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  1. Fulltext Insights into the genome structure and copy-number variation of Eimeria tenella
    Lim LS, Tay YL, Alias H, Wan KL, Dear PH
    BMC Genomics, 2012;13:389.
    PMID: 22889016 DOI: 10.1186/1471-2164-13-389
    Eimeria is a genus of parasites in the same phylum (Apicomplexa) as human parasites such as Toxoplasma, Cryptosporidium and the malaria parasite Plasmodium. As an apicomplexan whose life-cycle involves a single host, Eimeria is a convenient model for understanding this group of organisms. Although the genomes of the Apicomplexa are diverse, that of Eimeria is unique in being composed of large alternating blocks of sequence with very different characteristics - an arrangement seen in no other organism. This arrangement has impeded efforts to fully sequence the genome of Eimeria, which remains the last of the major apicomplexans to be fully analyzed. In order to increase the value of the genome sequence data and aid in the effort to gain a better understanding of the Eimeria tenella genome, we constructed a whole genome map for the parasite.
    Matched MeSH terms: Genome, Protozoan*
  2. Fulltext Population genomic structure and adaptation in the zoonotic malaria parasite Plasmodium knowlesi
    Assefa S, Lim C, Preston MD, Duffy CW, Nair MB, Adroub SA, et al.
    Proc Natl Acad Sci U S A, 2015 Oct 20;112(42):13027-32.
    PMID: 26438871 DOI: 10.1073/pnas.1509534112
    Malaria cases caused by the zoonotic parasite Plasmodium knowlesi are being increasingly reported throughout Southeast Asia and in travelers returning from the region. To test for evidence of signatures of selection or unusual population structure in this parasite, we surveyed genome sequence diversity in 48 clinical isolates recently sampled from Malaysian Borneo and in five lines maintained in laboratory rhesus macaques after isolation in the 1960s from Peninsular Malaysia and the Philippines. Overall genomewide nucleotide diversity (π = 6.03 × 10(-3)) was much higher than has been seen in worldwide samples of either of the major endemic malaria parasite species Plasmodium falciparum and Plasmodium vivax. A remarkable substructure is revealed within P. knowlesi, consisting of two major sympatric clusters of the clinical isolates and a third cluster comprising the laboratory isolates. There was deep differentiation between the two clusters of clinical isolates [mean genomewide fixation index (FST) = 0.21, with 9,293 SNPs having fixed differences of FST = 1.0]. This differentiation showed marked heterogeneity across the genome, with mean FST values of different chromosomes ranging from 0.08 to 0.34 and with further significant variation across regions within several chromosomes. Analysis of the largest cluster (cluster 1, 38 isolates) indicated long-term population growth, with negatively skewed allele frequency distributions (genomewide average Tajima's D = -1.35). Against this background there was evidence of balancing selection on particular genes, including the circumsporozoite protein (csp) gene, which had the top Tajima's D value (1.57), and scans of haplotype homozygosity implicate several genomic regions as being under recent positive selection.
    Matched MeSH terms: Genome, Protozoan*
  3. Fulltext Sequence analysis of the PIP5K locus in Eimeria maxima provides further evidence for eimerian genome plasticity and segmental organization
    Song BK, Pan MZ, Lau YL, Wan KL
    Genet. Mol. Res., 2014;13(3):5803-14.
    PMID: 25117339 DOI: 10.4238/2014.July.29.8
    Commercial flocks infected by Eimeria species parasites, including Eimeria maxima, have an increased risk of developing clinical or subclinical coccidiosis; an intestinal enteritis associated with increased mortality rates in poultry. Currently, infection control is largely based on chemotherapy or live vaccines; however, drug resistance is common and vaccines are relatively expensive. The development of new cost-effective intervention measures will benefit from unraveling the complex genetic mechanisms that underlie host-parasite interactions, including the identification and characterization of genes encoding proteins such as phosphatidylinositol 4-phosphate 5-kinase (PIP5K). We previously identified a PIP5K coding sequence within the E. maxima genome. In this study, we analyzed two bacterial artificial chromosome clones presenting a ~145-kb E. maxima (Weybridge strain) genomic region spanning the PIP5K gene locus. Sequence analysis revealed that ~95% of the simple sequence repeats detected were located within regions comparable to the previously described feature-rich segments of the Eimeria tenella genome. Comparative sequence analysis with the orthologous E. maxima (Houghton strain) region revealed a moderate level of conserved synteny. Unique segmental organizations and telomere-like repeats were also observed in both genomes. A number of incomplete transposable elements were detected and further scrutiny of these elements in both orthologous segments revealed interesting nesting events, which may play a role in facilitating genome plasticity in E. maxima. The current analysis provides more detailed information about the genome organization of E. maxima and may help to reveal genotypic differences that are important for expression of traits related to pathogenicity and virulence.
