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Parasite-specific CD8 T cell responses play a key role in mediating immunity against Theileria parva in cattle (Bos taurus), and there is evidence that efficient induction of these responses requires CD4 T cell responses. However, information on the antigenic specificity of the CD4 T cell response is lacking. The current study used a high-throughput system for Ag identification using CD4 T cells from immune animals to screen a library of ∼40,000 synthetic peptides representing 499 T. parva gene products. Use of CD4 T cells from 12 immune cattle, representing 12 MHC class II types, identified 26 Ags. Unlike CD8 T cell responses, which are focused on a few dominant Ags, multiple Ags were recognized by CD4 T cell responses of individual animals. The Ags had diverse properties, but included proteins encoded by two multimember gene families: five haloacid dehalogenases and five subtelomere-encoded variable secreted proteins. Most Ags had predicted signal peptides and/or were encoded by abundantly transcribed genes, but neither parameter on their own was reliable for predicting antigenicity. Mapping of the epitopes confirmed presentation by DR or DQ class II alleles and comparison of available T. parva genome sequences demonstrated that they included both conserved and polymorphic epitopes. Immunization of animals with vaccine vectors expressing two of the Ags demonstrated induction of CD4 T cell responses capable of recognizing parasitized cells. The results of this study provide detailed insight into the CD4 T cell responses induced by T. parva and identify Ags suitable for use in vaccine development.

The Human Microbiome Project (HMP) explored microbial communities of the human body in both healthy and disease states. Two phases of the HMP (HMP and iHMP) together generated >48TB of data (public and controlled access) from multiple, varied omics studies of both the microbiome and associated hosts. The Human Microbiome Project Data Coordination Center (HMPDACC) was established to provide a portal to access data and resources produced by the HMP. The HMPDACC provides a unified data repository, multi-faceted search functionality, analysis pipelines and standardized protocols to facilitate community use of HMP data. Recent efforts have been put toward making HMP data more findable, accessible, interoperable and reusable. HMPDACC resources are freely available at www.hmpdacc.org.

Parasite-specific CD8 T cell responses play a key role in mediating immunity against

Babesia bovis is a hemoprotozoan parasite of cattle that has a complex life cycle within vertebrate and invertebrate hosts. In the mammalian host, B. bovis undergoes asexual reproduction while in the tick midgut, gametes are induced, fuse, and form zygotes. The zygote infects tick gut epithelial cells and transform into kinetes that are released into the hemolymph and invade other tick tissues such as the ovaries, resulting in transovarial transmission to tick offspring. To compare gene regulation between different B. bovis life stages, we collected parasites infecting bovine erythrocytes and tick hemolymph. Total RNA samples were isolated, and multiplexed libraries sequenced using paired-end 100 cycle reads of a HiSeq 2500. The data was normalized using the TMM method and analysed for significant differential expression using the generalized linear model likelihood ratio test (GLM LRT) in edgeR. To validate our datasets, ten genes were selected using NormFinder. Genes that had no significant fold change between the blood and tick stages in the RNA-Seq datasets were tested by quantitative PCR to determine their suitability as "housekeeping" genes. The normalized RNA-Seq data revealed genes upregulated during infection of the mammalian host or tick vector and six upregulated genes were validated by quantitative PCR. These datasets can help identify useful targets for controlling bovine babesiosis.

