evaluated both in vitro and in vivo. The in vitro experiments demonstrated that Ethanamizuril effectively inhibits the sporulation of E. tenella oocysts in a dose-dependent manner and significantly reduces the sporozoite excystation rate. Furthermore, in vivo tests revealed that treatment with 10 mg/L Ethanamizuril in drinking water significantly decreased the copy number of first-generation and secondary-generation merozoites in the chicken cecum, indicating that it can inhibit the development of whole schizonts development. Moreover, treatment with Ethanamizuril demonstrated excellent protective efficacy with an anti-coccidial index (ACI) of 180, which was manifested through higher body weight gains, lighter cecal lesion, lower fecal oocyst shedding score and reduced liver index. Collectively, this study suggests that Ethanamizuril effectively treats E. tenella infection by inhibiting both endogenous and exogenous stages development.

Expression of IL-10 and TGF-ß1 in horses experimentally infected with T. equi merozoites is associated with antibody production but not modulation of pro-inflammatory responses.

Theileria equi (T. equi) is an apicomplexan parasite that causes severe hemolytic anemia in equids. Presently, there is inadequate knowledge of the immune responses induced by T. equi in equid hosts impeding understanding of the host parasite relationship and development of potent vaccines for control of T. equi infections. The objective of this study was to evaluate the host-parasite dynamics between T. equi merozoites and infected horses by assessing cytokine expression during primary and secondary parasite exposure, and to determine whether the pattern of expression correlated with clinical indicators of disease. Our findings showed that the expression of pro-inflammatory cytokines was very low and inconsistent during both primary and secondary infection. There was also no correlation between the symptoms observed during primary infection and expression of the cytokines. This suggests that the symptoms might have occurred primarily due to hemolysis and likely not the undesirable effects of pro-inflammatory responses. However, IL-10 and TGF-ß1 were highly expressed in both phases of infection, and their expression was linked to antibody production but not moderation of pro-inflammatory cytokine responses.

Global profiling of protein S-palmitoylation in the second-generation merozoites of Eimeria tenella.

The intracellular protozoan Eimeria tenella is responsible for avian coccidiosis which is characterized by host intestinal damage. During developmental cycle, E. tenella undergoes versatile transitional stages such as oocyst, sporozoites, merozoites, and gametocytes. These developmental transitions involve changes in cell shape and cell size requiring cytoskeletal remodeling and changes in membrane proteins, which may require transcriptional and translational regulations as well as post-translational modification of proteins. Palmitoylation is a post-translational modification (PTM) of protein that orchestrates protein targeting, folding, stability, regulated enzymatic activity and even epigenetic regulation of gene expression. Previous research revealed that protein palmitoylation play essential role in Toxoplasma gondii, Trypanosoma cruzi, Trichomonas vaginalis, and several Plasmodium parasites. Until now, there is little information on the enzymes related to palmitoylation and role of protein acylation or palmitoylation in E. tenella. Therefore, palmitome of the second-generation merozoite of E. tenella was investigated. We identified a total of 2569 palmitoyl-sites that were assigned to 2145 palmitoyl-peptides belonging to 1561 protein-groups that participated in biological processes including parasite morphology, motility and host cell invasion. In addition, RNA biosynthesis, protein biosynthesis, folding, proteasome-ubiquitin degradation, and enzymes involved in PTMs, carbohydrate metabolism, glycan biosynthesis, and mitochondrial respiratory chain as well as vesicle trafficking were identified. The study allowed us to decipher the broad influence of palmitoylation in E. tenella biology, and its potential roles in the pathobiology of E. tenella infection. Raw data are publicly available at iProX with the dataset identifier PXD045061.

In vitro infection of bovine erythrocytes with Theileria annulata merozoites as a key step in completing the T. annulata life cycle in vitro.