    Matched MeSH terms: Genome, Protozoan*
  4. Fulltext A reference genome and methylome for the Plasmodium knowlesi A1-H.1 line
    Benavente ED, de Sessions PF, Moon RW, Grainger M, Holder AA, Blackman MJ, et al.
    Int J Parasitol, 2018 03;48(3-4):191-196.
    PMID: 29258833 DOI: 10.1016/j.ijpara.2017.09.008
    Plasmodium knowlesi, a common parasite of macaques, is recognised as a significant cause of human malaria in Malaysia. The P. knowlesi A1H1 line has been adapted to continuous culture in human erythrocytes, successfully providing an in vitro model to study the parasite. We have assembled a reference genome for the PkA1-H.1 line using PacBio long read combined with Illumina short read sequence data. Compared with the H-strain reference, the new reference has improved genome coverage and a novel description of methylation sites. The PkA1-H.1 reference will enhance the capabilities of the in vitro model to improve the understanding of P. knowlesi infection in humans.
    Matched MeSH terms: Genome, Protozoan*
  5. Fulltext Deciphering the Draft Genome of Toxoplasma gondii RH Strain
    Lau YL, Lee WC, Gudimella R, Zhang G, Ching XT, Razali R, et al.
    PLoS One, 2016;11(6):e0157901.
    PMID: 27355363 DOI: 10.1371/journal.pone.0157901
    Toxoplasmosis is a widespread parasitic infection by Toxoplasma gondii, a parasite with at least three distinct clonal lineages. This article reports the whole genome sequencing and de novo assembly of T. gondii RH (type I representative strain), as well as genome-wide comparison across major T. gondii lineages. Genomic DNA was extracted from tachyzoites of T. gondii RH strain and its identity was verified by PCR and LAMP. Subsequently, whole genome sequencing was performed, followed by sequence filtering, genome assembly, gene annotation assignments, clustering of gene orthologs and phylogenetic tree construction. Genome comparison was done with the already archived genomes of T. gondii. From this study, the genome size of T. gondii RH strain was found to be 69.35Mb, with a mean GC content of 52%. The genome shares high similarity to the archived genomes of T. gondii GT1, ME49 and VEG strains. Nevertheless, 111 genes were found to be unique to T. gondii RH strain. Importantly, unique genes annotated to functions that are potentially critical for T. gondii virulence were found, which may explain the unique phenotypes of this particular strain. This report complements the genomic archive of T. gondii. Data obtained from this study contribute to better understanding of T. gondii and serve as a reference for future studies on this parasite.
    Matched MeSH terms: Genome, Protozoan*
  6. Fulltext Genomic analysis of the causative agents of coccidiosis in domestic chickens
    Reid AJ, Blake DP, Ansari HR, Billington K, Browne HP, Bryant J, et al.
    Genome Res, 2014 Oct;24(10):1676-85.
    PMID: 25015382 DOI: 10.1101/gr.168955.113
    Global production of chickens has trebled in the past two decades and they are now the most important source of dietary animal protein worldwide. Chickens are subject to many infectious diseases that reduce their performance and productivity. Coccidiosis, caused by apicomplexan protozoa of the genus Eimeria, is one of the most important poultry diseases. Understanding the biology of Eimeria parasites underpins development of new drugs and vaccines needed to improve global food security. We have produced annotated genome sequences of all seven species of Eimeria that infect domestic chickens, which reveal the full extent of previously described repeat-rich and repeat-poor regions and show that these parasites possess the most repeat-rich proteomes ever described. Furthermore, while no other apicomplexan has been found to possess retrotransposons, Eimeria is home to a family of chromoviruses. Analysis of Eimeria genes involved in basic biology and host-parasite interaction highlights adaptations to a relatively simple developmental life cycle and a complex array of co-expressed surface proteins involved in host cell binding.
