What is new in FungiDB: a web-based bioinformatics platform for omics-scale data analysis for fungal and oomycete species.

FungiDB (https://fungidb.org) serves as a valuable online resource that seamlessly integrates genomic and related large-scale data for a wide range of fungal and oomycete species. As an integral part of the VEuPathDB Bioinformatics Resource Center (https://veupathdb.org), FungiDB continually integrates both published and unpublished data addressing various aspects of fungal biology. Established in early 2011, the database has evolved to support 674 datasets. The datasets include over 300 genomes spanning various taxa (e.g. Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Mucoromycota, as well as Albuginales, Peronosporales, Pythiales, and Saprolegniales). In addition to genomic assemblies and annotation, over 300 extra datasets encompassing diverse information, such as expression and variation data, are also available. The resource also provides an intuitive web-based interface, facilitating comprehensive approaches to data mining and visualization. Users can test their hypotheses and navigate through omics-scale datasets using a built-in search strategy system. Moreover, FungiDB offers capabilities for private data analysis via the integrated VEuPathDB Galaxy platform. FungiDB also permits genome improvements by capturing expert knowledge through the User Comments system and the Apollo genome annotation editor for structural and functional gene curation. FungiDB facilitates data exploration and analysis and contributes to advancing research efforts by capturing expert knowledge for fungal and oomycete species.

"Evaluating the Benefits and Limits of Multiple Displacement Amplification with Whole-Genome Oxford Nanopore Sequencing".

Multiple Displacement Amplification (MDA) outperforms conventional PCR in long fragment and whole genome amplification which makes it attractive to couple with long-read sequencing of samples with limited quantities of DNA to obtain improved genome assemblies. Here, we explore the efficacy and limits of MDA for genome sequence assembly using Oxford Nanopore Technologies (ONT) rapid library preparations and minION sequencing. We successfully generated almost complete genome sequences for all organisms examined, including Cryptosporidium meleagridis, Staphylococcus aureus, Enterococcus faecium, and Escherichia coli, with the ability to generate high-quality data from samples starting with only 0.025 ng of total DNA. Controlled sheared DNA samples exhibited a distinct pattern of size-increase after MDA, which may be associated with the amplification of long, low-abundance fragments present in the assay, as well as generating concatemeric sequences during amplification. To address concatemers, we developed a computational pipeline (CADECT: Concatemer Detection Tool) to identify and remove putative concatemeric sequences. This study highlights the efficacy of MDA in generating high-quality genome assemblies from limited amounts of input DNA. Also, the CADECT pipeline effectively mitigated the impact of concatemeric sequences, enabling the assembly of contiguous sequences even in cases where the input genomic DNA was degraded. These results have significant implications for the study of organisms that are challenging to culture in vitro, such as Cryptosporidium, and for expediting critical results in clinical settings with limited quantities of available genomic DNA.

A new chromosome-level genome assembly and annotation of Cryptosporidium meleagridis.

Cryptosporidium spp. are medically and scientifically relevant protozoan parasites that cause severe diarrheal illness in infants and immunosuppressed populations as well as animals. Although most human Cryptosporidium infections are caused by C. parvum and C. hominis, there are several other human-infecting species including C. meleagridis, which is commonly observed in developing countries. Here, we polished and annotated a long-read genome sequence assembly for C. meleagridis TU1867, a species which infects birds and humans. The genome sequence was generated using a combination of whole genome amplification (WGA) and long-read Oxford Nanopore Technologies sequencing. The assembly was then polished with Illumina data. The chromosome-level genome assembly is 9.2 Mbp with a contig N50 of 1.1 Mb. Annotation revealed 3,923 protein-coding genes. A BUSCO analysis indicates a completeness of 96.6% (n=446), including 430 (96.4%) single-copy and 1 (0.224%) duplicated apicomplexan conserved gene(s). The new C. meleagridis genome assembly is nearly gap-free and provides a valuable new resource for the Cryptosporidium community and future studies on evolution and host-specificity.

Genomic and virulence analysis of in vitro cultured Cryptosporidium parvum.

