Background: Eimeria necatrix is one of the most pathogenic parasites, causing high mortality in chickens. Although its genome sequence has been published, the sequences and complete structures of its mRNA transcripts remain unclear, limiting exploration of novel biomarkers, drug targets and genetic functions in E. necatrix. Methods: Second-generation merozoites (MZ-2) of E. necatrix were collected using Percoll density gradients, and high-quality RNA was extracted from them. Single-molecule real-time (SMRT) sequencing and Illumina sequencing were combined to generate the transcripts of MZ-2. Combined with the SMRT sequencing data of sporozoites (SZ) collected in our previous study, the transcriptome and transcript structures of E. necatrix were studied. Results: SMRT sequencing yielded 21,923 consensus isoforms in MZ-2. A total of 17,151 novel isoforms of known genes and 3918 isoforms of novel genes were successfully identified. We also identified 2752 (SZ) and 3255 (MZ-2) alternative splicing (AS) events, 1705 (SZ) and 1874 (MZ-2) genes with alternative polyadenylation (APA) sites, 4019 (SZ) and 2588 (MZ-2) fusion transcripts, 159 (SZ) and 84 (MZ-2) putative transcription factors (TFs) and 3581 (SZ) and 2039 (MZ-2) long non-coding RNAs (lncRNAs). To validate fusion transcripts, reverse transcription-PCR was performed on 16 candidates, with an accuracy reaching up to 87.5%. Sanger sequencing of the PCR products further confirmed the authenticity of chimeric transcripts. Comparative analysis of transcript structures revealed a total of 3710 consensus isoforms, 815 AS events, 1139 genes with APA sites, 20 putative TFs and 352 lncRNAs in both SZ and MZ-2. Conclusions: We obtained many long-read isoforms in E. necatrix SZ and MZ-2, from which a series of lncRNAs, AS events, APA events and fusion transcripts were identified. Information on TFs will improve understanding of transcriptional regulation, and fusion event data will greatly improve draft versions of gene models in E. necatrix. This information offers insights into the mechanisms governing the development of E. necatrix and will aid in the development of novel strategies for coccidiosis control. Graphical

Background: Toxoplasmosis is a zoonosis with a worldwide presence that is caused by the intracellular parasite Toxoplasma gondii. Active regulation of apoptosis is an important immune mechanism by which host cells resist the growth of T. gondii or avoid excessive pathological damage induced by this parasite. Previous studies found that upregulated expression of microRNA-185 (miR-185) during T. gondii infection has a potential role in regulating the expression of the ARAF gene, which is reported to be associated with cell proliferation and apoptosis. Methods: The expression levels of miR-185 and the ARAF gene were evaluated by qPCR and Western blot, respectively, in mice tissues, porcine kidney epithelial cells (PK-15) and porcine alveolar macrophages (3D4/21) following infection with the T. gondii ToxoDB#9 and RH strains. The dual luciferase reporter assay was then used to verify the relationship between miR-185 and ARAF targets in PK-15 cells. PK-15 and 3D4/21 cell lines with stable knockout of the ARAF gene were established by CRISPR, and then the apoptosis rates of the cells following T. gondii infection were detected using cell flow cytometry assays. Simultaneously, the activities of cleaved caspase-3, as a key apoptosis executive protein, were detected by Western blot to evaluate the apoptosis levels of cells. Results: Infection with both the T. gondii ToxoDB#9 and RH strains induced an increased expression of miR-185 and a decreased expression of ARAF in mice tissues, PK-15 and 3D4/21 cells. MiR-185 mimic transfections showed a significantly negative correlation in expression levels between miR-185 and the ARAF gene. The dual luciferase reporter assay confirmed that ARAF was a target of miR-185. Functional investigation revealed that T. gondii infection induced the apoptosis of PK-15 and 3D4/21 cells, which could be inhibited by ARAF knockout or overexpression of miR-185. The expression levels of cleaved caspase-3 protein were significantly lower in cells with ARAF knockout than in normal cells, which were consistent with the results of the cell flow cytometry assays. Conclusions: Toxoplasma gondii infection could lead to the upregulation of miR-185 and the downregulation of ARAF, which was not related to the strain of T. gondii and the host cells. Toxoplasma gondii infection could regulate the apoptosis of host cells via the miR-185/ARAF axis, which represents an additional strategy used by T. gondii to counteract host-cell apoptosis in order to maintain survival and reproduce in the host cells. Graphical