    Matched MeSH terms: Genome, Protozoan*
  7. Fulltext Plasmodium knowlesi genome sequences from clinical isolates reveal extensive genomic dimorphism
    Pinheiro MM, Ahmed MA, Millar SB, Sanderson T, Otto TD, Lu WC, et al.
    PLoS One, 2015;10(4):e0121303.
    PMID: 25830531 DOI: 10.1371/journal.pone.0121303
    Plasmodium knowlesi is a newly described zoonosis that causes malaria in the human population that can be severe and fatal. The study of P. knowlesi parasites from human clinical isolates is relatively new and, in order to obtain maximum information from patient sample collections, we explored the possibility of generating P. knowlesi genome sequences from archived clinical isolates. Our patient sample collection consisted of frozen whole blood samples that contained excessive human DNA contamination and, in that form, were not suitable for parasite genome sequencing. We developed a method to reduce the amount of human DNA in the thawed blood samples in preparation for high throughput parasite genome sequencing using Illumina HiSeq and MiSeq sequencing platforms. Seven of fifteen samples processed had sufficiently pure P. knowlesi DNA for whole genome sequencing. The reads were mapped to the P. knowlesi H strain reference genome and an average mapping of 90% was obtained. Genes with low coverage were removed leaving 4623 genes for subsequent analyses. Previously we identified a DNA sequence dimorphism on a small fragment of the P. knowlesi normocyte binding protein xa gene on chromosome 14. We used the genome data to assemble full-length Pknbpxa sequences and discovered that the dimorphism extended along the gene. An in-house algorithm was developed to detect SNP sites co-associating with the dimorphism. More than half of the P. knowlesi genome was dimorphic, involving genes on all chromosomes and suggesting that two distinct types of P. knowlesi infect the human population in Sarawak, Malaysian Borneo. We use P. knowlesi clinical samples to demonstrate that Plasmodium DNA from archived patient samples can produce high quality genome data. We show that analyses, of even small numbers of difficult clinical malaria isolates, can generate comprehensive genomic information that will improve our understanding of malaria parasite diversity and pathobiology.
    Matched MeSH terms: Genome, Protozoan*
  8. In silico identification and validation of a novel hypothetical protein in Cryptosporidium hominis and virtual screening of inhibitors as therapeutics
    Shrivastava AK, Kumar S, Sahu PS, Mahapatra RK
    Parasitol Res, 2017 May;116(5):1533-1544.
    PMID: 28389892 DOI: 10.1007/s00436-017-5430-1
    Computational approaches to predict structure/function and other biological characteristics of proteins are becoming more common in comparison to the traditional methods in drug discovery. Cryptosporidiosis is a major zoonotic diarrheal disease particularly in children, which is caused primarily by Cryptosporidium hominis and Cryptosporidium parvum. Currently, there are no vaccines for cryptosporidiosis and recommended drugs are ineffective. With the availability of complete genome sequence of C. hominis, new targets have been recognized for the development of effective and better drugs and/or vaccines. We identified a unique hypothetical protein (TU502HP) in the C. hominis genome from the CryptoDB database. A three-dimensional model of the protein was generated using the Iterative Threading ASSEmbly Refinement server through an iterative threading method. Functional annotation and phylogenetic study of TU502HP protein revealed similarity with human transportin 3. The model is further subjected to a virtual screening study form the ZINC database compound library using the Dock Blaster server. A docking study through AutoDock software reported N-(3-chlorobenzyl)ethane-1,2-diamine as the best inhibitor in terms of docking score and binding energy. The reliability of the binding mode of the inhibitor is confirmed by a complex molecular dynamics simulation study using GROMACS software for 10 ns in the water environment. Furthermore, antigenic determinants of the protein were determined with the help of DNASTAR software. Our findings report a great potential in order to provide insights in the development of new drug(s) or vaccine(s) for treatment and prophylaxis of cryptosporidiosis among humans and animals.