Theileria parva is an economically important, intracellular, tick-transmitted parasite of cattle. A live vaccine against the parasite is effective against challenge from cattle-transmissible T. parva but not against genotypes originating from the African Cape buffalo, a major wildlife reservoir, prompting the need to characterize genome-wide variation within and between cattle- and buffalo-associated T. parva populations. Here, we describe a capture-based target enrichment approach that enables, for the first time, de novo assembly of nearly complete T. parva genomes derived from infected host cell lines. This approach has exceptionally high specificity and sensitivity and is successful for both cattle- and buffalo-derived T. parva parasites. De novo genome assemblies generated for cattle genotypes differ from the reference by ~54K single nucleotide polymorphisms (SNPs) throughout the 8.31 Mb genome, an average of 6.5 SNPs/kb. We report the first buffalo-derived T. parva genome, which is ~20 kb larger than the genome from the reference, cattle-derived, Muguga strain, and contains 25 new potential genes. The average non-synonymous nucleotide diversity (πN) per gene, between buffalo-derived T. parva and the Muguga strain, was 1.3%. This remarkably high level of genetic divergence is supported by an average Wright’s fixation index (FST), genome-wide, of 0.44, reflecting a degree of genetic differentiation between cattle- and buffalo-derived T. parva parasites more commonly seen between, rather than within, species. These findings present clear implications for vaccine development, further demonstrated by the ability to assemble nearly all known antigens in the buffalo-derived strain, which will be critical in design of next generation vaccines. The DNA capture approach used provides a clear advantage in specificity over alternative T. parva DNA enrichment methods used previously, such as those that utilize schizont purification, is less labor intensive, and enables in-depth comparative genomics in this apicomplexan parasite., Author summary An estimated 50 million cattle in sub-Saharan Africa are at risk of the deadly livestock disease East coast fever (ECF), caused by the parasite Theileria parva, which imposes tremendous economic hardship on smallholder farmers. An existing ECF vaccine protects against strains circulating among cattle, but not against T. parva derived from African Cape buffalo, its main wildlife carrier. Understanding this difference in protective efficacy requires characterization of the genetic diversity in T. parva strains associated with each mammalian host, a goal that has been hindered by the proliferation of T. parva in nucleated host cells, with much larger genomes. Here we adapted a sequence capture approach to target the whole parasite genome, enabling enrichment of parasite DNA over that of the host. Choices in protocol development resulted in nearly 100% parasite genome specificity and sensitivity, making this approach the most successful yet to generate T. parva genome sequence data in a high-throughput manner. The analyses uncovered a degree of genetic differentiation between cattle- and buffalo-derived genotypes that is akin to levels more commonly seen between species. This approach, which will enable an in-depth T. parva population genomics study from cattle and buffalo in the endemic regions, can easily be adapted to other intracellular pathogens.

Circumsporozoite protein (CSP) coats the Plasmodium falciparum sporozoite surface and is a major malaria subunit vaccine target. We measured epitope-specific reactivity to field-derived CSP haplotypes in serum samples from Malian adults and children on a custom peptide microarray. Compared to children, adults showed greater antibody responses and responses to more variants in regions proximal to and within the central repeat region. Children acquired short-lived immunity to an epitope proximal to the central repeat region but not to the central repeat region itself. This approach has the potential to differentiate immunodominant from protective epitope-specific responses when combined with longitudinal infection data.

Background The apicomplexan parasite Theileria parva causes a livestock disease called East coast fever (ECF), with millions of animals at risk in sub-Saharan East and Southern Africa, the geographic distribution of T. parva. Over a million bovines die each year of ECF, with a tremendous economic burden to pastoralists in endemic countries. Comprehensive, accurate parasite genome annotation can facilitate the discovery of novel chemotherapeutic targets for disease treatment, as well as elucidate the biology of the parasite. However, genome annotation remains a significant challenge because of limitations in the quality and quantity of the data being used to inform the location and function of protein-coding genes and, when RNA data are used, the underlying biological complexity of the processes involved in gene expression. Here, we apply our recently published RNAseq dataset derived from the schizont life-cycle stage of T. parva to update structural and functional gene annotations across the entire nuclear genome. Results The re-annotation effort lead to evidence-supported updates in over half of all protein-coding sequence (CDS) predictions, including exon changes, gene merges and gene splitting, an increase in average CDS length of approximately 50 base pairs, and the identification of 128 new genes. Among the new genes identified were those involved in N-glycosylation, a process previously thought not to exist in this organism and a potentially new chemotherapeutic target pathway for treating ECF. Alternatively-spliced genes were identified, and antisense and multi-gene family transcription were extensively characterized. Conclusions The process of re-annotation led to novel insights into the organization and expression profiles of protein-coding sequences in this parasite, and uncovered a minimal N-glycosylation pathway that changes our current understanding of the evolution of this post-translational modification in apicomplexan parasites.