Theileria annulata is a protozoan parasite with a complex life cycle involving a bovine host and a tick vector. It is transmitted by Hyalomma ticks and is the causative agent of tropical theileriosis, a debilitating and often fatal disease in southern Europe, northern Africa and large parts of Asia. Understanding the biology of different life cycle stages is critical for the control of tropical theileriosis and requires the use of experimental animals which poses an ethical concern. We present for the first time the in vitro infection of red blood cells (RBCs) with T. annulata differentiated schizonts. The Ankara cell line of T. annulata was cultured at 41 °C for nine days to induce merogony and subsequently incubated with purified RBCs for one to three days. Percentage of parasitized erythrocyte (PPE) over the short culture period was estimated by Giemsa staining (0.007-0.01%), Flow cytometry activated sorting (FACS) (0.02-1.1%) and observation of FACS sorted cells by confocal microscopy (0.05-0.4%). There was a significant difference in the PPE between FACS and the two other techniques (one-way ANOVA followed by Tukey test, P = 0.004) but no significant difference was observed between the confocal imaging and Giemsa staining methods (ANOVA one-way followed by Tukey test, P = 0.06). Importantly, all three complementary methods confirmed the invasion of RBCs by T. annulata merozoites in vitro. Although the experimental conditions will require further optimization to increase the PPE, the in vitro infection of RBCs by T. annulata merozoites is pivotal in paving the way for the eventual completion of the T. annulata life cycle in vitro when combined with artificial tick feeding.

Proteomics analysis of Toxoplasma gondii merozoites reveals regulatory proteins involved in sexual reproduction.

Sexual reproduction plays a crucial role in the transmission and life cycle of toxoplasmosis. The merozoites are the only developmental stage capable of differentiation into male and female gametes, thereby initiating sexual reproduction to form oocysts that are excreted into the environment. Hence, our study aimed to perform proteomic analyses of T. gondii Pru strain merozoites, a pre-sexual developmental stage in cat IECs, and tachyzoites, an asexual developmental stage, using the tandem mass tag (TMT) method in order to identify the differentially expressed proteins (DEPs) of merozoites. Proteins functions were subjected to cluster analysis, and DEPs were validated through the qPCR method. The results showed that a total of 106 proteins were identified, out of which 85 proteins had quantitative data. Among these, 15 proteins were differentially expressed within merozoites, with four exhibiting up-regulation and being closely associated with the material and energy metabolism as well as the cell division of T. gondii. Two novel DEPs, namely S8GHL5 and A0A125YP41, were identified, and their homologous family members have been demonstrated to play regulatory roles in oocyte maturation and spermatogenesis in other species. Therefore, they may potentially exhibit regulatory functions during the differentiation of micro- and macro-gametophytes at the initiation stage of sexual reproduction in T. gondii. In conclusion, our results showed that the metabolic and divisional activities in the merozoites surpass those in the tachyzoites, thereby providing structural, material, and energetic support for gametophytes development. The discovery of two novel DEPs associated with sexual reproduction represents a significant advancement in understanding Toxoplasma sexual reproduction initiation and oocyst formation.

Severity of Schistosoma haematobium co-infection with malaria in school-children is potentially modulated by host CD14 gene variants.

OBJECTIVE: Schistosomiasis remains a chronic disease of global importance, especially in many rural areas of the world where co-infection with Plasmodium falciparum is common. It is critical to decipher the role of single or co-infected disease scenarios on immune system regulation in such individuals and how such co-infections can either ameliorate or complicate immune response and the consequent disease outcome. First, 10 ml of urine samples, collected between 10:00 am and 2:00 pm, was filtered for diagnosis of schistosomiasis, while egg count, indicative of disease severity, was determined by microscopy. Furthermore, genomic DNA samples extracted from dried blood spots collected on filter paper from one hundred and forty-four Schistosoma haematobium-infected school-children was tested for P. falciparum parasite positivity by an allele-specific nested-PCR analysis of merozoite surface protein (msp)-1 and -2 genes and a real-time PCR assay. In addition, among P. falciparum parasite-positive individuals, we carried out a Taqman SNP genotyping assay to extrapolate the effect of host CD14 (-159 C/T; rs2569190) genetic variants on schistosome egg count. RESULTS: Of the 144 individuals recruited, P. falciparum parasite positivity with msp-1 gene were 34%, 43% and 55% for MAD20, RO33 and K1 alleles respectively. Of the co-infected individuals, CD14 genetic variants ranged from 18.8% vs 21.5%, 33.3% vs 44.4%, 9.7% vs 11.8% for single versus schistosome co-infection for the wild type (CC), heterozygous (CT) and mutant (TT) variants respectively. Though the mean egg count for co-infected individuals with CD14 wild type (33.7 eggs per 10 ml of urine) and heterozygote variants (37.5 eggs per 10 ml of urine) were lower than that of schistosome infection alone (52.48 and 48.08 eggs/10 ml of urine respectively), it lacked statistical significance (p-value 0.12 and 0.29), possibly reflecting the benefit of the CD14 activation in schistosome plus malaria co-infection and not schistosome infection alone. In addition, the lower mean egg count in co-infected individuals reveal the benefit of downstream Th1 immune response mitigated by CD14 innate activation that is absent in schistosome infection alone.