Recent advances in the in vitro cultivation of Cryptosporidium parvum using hollow fiber bioreactor technology (HFB) have permitted continuous growth of parasites that complete all life cycle stages. The method provides access to all stages of the parasite and provides a method for non-animal production of oocysts for use in clinical trials. Here we examined the effect of long-term (>20 months) in vitro culture on virulence-factors, genome conservation, and in vivo pathogenicity of the host by in vitro cultured parasites. We find low-level sequence variation that is consistent with that observed in calf-passaged parasites. Further using a calf model infection, oocysts obtained from the HFB caused diarrhea of the same volume, duration and oocyst shedding intensity as in vivo passaged parasites.

VEuPathDB: the eukaryotic pathogen, vector and host bioinformatics resource center in 2023.

The Eukaryotic Pathogen, Vector and Host Informatics Resource (VEuPathDB, https://veupathdb.org) is a Bioinformatics Resource Center funded by the National Institutes of Health with additional funding from the Wellcome Trust. VEuPathDB supports >600 organisms that comprise invertebrate vectors, eukaryotic pathogens (protists and fungi) and relevant free-living or non-pathogenic species or hosts. Since 2004, VEuPathDB has analyzed omics data from the public domain using contemporary bioinformatic workflows, including orthology predictions via OrthoMCL, and integrated the analysis results with analysis tools, visualizations, and advanced search capabilities. The unique data mining platform coupled with >3000 pre-analyzed data sets facilitates the exploration of pertinent omics data in support of hypothesis driven research. Comparisons are easily made across data sets, data types and organisms. A Galaxy workspace offers the opportunity for the analysis of private large-scale datasets and for porting to VEuPathDB for comparisons with integrated data. The MapVEu tool provides a platform for exploration of spatially resolved data such as vector surveillance and insecticide resistance monitoring. To address the growing body of omics data and advances in laboratory techniques, VEuPathDB has added several new data types, searches and features, improved the Galaxy workspace environment, redesigned the MapVEu interface and updated the infrastructure to accommodate these changes.

Genetic crosses within and between species of Cryptosporidium.

Parasites and their hosts are engaged in reciprocal coevolution that balances competing mechanisms of virulence, resistance, and evasion. This often leads to host specificity, but genomic reassortment between different strains can enable parasites to jump host barriers and conquer new niches. In the apicomplexan parasite Cryptosporidium, genetic exchange has been hypothesized to play a prominent role in adaptation to humans. The sexual lifecycle of the parasite provides a potential mechanism for such exchange; however, the boundaries of Cryptosporidium sex are currently undefined. To explore this experimentally, we established a model for genetic crosses. Drug resistance was engineered using a mutated phenylalanyl tRNA synthetase gene and marking strains with this and the previously used Neo transgene enabled selection of recombinant progeny. This is highly efficient, and genomic recombination is evident and can be continuously monitored in real time by drug resistance, flow cytometry, and PCR mapping. Using this approach, multiple loci can now be modified with ease. We demonstrate that essential genes can be ablated by crossing a Cre recombinase driver strain with floxed strains. We further find that genetic crosses are also feasible between species. Crossing Cryptosporidium parvum, a parasite of cattle and humans, and Cryptosporidium tyzzeri a mouse parasite resulted in progeny with a recombinant genome derived from both species that continues to vigorously replicate sexually. These experiments have important fundamental and translational implications for the evolution of Cryptosporidium and open the door to reverse- and forward-genetic analysis of parasite biology and host specificity.

Massive invasion of organellar DNA drives nuclear genome evolution in Toxoplasma.