Babesia microti, a tick-transmitted, intraerythrocytic protozoan parasite circulating mainly among small mammals, is the primary cause of human babesiosis. While most cases are transmitted by Ixodes ticks, the disease may also be transmitted through blood transfusion and perinatally. A comprehensive analysis of genome composition, genetic diversity, and gene expression profiling of seven B. microti isolates revealed that genetic variation in isolates from the Northeast United States is almost exclusively associated with genes encoding the surface proteome and secretome of the parasite. Furthermore, we found that polymorphism is restricted to a small number of genes, which are highly expressed during infection. In order to identify pathogen-encoded factors involved in host-parasite interactions, we screened a proteome array comprised of 174 B. microti proteins, including several predicted members of the parasite secretome. Using this immuno-proteomic approach we identified several novel antigens that trigger strong host immune responses during the onset of infection. The genomic and immunological data presented herein provide the first insights into the determinants of B. microti interaction with its mammalian hosts and their relevance for understanding the selective pressures acting on parasite evolution.

Human cryptosporidiosis is caused primarily by Cryptosporidium hominis, C. parvum and C. meleagridis. To accelerate research on parasites in the genus Cryptosporidium, we generated annotated, draft genome sequences of human C. hominis isolates TU502_2012 and UKH1, C. meleagridis UKMEL1, also isolated from a human patient, and the avian parasite C. baileyi TAMU-09Q1. The annotation of the genome sequences relied in part on RNAseq data generated from the oocyst stage of both C. hominis and C. baileyi. The genome assembly of C. hominis is significantly more complete and less fragmented than that available previously, which enabled the generation of a much-improved gene set for this species, with an increase in average gene length of 500 bp relative to the protein-encoding genes in the 2004 C. hominis annotation. Our results reveal that the genomes of C. hominis and C. parvum are very similar in both gene density and average gene length. These data should prove a valuable resource for the Cryptosporidium research community., The release of the draft genome sequence, and corresponding annotation, of Cryptosporidium baileyi, C. hominis isolates TU502_2012 and UKH1, and C. meleagridis, will accelerate research on Cryptosporidium parasites.

Human cryptosporidiosis, caused primarily by Cryptosporidium hominis and a subset of Cryptosporidium parvum, is a major cause of moderate-to-severe diarrhea in children under 5 years of age in developing countries and can lead to nutritional stunting and death. Cryptosporidiosis is particularly severe and potentially lethal in immunocompromised hosts. Biological and technical challenges have impeded traditional vaccinology approaches to identify novel targets for the development of vaccines against C. hominis, the predominant species associated with human disease. We deemed that the existence of genomic resources for multiple species in the genus, including a much-improved genome assembly and annotation for C. hominis, makes a reverse vaccinology approach feasible. To this end, we sought to generate a searchable online resource, termed C. hominis gene catalog, which registers all C. hominis genes and their properties relevant for the identification and prioritization of candidate vaccine antigens, including physical attributes, properties related to antigenic potential and expression data. Using bioinformatic approaches, we identified ∼400 C. hominis genes containing properties typical of surface-exposed antigens, such as predicted glycosylphosphatidylinositol (GPI)-anchor motifs, multiple transmembrane motifs and/or signal peptides targeting the encoded protein to the secretory pathway. This set can be narrowed further, e.g. by focusing on potential GPI-anchored proteins lacking homologs in the human genome, but with homologs in the other Cryptosporidium species for which genomic data are available, and with low amino acid polymorphism. Additional selection criteria related to recombinant expression and purification include minimizing predicted post-translation modifications and potential disulfide bonds. Forty proteins satisfying these criteria were selected from 3745 proteins in the updated C. hominis annotation. The immunogenic potential of a few of these is currently being tested.Database URL: http://cryptogc.igs.umaryland.edu.