Identification of Eimeria tenella sporozoite immunodominant mimotopes by random phage-display peptide libraries-a proof of concept study.

Introduction: Coccidiosis, caused by parasites of numerous Eimeria species, has long been recognized as an economically significant disease in the chicken industry worldwide. The rise of anti-coccidian resistance has driven a search for other parasite management techniques. Recombinant antigen vaccination presents a highly feasible alternative. Properly identifying antigens that might trigger a potent immune response is one of the major obstacles to creating a viable genetically modified vaccine. Methods: This study evaluated a reverse immunology approach for the identification of B-cell epitopes. Antisera from rabbits and hens inoculated with whole-sporozoites of E. tenella were used to identify Western blot antigens. The rabbit IgG fraction from the anti-sporozoite serum exhibited the highest reactogenicity; consequently, it was purified and utilized to screen two random Phage-display peptide libraries (12 mer and c7c mer). After three panning rounds, 20 clones from each library were randomly selected, their nucleotide sequences acquired, and their reactivity to anti-sporozoite E. tenella serum assessed. The selected peptide clones inferred amino acid sequences matched numerous E. tenella proteins. Results and Conclusions: The extracellular domain of the epidermal growth factor-like (EGF-like) repeats, and the thrombospondin type-I (TSP-1) repeats of E. tenella micronemal protein 4 (EtMIC4) matched with the c7c mer selected clones CNTGSPYEC (2/20) and CMSTGLSSC (1/20) respectively. The clone CSISSLTHC that matched with a conserved hypothetical protein of E. tenella was widely selected (3/20). Selected clones from the 12-mer phage display library AGHTTQFNSKTT (7/20), GPNSAFWAGSER (2/20) and HFAYWWNGVRGP (8/20) showed similarities with a cullin homolog, elongation factor-2 and beta-dynein chain a putative E. tenella protein, respectively. Four immunodominant clones were previously selected and used to immunize rabbits. By ELISA and Western blot, all rabbit anti-clone serums detected E. tenella native antigens. Discussion: Thus, selected phagotopes contained recombinant E. tenella antigen peptides. Using antibodies against E. tenella sporozoites, this study demonstrated the feasibility of screening Phage-display random peptide libraries for true immunotopes. In addition, this study looked at an approach for finding novel candidates that could be used as an E. tenella recombinant epitope-based vaccine.

Erythrocyte membrane with CLIPPKF as biomimetic nanodecoy traps merozoites and attaches to infected red blood cells to prevent Plasmodium infection.

BACKGROUND: Malaria remains a serious threat to global public health. With poor efficacies of vaccines and the emergence of drug resistance, novel strategies to control malaria are urgently needed. RESULTS: We developed erythrocyte membrane-camouflaged nanoparticles loaded with artemether based on the growth characteristics of Plasmodium. The nanoparticles could capture the merozoites to inhibit them from repeatedly infecting normal erythrocytes, owing to the interactions between merozoites and heparin-like molecules on the erythrocyte membrane. Modification with a phosphatidylserine-targeting peptide (CLIPPKF) improved the drug accumulation in infected red blood cells (iRBCs) from the externalized phosphatidylserine induced by Plasmodium infection. In Plasmodium berghei ANKA strain (pbANKA)-infected C57BL/6 mice, the nanoparticles significantly attenuated Plasmodium-induced inflammation, apoptosis, and anemia. We observed reduced weight variation and prolonged survival time in pbANKA-challenged mice, and the nanoparticles showed good biocompatibility and negligible cytotoxicity. CONCLUSION: Erythrocyte membrane-camouflaged nanoparticles loaded with artemether were shown to provide safe and effective protection against Plasmodium infection.

Phenotypic characterization of Ghanaian P. falciparum clinical isolates reveals a homogenous parasite population.