Toxoplasma gondii is a zoonotic protist pathogen that infects up to one third of the human population. This apicomplexan parasite contains three genome sequences: nuclear (65 Mb); plastid organellar, ptDNA (35 kb); and mitochondrial organellar, mtDNA (5.9 kb of non-repetitive sequence). We find that the nuclear genome contains a significant amount of NUMTs (nuclear integrants of mitochondrial DNA) and NUPTs (nuclear integrants of plastid DNA) that are continuously acquired and represent a significant source of intraspecific genetic variation. NUOT (nuclear DNA of organellar origin) accretion has generated 1.6% of the extant T. gondii ME49 nuclear genome-the highest fraction ever reported in any organism. NUOTs are primarily found in organisms that retain the non-homologous end-joining repair pathway. Significant movement of organellar DNA was experimentally captured via amplicon sequencing of a CRISPR-induced double-strand break in non-homologous end-joining repair competent, but not ku80 mutant, Toxoplasma parasites. Comparisons with Neospora caninum, a species that diverged from Toxoplasma ~28 mya, revealed that the movement and fixation of five NUMTs predates the split of the two genera. This unexpected level of NUMT conservation suggests evolutionary constraint for cellular function. Most NUMT insertions reside within (60%) or nearby genes (23% within 1.5 kb), and reporter assays indicate that some NUMTs have the ability to function as cis-regulatory elements modulating gene expression. Together, these findings portray a role for organellar sequence insertion in dynamically shaping the genomic architecture and likely contributing to adaptation and phenotypic changes in this important human pathogen.

Genetic crosses within and between species of Cryptosporidium.

Parasites and their hosts are engaged in rapid coevolution that balances competing mechanisms of virulence, resistance, and evasion. This often leads to host specificity, but genomic reassortment between different strains can enable parasites to jump host barriers and conquer new niches. In the apicomplexan parasite Cryptosporidium genetic exchange has been hypothesized to play a prominent role in adaptation to humans. The sexual lifecycle of the parasite provides a potential mechanism for such exchange; however, the boundaries of Cryptosporidium sex are currently undefined. To explore this experimentally, we established a model for genetic crosses. Drug resistance was engineered using a mutated phenylalanyl tRNA synthetase gene and marking strains with this and the previously used Neo transgene enabled selection of recombinant progeny. This is highly efficient, and genomic recombination is evident and can be continuously monitored in real time by drug resistance, flow cytometry, and PCR mapping. Using this approach multiple loci can now be modified with ease. We demonstrate that essential genes can be ablated by crossing a Cre recombinase driver strain with floxed strains. We further find that genetic crosses are also feasible between species. Crossing C. parvum, a parasite of cattle and humans, and C. tyzzeri a mouse parasite resulted in progeny with a recombinant genome derived from both species that continues to vigorously replicate sexually. These experiments have important fundamental and translational implications for the evolution of Cryptosporidium and open the door to reverse- and forward- genetic analysis of parasite biology and host specificity.

New T2T assembly of Cryptosporidium parvum IOWA annotated with reference genome gene identifiers.

Cryptosporidium parvum is a significant pathogen causing gastrointestinal infections in humans and animals, that is spread through the ingestion of contaminated food and water. Despite its global impact on public health, generating a C. parvum genome sequence has always been challenging due to a lack of in vitro cultivation systems and challenging sub-telomeric gene families. A gapless telomere to telomere genome assembly has been created for Cryptosporidium parvum IOWA obtained from Bunch Grass Farms, named here as CpBGF. There are 8 chromosomes that total 9,259,183 bp. The new hybrid assembly which was generated with Illumina and Oxford Nanopore resolves complex sub-telomeric regions of chromosomes 1, 7 and 8. To facilitate ease of use and consistency with the literature, whenever possible, chromosomes have been oriented and genes in this annotation have been given the same gene IDs used in the current reference genome sequence generated in 2004. The annotation of this assembly utilized considerable RNA expression evidence, thus, untranslated regions, long noncoding RNAs and antisense RNAs are annotated. The CpBGF genome assembly serves as a valuable resource for understanding the biology, pathogenesis, and transmission of C. parvum, and it facilitates the development of diagnostics, drugs, and vaccines against cryptosporidiosis.

Massive invasion of organellar DNA drives nuclear genome evolution in Toxoplasma.

