Bovine Piroplasma Populations in the Philippines Characterized Using Targeted Amplicon Deep Sequencing.

Molecular assays and capillary electrophoresis sequencing have been used to identify parasites in livestock. The low sample capacity, which increases labor and processing time, is one drawback. Targeted amplicon sequencing (Ampliseq) uses the fast and large sample capacity platform to identify parasites in the target host, overcoming this limitation. DNA was extracted from 162 whole blood samples collected from cattle in three provinces in the Philippines. Using Illumina's Miseq platform, the V4 hypervariable region of the piroplasma 18S rRNA gene was amplified and sequenced. The AMPtk pipeline was used to obtain distinct amplicon sequence variants (ASVs) and the NCBI BLAST non-redundant database was used to assign taxonomy. In total, 95 (58.64%) samples were positive for piroplasma. Using the AMPTk pipeline, 2179 ASVs were obtained. A total of 79 distinct ASVs were obtained after clustering and filtering, which belonged to genera Babesia (n = 58), Theileria (n = 17), Hepatozoon (n = 2), and Sarcocystis (n = 2). The ASV top hits were composed of 10 species: Babesia bovis, B. bigemina, Theileria orientalis, Babesia sp., Hepatozoon canis, Sarcocystis cruzi, T. annulata, T. equi, T. mutans, and Theileria sp. Thung Song. The results generated in this study demonstrated the applicability of Ampliseq in detecting piroplasmid parasites infecting cattle in the Philippines.

Chromosome-level genome assembly of Babesia caballi reveals diversity of multigene families among Babesia species.

BACKGROUND: Babesia caballi is an intraerythrocytic parasite from the phylum Apicomplexa, capable of infecting equids and causing equine piroplasmosis. However, since there is limited genome information available on B. caballi, molecular mechanisms involved in host specificity and pathogenicity of this species have not been fully elucidated yet. RESULTS: Genomic DNA from a B. caballi subclone was purified and sequenced using both Illumina and Nanopore technologies. The resulting assembled sequence consisted of nine contigs with a size of 12.9 Mbp, rendering a total of 5,910 protein-coding genes. The phylogenetic tree of Apicomplexan species was reconstructed using 263 orthologous genes. We identified 481 ves1-like genes and named "ves1c". In contrast, expansion of the major facilitator superfamily (mfs) observed in closely related B. bigemina and B. ovata species was not found in B. caballi. A set of repetitive units containing an open reading frame with a size of 297 bp was also identified. CONCLUSIONS: We present a chromosome-level genome assembly of B. caballi. Our genomic data may contribute to estimating gene expansion events involving multigene families and exploring the evolution of species from this genus.

Single Cell Transcriptomes of In Vitro Bradyzoite Infected Cells Reveals Toxoplasma gondii Stage Dependent Host Cell Alterations.

Toxoplasma gondii bradyzoites establish chronic infections within their host cells. Recent studies have demonstrated that several parasite effector proteins are translocated to host cells during the bradyzoite stage of chronic infection. To understand the interaction between host cells and bradyzoites at the transcriptomic landscape level, we utilized single-cell RNA-sequencing (scRNA-Seq) to characterize the bradyzoite-induced host cell response. Distinct gene expression profiles were observed in infected host, cells with low parasite mapped reads, and mock (non-exposed) control cells. Gene set enrichment analysis showed that c-Myc and NF-κB signaling and energy metabolic pathways were upregulated by infection. Type I and II interferon response pathways were upregulated in cells with low parasite mapped reads compared to the non-exposed host control cells, and this upregulation effect was reversed in infected cells. Differences were observed in the host cells depending on the differentiation status of the parasites, as determined by BAG1 and SAG1 expression. NF-κB, inflammatory response pathways, and IFN-γ response pathways were downregulated in host cells containing T. gondiiBAG1+/SAG1-, whereas this downregulation effect was reversed in case of T. gondiiBAG1-/SAG1+. We also identified two distinct host cell subsets that contained T. gondiiBAG1+/SAG1-, one of which displayed distinct transcriptomes with upregulated c-Myc expression. Overall, these data clearly demonstrate that host cell transcriptional alteration by bradyzoite infection is different from that of tachyzoite infection, indicating fine-tuning of the host immune response.

TSS-seq of Toxoplasma gondii sporozoites revealed a novel motif in stage-specific promoters.

Toxoplasma gondii is one of the most common zoonotic protozoan parasites. It has three major infectious stages: rapidly multiplying tachyzoites (Tz), slowly replicating bradyzoites (Bz) and a resting/free-living stage, sporozoites (Sz). The regulatory mechanisms governing stage-specific gene expression are not fully understood. Few transcriptional start sites (TSS) are known for Sz. In this study, we obtained TSS of Sz using an oligo-capping method and RNA-seq analysis. We identified 1,043,503 TSS in the Sz transcriptome. These defined 38,973 TSS clusters, of which, 11,925 were expressed in Sz and 1535 TSS differentially expressed in Sz. Based on these data, we defined promoter regions and novel sporozoite stage-specific motifs using MEME. TGTANNTACA was distributed around -55 to -75 regions from each TSS. Interestingly, the same motif was reported in another apicomplexan, Plasmodium berghei, as a cis-element of female-specific gametocyte genes, implying the presence of common regulatory machinery. Further comparative analysis should better define the distribution and function of these elements in other members of this important parasitic phylum.