Background: Erythrocyte invasion by P. falciparum involves functionally overlapping interactions between the parasite's ligands and the erythrocyte surface receptors. While some P. falciparum isolates necessarily engage the sialic acid (SA) moieties of the erythrocytes during the invasion, others use ligands whose binding is independent of SA for successful invasion. Deciphering the major pathway used by P. falciparum clinical isolates represent a key step toward developing an efficient blood stage malaria vaccine. Methods: We collected a total of 156 malaria-infected samples from Ghanaian children aged 2 to 14 years and used a two-color flow cytometry-based invasion assay to assess the invasion phenotype diversity of Ghanaian P. falciparum clinical isolates. Anti-human CR1 antibodies were used to determine the relative contribution of the PfRh4-CR1 interaction in the parasites invasion phenotype and RT-qPCR was used to assess the expression levels of key invasion-related ligands. Results: Our findings show no clear association between demographic or clinical data and existing reports on the malaria transmission intensity. The complete invasion data obtained for 156 isolates, showed the predominance of SA-independent pathways in Ghanaian clinical isolates. Isolates from Hohoe and Navrongo had the highest diversity in invasion profile. Our data also confirmed that the PfRh4-CR1 mediated alternative pathway is important in Ghanaian clinical isolates. Furthermore, the transcript levels of ten invasion-related genes obtained in the study showed little variations in gene expression profiles within and between parasite populations across sites. Conclusion: Our data suggest a low level of phenotypic diversity in Ghanaian clinical isolates across areas of varying endemicity and further highlight its importance in the quest for new intervention strategies, such as the investigation of blood-stage vaccine targets, particularly those targeting specific pathways and able to trigger the stimulation of broadly neutralizing invasion antibodies.

Improvement of the antibody-dependent respiratory burst assay for assessing protective immune responses to malaria.

The antibody-dependent respiratory burst (ADRB) assay is a sensitive isoluminol-based chemiluminescence (CL) functional assay designed to assess the capacity of opsonizing antibodies against merozoites to induce neutrophil respiratory burst. ADRB was shown to measure protective immunity against malaria in endemic areas, but the assay needed further improvement to ensure better sensitivity and reproducibility. Here, we adjusted parameters such as the freezing-thawing procedure of merozoites, merozoites's concentration and the buffer solution's pH, and we used the improved assay to measure ADRB activity of 207 sera from 97 and 110 individuals living, respectively, in Dielmo and Ndiop villages with differing malaria endemicity. The improvement led to increased CL intensity and assay sensitivity, and a higher reproducibility. In both areas, ADRB activity correlated with malaria endemicity and individual's age discriminated groups with and without clinical malaria episodes, and significantly correlated with in vivo clinical protection from Plasmodium falciparum malaria. Our results demonstrate that the improved ADRB assay can be valuably used to assess acquired immunity during monitoring by control programmes and/or clinical trials.

Theileria equi RAP-1a and RAP-1b proteins contain immunoreactive epitopes and are suitable candidates for vaccine and diagnostics development.

Theileria equi is an obligate intracellular protozoan parasite that causes severe hemolytic anaemia in most equid species. Similar to other apicomplexan parasites, T. equi contains rhoptries whose contents have been implicated in host cell invasion and formation of the parasitophorous vacuole that is crucial for survival of the species within cells. Despite their importance, the composition of T. equi rhoptries and their role(s) in host cell invasion remain unexplored. To gain insight into these issues, we evaluated the expression, immunogenicity, and functional roles of two T. equi rhoptry-associated proteins abbreviated as RAP-1a and RAP-1b. The full-length RAP-1a protein was expressed to perform the analysis but our efforts to express the full-length RAP-1b protein failed due to an unknown reason. We therefore generated synthetic immunogenic peptides that map onto the N- and C-termini of the RAP-1b protein as an alternative approach. Our findings show that both proteins are expressed in the extracellular and intra-erythrocytic merozoite stages of T. equi. Serological analyses show that T. equi-infected horses mount antibody responses that recognise both proteins and correlate with a decrease in T. equi load in both acutely and persistently infected horses. In vitro neutralisation studies show that the T. equi RAP-1a protein contains neutralisation-sensitive epitopes as antibodies developed against the protein significantly inhibited the parasites from invading equine erythrocytes. Conversely, antibodies developed against the RAP-1b synthetic peptides did not neutralise parasite invasion, showing that the protein regions on which the peptides were based are not required for T. equi invasion. Overall, the data shows that T. equi rhoptries and their contents are involved in invasion of host cells and supports T. equi RAP-1 proteins as candidates for developing novel serodiagnosis tools and vaccines.