Toxoplasma gondii is a zoonotic protist pathogen that infects up to 1/3 of the human population. This apicomplexan parasite contains three genome sequences: nuclear (63 Mb); plastid organellar, ptDNA (35 kb); and mitochondrial organellar, mtDNA (5.9 kb of non-repetitive sequence). We find that the nuclear genome contains a significant amount of NUMTs (nuclear DNA of mitochondrial origin) and NUPTs (nuclear DNA of plastid origin) that are continuously acquired and represent a significant source of intraspecific genetic variation. NUOT (nuclear DNA of organellar origin) accretion has generated 1.6% of the extant T. gondii ME49 nuclear genome; the highest fraction ever reported in any organism. NUOTs are primarily found in organisms that retain the non-homologous end-joining repair pathway. Significant movement of organellar DNA was experimentally captured via amplicon sequencing of a CRISPR-induced double-strand break in non-homologous end-joining repair competent, but not ku80 mutant, Toxoplasma parasites. Comparisons with Neospora caninum, a species that diverged from Toxoplasma ~28 MY ago, revealed that the movement and fixation of 5 NUMTs predates the split of the two genera. This unexpected level of NUMT conservation suggests evolutionary constraint for cellular function. Most NUMT insertions reside within (60%) or nearby genes (23% within 1.5 kb) and reporter assays indicate that some NUMTs have the ability to function as cis-regulatory elements modulating gene expression. Together these findings portray a role for organellar sequence insertion in dynamically shaping the genomic architecture and likely contributing to adaptation and phenotypic changes in this important human pathogen.

TriTrypDB: An integrated functional genomics resource for kinetoplastida.

Parasitic diseases caused by kinetoplastid parasites are a burden to public health throughout tropical and subtropical regions of the world. TriTrypDB (https://tritrypdb.org) is a free online resource for data mining of genomic and functional data from these kinetoplastid parasites and is part of the VEuPathDB Bioinformatics Resource Center (https://veupathdb.org). As of release 59, TriTrypDB hosts 83 kinetoplastid genomes, nine of which, including Trypanosoma brucei brucei TREU927, Trypanosoma cruzi CL Brener and Leishmania major Friedlin, undergo manual curation by integrating information from scientific publications, high-throughput assays and user submitted comments. TriTrypDB also integrates transcriptomic, proteomic, epigenomic, population-level and isolate data, functional information from genome-wide RNAi knock-down and fluorescent tagging, and results from automated bioinformatics analysis pipelines. TriTrypDB offers a user-friendly web interface embedded with a genome browser, search strategy system and bioinformatics tools to support custom in silico experiments that leverage integrated data. A Galaxy workspace enables users to analyze their private data (e.g., RNA-sequencing, variant calling, etc.) and explore their results privately in the context of publicly available information in the database. The recent addition of an annotation platform based on Apollo enables users to provide both functional and structural changes that will appear as 'community annotations' immediately and, pending curatorial review, will be integrated into the official genome annotation.

MaHPIC malaria systems biology data from Plasmodium cynomolgi sporozoite longitudinal infections in macaques.

Plasmodium cynomolgi causes zoonotic malarial infections in Southeast Asia and this parasite species is important as a model for Plasmodium vivax and Plasmodium ovale. Each of these species produces hypnozoites in the liver, which can cause relapsing infections in the blood. Here we present methods and data generated from iterative longitudinal systems biology infection experiments designed and performed by the Malaria Host-Pathogen Interaction Center (MaHPIC) to delve deeper into the biology, pathogenesis, and immune responses of P. cynomolgi in the Macaca mulatta host. Infections were initiated by sporozoite inoculation. Blood and bone marrow samples were collected at defined timepoints for biological and computational experiments and integrative analyses revolving around primary illness, relapse illness, and subsequent disease and immune response patterns. Parasitological, clinical, haematological, immune response, and -omic datasets (transcriptomics, proteomics, metabolomics, and lipidomics) including metadata and computational results have been deposited in public repositories. The scope and depth of these datasets are unprecedented in studies of malaria, and they are projected to be a F.A.I.R., reliable data resource for decades.

Plasmodium knowlesi Cytoadhesion Involves SICA Variant Proteins.