Additional file 1: Supplemental Figure S1. Architecture of the Theileria parva Muguga genome and associated new structural annotation and RNAseq expression data. Supplemental Figure S2. Updated gene annotation efforts in the Theileria parva genome reveal the existence of many genes that overlap adjacent genes at either UTR or CDS sequences. Supplemental Figure S3. RNA-seq reads that map to introns in the Theileria parva Muguga genome support the existence of genes where a subset of introns are not spliced. Supplemental Figure S4. Histogram of mRNA length across all annotated transcripts in the current T. parva Muguga annotation. Supplementary Fig.ure S5. Types of transcription initiated in Theileria parva. Supplemental Figure S7. The expression distribution of three selected gene families in T. parva Muguga (SVSP = subtelomeric variable secreted protein gene family; Tpr = T. parva repeat gene family; TashAT = Theileria annulata schizont AT hook gene family). Supplementary Figure S8. Theileria PIN1 is an example of a parasite-secreted protein that plays a role in host transformation. Supplemental Figure S9. The expression N-glycosylation pathway components in the sporozoite and schizont life cycle stages of T. parva Muguga. Supplemental Figure S10. A representation of the relative weights of each evidence in each EVM prediction tested. Supplemental Figure S11. The percentage of validated genes, or coding exons correctly predicted by EVM with each evidence combination. Supplemental Figure S12. A comparison of the prediction accuracy of each gene predictor used in this study. Supplemental Table S1. RNAseq read counts, length and GC content of each T. parva chromosome. Supplemental Table S2. A comparison of genome characteristics of T. parva Muguga to several other piroplasms and Plasmodium falciparum 3D7. Supplemental Table S3. A list of the expression levels (RPKM = reads per kilobase of transcript per million reads) of known T. parva antigens. Supplemental Table S4. A list of the most highly-expressed genes in the T. parva schizont RNAseq dataset. Supplemental Table S5. A table of key T. parva genes with reads per kilobase of transcript per million reads of zero. (Tp = Theileria parva Muguga; Pf = Plasmodium falciparum 3D7). Supplemental Table S6. A description of the top 20 largest multi-gene families defined by OrthoMCL in T. parva Muguga and their conservation in T. annulata (Ta), T. orientalis (To), and T. equi (Te), as defined by Jaccard-filtered clusters of orthologous genes. Supplemental Table S7. Summary of the top-ranked Phyre2 hits for each proposed Alg homolog discussed in this study. Supplemental Table S8. The exon distribution of the validation and training sets used for gene prediction.

Additional file 1: Supplemental Figure S1. Architecture of the Theileria parva Muguga genome and associated new structural annotation and RNAseq expression data. Supplemental Figure S2. Updated gene annotation efforts in the Theileria parva genome reveal the existence of many genes that overlap adjacent genes at either UTR or CDS sequences. Supplemental Figure S3. RNA-seq reads that map to introns in the Theileria parva Muguga genome support the existence of genes where a subset of introns are not spliced. Supplemental Figure S4. Histogram of mRNA length across all annotated transcripts in the current T. parva Muguga annotation. Supplementary Fig.ure S5. Types of transcription initiated in Theileria parva. Supplemental Figure S7. The expression distribution of three selected gene families in T. parva Muguga (SVSP = subtelomeric variable secreted protein gene family; Tpr = T. parva repeat gene family; TashAT = Theileria annulata schizont AT hook gene family). Supplementary Figure S8. Theileria PIN1 is an example of a parasite-secreted protein that plays a role in host transformation. Supplemental Figure S9. The expression N-glycosylation pathway components in the sporozoite and schizont life cycle stages of T. parva Muguga. Supplemental Figure S10. A representation of the relative weights of each evidence in each EVM prediction tested. Supplemental Figure S11. The percentage of validated genes, or coding exons correctly predicted by EVM with each evidence combination. Supplemental Figure S12. A comparison of the prediction accuracy of each gene predictor used in this study. Supplemental Table S1. RNAseq read counts, length and GC content of each T. parva chromosome. Supplemental Table S2. A comparison of genome characteristics of T. parva Muguga to several other piroplasms and Plasmodium falciparum 3D7. Supplemental Table S3. A list of the expression levels (RPKM = reads per kilobase of transcript per million reads) of known T. parva antigens. Supplemental Table S4. A list of the most highly-expressed genes in the T. parva schizont RNAseq dataset. Supplemental Table S5. A table of key T. parva genes with reads per kilobase of transcript per million reads of zero. (Tp = Theileria parva Muguga; Pf = Plasmodium falciparum 3D7). Supplemental Table S6. A description of the top 20 largest multi-gene families defined by OrthoMCL in T. parva Muguga and their conservation in T. annulata (Ta), T. orientalis (To), and T. equi (Te), as defined by Jaccard-filtered clusters of orthologous genes. Supplemental Table S7. Summary of the top-ranked Phyre2 hits for each proposed Alg homolog discussed in this study. Supplemental Table S8. The exon distribution of the validation and training sets used for gene prediction.