Recent achievements and doors opened for coccidian parasite research and development through transcriptomics of enteric sexual stages.

The Coccidia is the largest group of parasites within the Apicomplexa, a phylum of unicellular, obligate parasites characterized by the possession of an apical complex of organelles and structures in the asexual stages of their life cycles, as well as by a sexual reproductive phase that occurs enterically in host animals. Coccidian sexual reproduction involves morphologically distinct microgametes and macrogametes that combine to form a diploid zygote and, ultimately, following meiosis and mitosis, haploid, infectious sporozoites, inside sporocysts within an oocyst. Recent transcriptomic analyses have identified genes involved in coccidian sexual stage development and reproduction, including genes encoding for microgamete- and macrogamete-specific proteins with roles in gamete motility, fusion and fertilization, and in the formation of the resilient oocyst wall that allows coccidians to persist for long periods in the environment. Transcriptomics has also provided important clues about the regulation of gene expression in the transformation of parasites from one developmental stage to the next, a complex sequence of events that may involve transcription factors such as the apicomplexan Apetala2 (ApiAP2) family, alternative splicing, regulatory RNAs and MORC (a microrchida homologue and regulator of sexual stage development in Toxoplasma gondii). The molecular dissection of coccidian sexual development and reproduction by transcriptomic analyses may lead to the development of novel transmission-blocking strategies.

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.

Endogenous Developmental Cycle of the Human Coccidian Cyclospora cayetanensis.

Cyclospora cayetanensis is a coccidian parasite of humans of known and growing importance. However, we are surprisingly naïve as to our understanding of how to diagnose it and how it develops inside the human body. Here we provide details of the developmental stages of C. cayetanensis in the gallbladder of a 33-yr-old male with human immunodeficiency virus. The gallbladder was removed surgically in 2001 because of severe abdominal pain. For the present study, the archived paraffin block of gallbladder was processed for light microscopy and transmission electron microscopy (TEM). Histological sections were examined after staining with hematoxylin and eosin (HE) or using the periodic acid Schiff (PAS) reaction. Immature and mature asexual stages, gamonts, and oocysts were seen in epithelial cells, both in the superficial epithelium and in glands. The merozoites were present singly, in pairs, and 3 or more in a single parasitophorous vacuole in the host cytoplasm. Up to 6 nuclei were seen in immature schizonts without evidence of merozoite formation. Mature schizonts were 7.6 × 5.1 µm and contained up to 10, 3-4 µm long merozoites. Merozoites were 0.6 to 2.0 µm wide, and their shape varied from pear-shaped to slender. Merozoites were generally PAS-positive; however, some were intensely positive, some had only minute granules, while others were PAS-negative. The microgamonts (male) were 6.6 × 5.2 µm and contained fewer than 20 microgametes around a residual body. The microgametes were up to 2 µm long and were flagellated. Macrogamonts (female) contained distinctive eosinophilic wall-forming bodies that varied in size and were less than 1 µm in HE-stained sections. Macrogamonts were 5.8-6.5 × 5.3-6.5 µm. Oocysts in sections were unsporulated and had a diameter of 5.7-7.5 µm. The TEM examination confirmed the histologic findings. The DNA extracted from paraffin sections was confirmed as C. cayetanensis with real-time PCR. The detailed description of the life cycle stages of C. cayetanensis reported here in an immunosuppressed patient could facilitate histopathologic diagnosis of this parasite. We have shown that the parasite's development more closely resembles that of Cystoisospora than Eimeria and that the parasite has multiple nuclei per immature meront indicating schizogony, and we have undermined evidence for a Type II meront.

Protein S-Palmitoylation Is Responsive to External Signals and Plays a Regulatory Role in Microneme Secretion in Plasmodium falciparum Merozoites.