Plasmodium knowlesi poses a health threat throughout Southeast Asian communities and currently causes most cases of malaria in Malaysia. This zoonotic parasite species has been studied in Macaca mulatta (rhesus monkeys) as a model for severe malarial infections, chronicity, and antigenic variation. The phenomenon of Plasmodium antigenic variation was first recognized during rhesus monkey infections. Plasmodium-encoded variant proteins were first discovered in this species and found to be expressed at the surface of infected erythrocytes, and then named the Schizont-Infected Cell Agglutination (SICA) antigens. SICA expression was shown to be spleen dependent, as SICA expression is lost after P. knowlesi is passaged in splenectomized rhesus. Here we present data from longitudinal P. knowlesi infections in rhesus with the most comprehensive analysis to date of clinical parameters and infected red blood cell sequestration in the vasculature of tissues from 22 organs. Based on the histopathological analysis of 22 tissue types from 11 rhesus monkeys, we show a comparative distribution of parasitized erythrocytes and the degree of margination of the infected erythrocytes with the endothelium. Interestingly, there was a significantly higher burden of parasites in the gastrointestinal tissues, and extensive margination of the parasites along the endothelium, which may help explain gastrointestinal symptoms frequently reported by patients with P. knowlesi malarial infections. Moreover, this margination was not observed in splenectomized rhesus that were infected with parasites not expressing the SICA proteins. This work provides data that directly supports the view that a subpopulation of P. knowlesi parasites cytoadheres and sequesters, likely via SICA variant antigens acting as ligands. This process is akin to the cytoadhesive function of the related variant antigen proteins, namely Erythrocyte Membrane Protein-1, expressed by Plasmodium falciparum.

Small and intermediate size structural RNAs in the unicellular parasite Cryptosporidium parvum as revealed by sRNA-seq and comparative genomics.

Small and intermediate-size noncoding RNAs (sRNAs and is-ncRNAs) have been shown to play important regulatory roles in the development of several eukaryotic organisms. However, they have not been thoroughly explored in Cryptosporidium parvum, an obligate zoonotic protist parasite responsible for the diarrhoeal disease cryptosporidiosis. Using Illumina sequencing of a small RNA library, a systematic identification of novel small and is-ncRNAs was performed in C. parvum excysted sporozoites. A total of 79 novel is-ncRNA candidates, including antisense, intergenic and intronic is-ncRNAs, were identified, including 7 new small nucleolar RNAs (snoRNAs). Expression of select novel is-ncRNAs was confirmed by RT-PCR. Phylogenetic conservation was analysed using covariance models (CMs) in related Cryptosporidium and apicomplexan parasite genome sequences. A potential new type of small ncRNA derived from tRNA fragments was observed. Overall, a deep profiling analysis of novel is-ncRNAs in C. parvum and related species revealed structural features and conservation of these novel is-ncRNAs. Covariance models can be used to detect is-ncRNA genes in other closely related parasites. These findings provide important new sequences for additional functional characterization of novel is-ncRNAs in the protist pathogen C. parvum.

Malaria disrupts the rhesus macaque gut microbiome.

Previous studies have suggested that a relationship exists between severity and transmissibility of malaria and variations in the gut microbiome, yet only limited information exists on the temporal dynamics of the gut microbial community during a malarial infection. Here, using a rhesus macaque model of relapsing malaria, we investigate how malaria affects the gut microbiome. In this study, we performed 16S sequencing on DNA isolated from rectal swabs of rhesus macaques over the course of an experimental malarial infection with Plasmodium cynomolgi and analyzed gut bacterial taxa abundance across primary and relapsing infections. We also performed metabolomics on blood plasma from the animals at the same timepoints and investigated changes in metabolic pathways over time. Members of Proteobacteria (family Helicobacteraceae) increased dramatically in relative abundance in the animal's gut microbiome during peak infection while Firmicutes (family Lactobacillaceae and Ruminococcaceae), Bacteroidetes (family Prevotellaceae) and Spirochaetes amongst others decreased compared to baseline levels. Alpha diversity metrics indicated decreased microbiome diversity at the peak of parasitemia, followed by restoration of diversity post-treatment. Comparison with healthy subjects suggested that the rectal microbiome during acute malaria is enriched with commensal bacteria typically found in the healthy animal's mucosa. Significant changes in the tryptophan-kynurenine immunomodulatory pathway were detected at peak infection with P. cynomolgi, a finding that has been described previously in the context of P. vivax infections in humans. During relapses, which have been shown to be associated with less inflammation and clinical severity, we observed minimal disruption to the gut microbiome, despite parasites being present. Altogether, these data suggest that the metabolic shift occurring during acute infection is associated with a concomitant shift in the gut microbiome, which is reversed post-treatment.