    Matched MeSH terms: Genome, Protozoan/genetics
  9. Fulltext Updates on k13 mutant alleles for artemisinin resistance in Plasmodium falciparum
    Zaw MT, Emran NA, Lin Z
    J Microbiol Immunol Infect, 2018 Apr;51(2):159-165.
    PMID: 28711439 DOI: 10.1016/j.jmii.2017.06.009
    BACKGROUND: In the fight against malaria caused by Plasmodium falciparum, the successes achieved by artemisinin were endangered by resistance of the parasites to the drug. Whole genome sequencing approach on artemisinin resistant parasite line discovered k13 gene associated with drug resistance. In vitro and in vivo studies indicated mutations in the k13 gene were linked to the artemisinin resistance.

    METHODOLOGY: The literatures published after April, 2015 up to December, 2016 on k13 mutant alleles for artemisinin resistance in Plasmodium falciparum and relevant literatures were comprehensively reviewed.

    RESULTS: To date, 13 non-synonymous mutations of k13 gene have been observed to have slow parasite clearance. Worldwide mapping of k13 mutant alleles have shown mutants associated with artemisinin resistance were confined to southeast Asia and China and did not invade to African countries. Although in vitro ring stage survival assay of 0-3 h was a recently developed assay, it was useful for rapid detection of artemisinin resistance associated k13 allelic marker in the parasite. Recently, dissemination of k13 mutant alleles was recommended to be investigated by identity of haplotypes. Significant characteristics of well described alleles in the reports were mentioned in this review for the benefit of future studies.

    CONCLUSION: According to the updates in the review, it can be concluded artemisinin resistance does not disseminate to India and African countries within short period whereas regular tracking of these mutants is necessary.

    Matched MeSH terms: Genome, Protozoan/genetics
  10. Fulltext A molecular barcode to inform the geographical origin and transmission dynamics of Plasmodium vivax malaria
    Diez Benavente E, Campos M, Phelan J, Nolder D, Dombrowski JG, Marinho CRF, et al.
    PLoS Genet, 2020 02;16(2):e1008576.
    PMID: 32053607 DOI: 10.1371/journal.pgen.1008576
    Although Plasmodium vivax parasites are the predominant cause of malaria outside of sub-Saharan Africa, they not always prioritised by elimination programmes. P. vivax is resilient and poses challenges through its ability to re-emerge from dormancy in the human liver. With observed growing drug-resistance and the increasing reports of life-threatening infections, new tools to inform elimination efforts are needed. In order to halt transmission, we need to better understand the dynamics of transmission, the movement of parasites, and the reservoirs of infection in order to design targeted interventions. The use of molecular genetics and epidemiology for tracking and studying malaria parasite populations has been applied successfully in P. falciparum species and here we sought to develop a molecular genetic tool for P. vivax. By assembling the largest set of P. vivax whole genome sequences (n = 433) spanning 17 countries, and applying a machine learning approach, we created a 71 SNP barcode with high predictive ability to identify geographic origin (91.4%). Further, due to the inclusion of markers for within population variability, the barcode may also distinguish local transmission networks. By using P. vivax data from a low-transmission setting in Malaysia, we demonstrate the potential ability to infer outbreak events. By characterising the barcoding SNP genotypes in P. vivax DNA sourced from UK travellers (n = 132) to ten malaria endemic countries predominantly not used in the barcode construction, we correctly predicted the geographic region of infection origin. Overall, the 71 SNP barcode outperforms previously published genotyping methods and when rolled-out within new portable platforms, is likely to be an invaluable tool for informing targeted interventions towards elimination of this resilient human malaria.
    Matched MeSH terms: Genome, Protozoan/genetics*
  11. Fulltext Characterisation of full-length cDNA sequences provides insights into the Eimeria tenella transcriptome
    Amiruddin N, Lee XW, Blake DP, Suzuki Y, Tay YL, Lim LS, et al.
    BMC Genomics, 2012 Jan 13;13:21.