Protein S-palmitoylation is an important post-translational modification (PTM) in blood stages of the malaria parasite, Plasmodium falciparum. S-palmitoylation refers to reversible covalent modification of cysteine residues of proteins by saturated fatty acids. In vivo, palmitoylation is regulated by concerted activities of DHHC palmitoyl acyl transferases (DHHC PATs) and acyl protein thioesterases (APTs), which are enzymes responsible for protein palmitoylation and depalmitoylation, respectively. Here, we investigate the role of protein palmitoylation in red blood cell (RBC) invasion by P. falciparum merozoites. We demonstrate for the first time that free merozoites require PAT activity for microneme secretion in response to exposure to the physiologically relevant low [K+] environment, characteristic of blood plasma. We have adapted copper catalyzed alkyne azide chemistry (CuAAC) to image palmitoylation in merozoites and found that exposure to low [K+] activates PAT activity in merozoites. Moreover, using acyl biotin exchange chemistry (ABE) and confocal imaging, we demonstrate that a calcium dependent protein kinase, PfCDPK1, an essential regulator of key invasion processes such as motility and microneme secretion, undergoes dynamic palmitoylation and localizes to the merozoite membrane. Treatment of merozoites with the PAT inhibitor, 2-bromopalmitate (2-BP), effectively inhibits microneme secretion and RBC invasion by the parasite, thus opening the possibility of targeting P. falciparum PATs for antimalarial drug discovery to inhibit blood stage growth of malaria parasites.

Proteomic analysis of the second-generation merozoites of Eimeria tenella under nitromezuril and ethanamizuril stress.

BACKGROUND: Eimeria tenella is a highly pathogenic coccidian that causes avian coccidiosis. Both nitromezuril (NZL) and ethanamizuril (EZL) are novel triazine compounds with high anticoccidial activity, but the mechanisms of their action are still unclear. This study explored the response of E. tenella to NZL and EZL by the study of changes in protein composition of the second-generation merozoites. METHODS: Label-free quantification (LFQ) proteomics of the second-generation merozoites of E. tenella following NZL and EZL treatment were studied by LC-MS/MS to explore the mechanisms of action. The identified proteins were annotated and analyzed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and protein-protein interaction (PPI) networks analysis. RESULTS: A total of 1430 proteins were identified by LC-MS/MS, of which 375 were considered as differential proteins in response to drug treatment (DPs). There were 26 only found in the NZL treatment group (N-group), 63 exclusive to the EZL treatment group (E-group), and 80 proteins were present in both drug groups. In addition, among the DPs, the abundant proteins with significantly altered expression in response to drug treatment (SDPs) were found compared with the C-group, of which 49 were upregulated and 51 were downregulated in the N-group, and 66 upregulated and 79 downregulated in the E-group. Many upregulated proteins after drug treatment were involved in transcription and protein metabolism, and surface antigen proteins (SAGs) were among the largest proportion of the downregulated SDPs. Results showed the top two enriched GO terms and the top one enriched pathway treated with EZL and NZL were related, which indicated that these two compounds had similar modes of action. CONCLUSIONS: LFQ proteomic analysis is a feasible method for screening drug-related proteins. Drug treatment affected transcription and protein metabolism, and SAGs were also affected significantly. This study provided new insights into the effects of triazine anticoccidials against E. tenella.

Eukaryotic elongation factor 2 is involved in the anticoccidial action of diclazuril in the second-generation merozoites of Eimeria tenella.

Eimeria tenella, an obligate intracellular parasite, can actively invade the cecal epithelial cells of chickens and cause severe enteric disease. Eukaryotic elongation factor 2 (eEF2) plays a major role in protein synthesis and cell survival. This study aims to explore the exact mechanisms underlying diclazuril inhibition in second-generation merozoites of E. tenella. The eEF2 cDNA of the second-generation merozoites of E. tenella (EtEF2) was cloned by reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Diclazuril-induced expression profiles of EtEF2 were also analyzed. The cloned full-length cDNA (2893 bp) of the EtEF2 nucleotide sequence encompassed a 2499 bp open reading frame (ORF) that encoded a polypeptide of 832 residues with an estimated molecular mass of 93.12 kDa and a theoretical isoelectric point of 5.99. The EtEF2 nucleotide sequence was submitted to the GenBank database with the accession number KF188423. The EtEF2 protein sequence shared 99 % homology with the eEF2 sequence of Toxoplasma gondii (GenBank XP_002367778.1). The GTPase activity domain and ADP-ribosylation domain were conserved signature sequences of the eEF2 gene family. The changes in the transcriptional and translational levels of EtEF2 were detected through quantitative real-time PCR and Western blot analyses. The mRNA expression level of EtEF2 was 2.706 fold increases and the protein level of EtEF2 was increased 67.31 % under diclazuril treatment. In addition, the localization of EtEF2 was investigated through immunofluorescence assay. Experimental results demonstrated that EtEF2 was distributed primarily in the cytoplasm of second-generation merozoites, and its fluorescence intensity was enhanced after diclazuril treatment. These findings indicated that EtEF2 may have an important role in understanding the signaling mechanism underlying the anticoccidial action of diclazuril and could be a promising target for novel drug exploration.