VEuPathDB: the eukaryotic pathogen, vector and host bioinformatics resource center.

The Eukaryotic Pathogen, Vector and Host Informatics Resource (VEuPathDB, https://veupathdb.org) represents the 2019 merger of VectorBase with the EuPathDB projects. As a Bioinformatics Resource Center funded by the National Institutes of Health, with additional support from the Welllcome Trust, VEuPathDB supports >500 organisms comprising invertebrate vectors, eukaryotic pathogens (protists and fungi) and relevant free-living or non-pathogenic species or hosts. Designed to empower researchers with access to Omics data and bioinformatic analyses, VEuPathDB projects integrate >1700 pre-analysed datasets (and associated metadata) with advanced search capabilities, visualizations, and analysis tools in a graphic interface. Diverse data types are analysed with standardized workflows including an in-house OrthoMCL algorithm for predicting orthology. Comparisons are easily made across datasets, data types and organisms in this unique data mining platform. A new site-wide search facilitates access for both experienced and novice users. Upgraded infrastructure and workflows support numerous updates to the web interface, tools, searches and strategies, and Galaxy workspace where users can privately analyse their own data. Forthcoming upgrades include cloud-ready application architecture, expanded support for the Galaxy workspace, tools for interrogating host-pathogen interactions, and improved interactions with affiliated databases (ClinEpiDB, MicrobiomeDB) and other scientific resources, and increased interoperability with the Bacterial & Viral BRC.

Long-read assembly and comparative evidence-based reanalysis of Cryptosporidium genome sequences reveal expanded transporter repertoire and duplication of entire chromosome ends including subtelomeric regions.

Cryptosporidiosis is a leading cause of waterborne diarrheal disease globally and an important contributor to mortality in infants and the immunosuppressed. Despite its importance, the Cryptosporidium community has only had access to a good, but incomplete, Cryptosporidium parvum IOWA reference genome sequence. Incomplete reference sequences hamper annotation, experimental design, and interpretation. We have generated a new C. parvum IOWA genome assembly supported by Pacific Biosciences (PacBio) and Oxford Nanopore long-read technologies and a new comparative and consistent genome annotation for three closely related species: C. parvum, Cryptosporidium hominis, and Cryptosporidium tyzzeri We made 1926 C. parvum annotation updates based on experimental evidence. They include new transporters, ncRNAs, introns, and altered gene structures. The new assembly and annotation revealed a complete Dnmt2 methylase ortholog. Comparative annotation between C. parvum, C. hominis, and C. tyzzeri revealed that most "missing" orthologs are found, suggesting that the biological differences between the species must result from gene copy number variation, differences in gene regulation, and single-nucleotide variants (SNVs). Using the new assembly and annotation as reference, 190 genes are identified as evolving under positive selection, including many not detected previously. The new C. parvum IOWA reference genome assembly is larger, gap free, and lacks ambiguous bases. This chromosomal assembly recovers all 16 chromosome ends, 13 of which are contiguously assembled. The three remaining chromosome ends are provisionally placed. These ends represent duplication of entire chromosome ends including subtelomeric regions revealing a new level of genome plasticity that will both inform and impact future research.

Clinical recovery of Macaca fascicularis infected with Plasmodium knowlesi.