Background: Toxoplasma gondiiis an obligate intracellular protozoan parasite that can cause a geographically widespread zoonosis. Our previous splenocyte microRNA profile analyses of pig infected withT. gondiirevealed that the coordination of a large number of miRNAs regulates the host immune response during infection. However, the functions of other miRNAs involved in the immune regulation duringT. gondiiinfection are not yet known.Methods: Clustering analysis was performed byK-means, self-organizing map (SOM), and hierarchical clustering to obtain miRNA groups with the similar expression patterns. Then, the target genes of the miRNA group in each subcluster were further analyzed for functional enrichment by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway to recognize the key signaling molecules and the regulatory signatures of the innate and adaptive immune responses of the host duringT. gondiiinfection.Results: A total of 252 miRNAs were successfully divided into 22 subclusters byK-means clustering (designated as K1–K22), 29 subclusters by SOM clustering (designated as SOM1–SOM29), and six subclusters by hierarchical clustering (designated as H1–H6) based on their dynamic expression levels in the different infection stages. A total of 634, 660, and 477 GO terms, 15, 26, and 14 KEGG pathways, and 16, 15, and 7 Reactome pathways were significantly enriched byK-means, SOM, and hierarchical clustering, respectively. Of note, up to 22 miRNAs mainly showing downregulated expression at 50 days post-infection (dpi) were grouped into one subcluster (namely subcluster H3-K17-SOM1) through the three algorithms. Functional analysis revealed that a large group of immunomodulatory signaling molecules were controlled by the different miRNA groups to regulate multiple immune processes, for instance, IL-1-mediated cellular response and Th1/Th2 cell differentiation partly depending on Notch signaling transduction for subclusters K1 and K2, innate immune response involved in neutrophil degranulation and TLR4 cascade signaling for subcluster K15, B cell activation for subclusters SOM17, SOM1, and SOM25, leukocyte migration, and chemokine activity for subcluster SOM9, cytokine–cytokine receptor interaction for subcluster H2, and interleukin production, chemotaxis of immune cells, chemokine signaling pathway, and C-type lectin receptor signaling pathway for subcluster H3-K17-SOM1.Conclusions: Cluster analysis of splenocyte microRNAs in the pig revealed key regulatory properties of subcluster miRNA molecules and important features in the immune regulation induced by acute and chronicT. gondiiinfection. These results contribute new insight into the identification of physiological immune responses and maintenance of tolerance in pig spleen tissues duringT. gondiiinfection.Graphical

Background: Eimeria coccidiosis is a significant intestinal parasitic disease, which can lead to weight loss, disease and even death of many animals. At present, there is no information about the prevalence of Eimeria among the world’s endangered species of Père David’s deer (Elaphurus davidianus). Therefore, the purpose of this study is to identify an unknown Eimeria genus in the Père David’s deer in Dafeng Milu National Nature Reserve, China. Results: A new Eimeria species is described from Père David’s deer. Sporulated oocysts (n = 54) are pyriform, with a rough, yellowish brown, 2-layered oocyst wall (2.5 μm thick). A numerous small granules are dispersed randomly on the wall. Oocysts measured 41.2 (39.2–42.8) μm × 29.5 (27.9–30.5) μm, oocyst length/width (L/W) ratio, 1.4. Oocyst residuum, a polar granule and a polar cap are absent. The micropyle (3.5 μm wide) is present. Sporocysts are spindle shaped, 18.2 (16.5–20.0) μm × 10.5 (9.8–11.9) μm, sporocyst L/W ratio, 1.7 (1.5–1.9). A thin convex Stieda body is present and the sporocyst residuum is composed of numerous small granules less than 2.0 μm in diameter dispersed randomly. Each sporocyst contained 2 comma-shaped sporozoites in head-to-tail arrangement. A nucleus is located immediately anterior to the posterior, strong refractive and subspherical refractile body (~ 8 μm). Molecular analysis was conducted at the 18S, ITS-1 and COI loci. Conclusion: Based on the morphological and molecular data, this isolate is a new species of coccidian parasite, which is named Eimeria davidianusi after its host, the Père David’s deer (Elaphurus davidianus).