    PMID: 22244352 DOI: 10.1186/1471-2164-13-21
    BACKGROUND: Eimeria tenella is an apicomplexan parasite that causes coccidiosis in the domestic fowl. Infection with this parasite is diagnosed frequently in intensively reared poultry and its control is usually accorded a high priority, especially in chickens raised for meat. Prophylactic chemotherapy has been the primary method used for the control of coccidiosis. However, drug efficacy can be compromised by drug-resistant parasites and the lack of new drugs highlights demands for alternative control strategies including vaccination. In the long term, sustainable control of coccidiosis will most likely be achieved through integrated drug and vaccination programmes. Characterisation of the E. tenella transcriptome may provide a better understanding of the biology of the parasite and aid in the development of a more effective control for coccidiosis.

    RESULTS: More than 15,000 partial sequences were generated from the 5' and 3' ends of clones randomly selected from an E. tenella second generation merozoite full-length cDNA library. Clustering of these sequences produced 1,529 unique transcripts (UTs). Based on the transcript assembly and subsequently primer walking, 433 full-length cDNA sequences were successfully generated. These sequences varied in length, ranging from 441 bp to 3,083 bp, with an average size of 1,647 bp. Simple sequence repeat (SSR) analysis identified CAG as the most abundant trinucleotide motif, while codon usage analysis revealed that the ten most infrequently used codons in E. tenella are UAU, UGU, GUA, CAU, AUA, CGA, UUA, CUA, CGU and AGU. Subsequent analysis of the E. tenella complete coding sequences identified 25 putative secretory and 60 putative surface proteins, all of which are now rational candidates for development as recombinant vaccines or drug targets in the effort to control avian coccidiosis.

    CONCLUSIONS: This paper describes the generation and characterisation of full-length cDNA sequences from E. tenella second generation merozoites and provides new insights into the E. tenella transcriptome. The data generated will be useful for the development and validation of diagnostic and control strategies for coccidiosis and will be of value in annotation of the E. tenella genome sequence.

    Matched MeSH terms: Genome, Protozoan
  12. Fulltext Plasmodium knowlesi: reservoir hosts and tracking the emergence in humans and macaques
    Lee KS, Divis PC, Zakaria SK, Matusop A, Julin RA, Conway DJ, et al.
    PLoS Pathog, 2011 Apr;7(4):e1002015.
    PMID: 21490952 DOI: 10.1371/journal.ppat.1002015
    Plasmodium knowlesi, a malaria parasite originally thought to be restricted to macaques in Southeast Asia, has recently been recognized as a significant cause of human malaria. Unlike the benign and morphologically similar P. malariae, these parasites can lead to fatal infections. Malaria parasites, including P. knowlesi, have not yet been detected in macaques of the Kapit Division of Malaysian Borneo, where the majority of human knowlesi malaria cases have been reported. In order to extend our understanding of the epidemiology and evolutionary history of P. knowlesi, we examined 108 wild macaques for malaria parasites and sequenced the circumsporozoite protein (csp) gene and mitochondrial (mt) DNA of P. knowlesi isolates derived from macaques and humans. We detected five species of Plasmodium (P. knowlesi, P. inui, P. cynomolgi, P. fieldi and P. coatneyi) in the long-tailed and pig-tailed macaques, and an extremely high prevalence of P. inui and P. knowlesi. Macaques had a higher number of P. knowlesi genotypes per infection than humans, and some diverse alleles of the P. knowlesi csp gene and certain mtDNA haplotypes were shared between both hosts. Analyses of DNA sequence data indicate that there are no mtDNA lineages associated exclusively with either host. Furthermore, our analyses of the mtDNA data reveal that P. knowlesi is derived from an ancestral parasite population that existed prior to human settlement in Southeast Asia, and underwent significant population expansion approximately 30,000-40,000 years ago. Our results indicate that human infections with P. knowlesi are not newly emergent in Southeast Asia and that knowlesi malaria is primarily a zoonosis with wild macaques as the reservoir hosts. However, ongoing ecological changes resulting from deforestation, with an associated increase in the human population, could enable this pathogenic species of Plasmodium to switch to humans as the preferred host.
    Matched MeSH terms: Genome, Protozoan
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