BACKGROUND: Kra monkeys (Macaca fascicularis), a natural host of Plasmodium knowlesi, control parasitaemia caused by this parasite species and escape death without treatment. Knowledge of the disease progression and resilience in kra monkeys will aid the effective use of this species to study mechanisms of resilience to malaria. This longitudinal study aimed to define clinical, physiological and pathological changes in kra monkeys infected with P. knowlesi, which could explain their resilient phenotype. METHODS: Kra monkeys (n = 15, male, young adults) were infected intravenously with cryopreserved P. knowlesi sporozoites and the resulting parasitaemias were monitored daily. Complete blood counts, reticulocyte counts, blood chemistry and physiological telemetry data (n = 7) were acquired as described prior to infection to establish baseline values and then daily after inoculation for up to 50 days. Bone marrow aspirates, plasma samples, and 22 tissue samples were collected at specific time points to evaluate longitudinal clinical, physiological and pathological effects of P. knowlesi infections during acute and chronic infections. RESULTS: As expected, the kra monkeys controlled acute infections and remained with low-level, persistent parasitaemias without anti-malarial intervention. Unexpectedly, early in the infection, fevers developed, which ultimately returned to baseline, as well as mild to moderate thrombocytopenia, and moderate to severe anaemia. Mathematical modelling and the reticulocyte production index indicated that the anaemia was largely due to the removal of uninfected erythrocytes and not impaired production of erythrocytes. Mild tissue damage was observed, and tissue parasite load was associated with tissue damage even though parasite accumulation in the tissues was generally low. CONCLUSIONS: Kra monkeys experimentally infected with P. knowlesi sporozoites presented with multiple clinical signs of malaria that varied in severity among individuals. Overall, the animals shared common mechanisms of resilience characterized by controlling parasitaemia 3-5 days after patency, and controlling fever, coupled with physiological and bone marrow responses to compensate for anaemia. Together, these responses likely minimized tissue damage while supporting the establishment of chronic infections, which may be important for transmission in natural endemic settings. These results provide new foundational insights into malaria pathogenesis and resilience in kra monkeys, which may improve understanding of human infections.

Challenges for Cryptosporidium Population Studies.

Cryptosporidiosis is ranked sixth in the list of the most important food-borne parasites globally, and it is an important contributor to mortality in infants and the immunosuppressed. Recently, the number of genome sequences available for this parasite has increased drastically. The majority of the sequences are derived from population studies of Cryptosporidium parvum and Cryptosporidium hominis, the most important species causing disease in humans. Work with this parasite is challenging since it lacks an optimal, prolonged, in vitro culture system, which accurately reproduces the in vivo life cycle. This obstacle makes the cloning of isolates nearly impossible. Thus, patient isolates that are sequenced represent a population or, at times, mixed infections. Oocysts, the lifecycle stage currently used for sequencing, must be considered a population even if the sequence is derived from single-cell sequencing of a single oocyst because each oocyst contains four haploid meiotic progeny (sporozoites). Additionally, the community does not yet have a set of universal markers for strain typing that are distributed across all chromosomes. These variables pose challenges for population studies and require careful analyses to avoid biased interpretation. This review presents an overview of existing population studies, challenges, and potential solutions to facilitate future population analyses.

A novel fragmented mitochondrial genome in the protist pathogen Toxoplasma gondii and related tissue coccidia.

Mitochondrial genome content and structure vary widely across the eukaryotic tree of life, with protists displaying extreme examples. Apicomplexan and dinoflagellate protists have evolved highly reduced mitochondrial genome sequences, mtDNA, consisting of only three cytochrome genes and fragmented rRNA genes. Here, we report the independent evolution of fragmented cytochrome genes in Toxoplasma and related tissue coccidia and evolution of a novel genome architecture consisting minimally of 21 sequence blocks (SBs) totaling 5.9 kb that exist as nonrandom concatemers. Single-molecule Nanopore reads consisting entirely of SBs ranging from 0.1 to 23.6 kb reveal both whole and fragmented cytochrome genes. Full-length cytochrome transcripts including a divergent coxIII are detected. The topology of the mitochondrial genome remains an enigma. Analysis of a cob point mutation reveals that homoplasmy of SBs is maintained. Tissue coccidia are important pathogens of man and animals, and the mitochondrion represents an important therapeutic target. The mtDNA sequence has been elucidated, but a definitive genome architecture remains elusive.