Sero-diagnostic potential of Plasmodium falciparum recombinant merozoite surface protein (MSP)-3 expressed in silkworm.

Plasmodium falciparum is a blood protozoan parasite, transmitted by Anopheles mosquitoes vectors, that can cause morbidity and even leads to mortality in tropical countries. Strategies are directed to combat malaria including development of diagnostic tools, serological markers and vaccinations. A target under intensive studies is Merozoite Surface Protein (MSP)-3. The aim of this study is to express and purify recombinant MSP3 of P. falciparum (rPfMSP3) using silkworm expression system as a host for its large-scale production and to investigate its potential effectiveness for sero-diagnosis. The rPfMSP3 formed oligomers in a blue-native PAGE and its N-glycosylation was confirmed by periodic acid-Schiff staining and PNGase F treatment. The amyloid-like morphology of the rPfMSP3 oligomers was observed. Enzyme-linked immunosorbent assay showed that 60-70% of human samples from subjects living in malaria endemic areas in Indonesia detected the rPfMSP3. Western blot results showed that the rPfMSP3 was recognized by a malaria infected human serum but not by an uninfected human serum. The rPfMSP3 was successfully expressed in silkworm as a soluble protein and has the potential to be used in serological measurement for detecting PfMSP3-specific antibodies in sera from individuals living in endemic areas.

New Observations Allowing the Differentiation of Late Asexual Stages of Cystoisospora canis from Developing Microgamonts in the Intestines of Experimentally Infected Dogs.

The coccidian parasite Cystoisospora canis (syn. Isospora canis) can cause clinical disease in dogs. Three generations of meronts are reported to occur in the small intestine of experimentally infected dogs before gametogony and oocyst formation. Oocyst excretion in the feces occurs at 9 to 11 days post-inoculation (PI). We examined the late asexual and sexual development of C. canis in 2 dogs necropsied 10 days after oral inoculation with 100,000 sporulated C. canis oocysts; both dogs had excreted oocysts 9 days PI. Asexual and sexual stages were seen in the lamina propria, throughout the small intestine in sections stained with hematoxylin and eosin from both dogs. In other studies of the C. canis life cycle, little attention has been given to distinguishing the last asexual generation of meronts and early microgamonts that can appear similar due to their stage of maturation and both having multiple nuclei. Here we report newly identified features of developing meronts and microgamonts and their distinction from each other by using sections processed using the periodic acid-Schiff (PAS) reaction. Using this method, we demonstrated that PAS-positive granules could be used to identify microgamonts and differentiate them from developing meront stages. These findings will aid pathologists and others in properly identifying coccidial parasites, in determining the cause of microscopic lesions in intestinal tissue, and in accurately identifying etiological agents.

Breadth of Functional Antibodies Is Associated With Plasmodium falciparum Merozoite Phagocytosis and Protection Against Febrile Malaria.

BACKGROUND: The specific targets of functional antibodies against Plasmodium falciparum merozoites remain largely unexplored and, more importantly, their relevance to naturally acquired immunity in longitudinal cohort studies (LCSs) is yet to be tested. METHODS: Functionality of immunoglobulin G (IgG) antibodies against 24 merozoite antigens was determined at the baseline of an LCS in Ghana using a bead-based opsonic phagocytosis assay (BPA). Antigen-specific IgG3 subclass antibodies were quantified in the same samples by the Luminex multiplex system. RESULTS: A wide range of BPA activity was observed across the different antigens. High BPA responses of nMSP3K1, GLURP-R2, MSP23D7, MSP119k, and PfRh2-2030 coupled beads were significantly associated with a higher probability of children not experiencing febrile malaria. Children with high breadth of functional antibodies against these antigens together with cMSP33D7 had a significantly reduced risk of febrile malaria (adjusted hazard ratio, 0.36 [95% confidence interval, .18-.72]; P = .004). Five of the 6 BPA activities significantly (likelihood ratio rest, P ≤ .05) contributed to the protective immunity observed with the IgG3 antibodies. CONCLUSIONS: The development of BPA allowed profiling of functional antibodies in an LCS. Identification of targets of opsonic phagocytosis may have implications in the development of a subunit malaria vaccine.