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Background: Toxoplasma gondii is an obligate intracellular parasite that infects humans and other warm-blooded animals. Previous quantitative proteomic analyses of infected host cells revealed that the expression of many host proteins is modulated by T. gondii infection. However, at present limited data are available on the differentially expressed miRNAs (DEMs) associated with the pathology and host immune responses induced by acute and chronic infection with T. gondii in pigs in vivo. In this study, high-throughput sequencing was used to investigate expression profiles of spleen miRNAs at 10, 25 and 50 days post-infection (DPI) in pigs infected with Chinese I genotype strain T. gondii isolated from a dead pig. Results: When compared to the control group, 34, 6 and 86 DEMs were found in spleens of infected pigs at 10, 25 and 50 DPI, respectively. Gene Ontology (GO) enrichment analysis of the target genes of DEMs showed that no GO terms were enriched at 25 DPI, whereas 28 and 241 GO terms, of which two and 215 were sample-specific, were significantly enriched at 10 and 50 DPI, respectively. The top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the target genes of DEMs included signal transduction, immune system, metabolism and diseases. miRNA–gene network analysis revealed that the DEMs played important roles in the host immune response to T. gondii infection by modulating expression levels of cellular immunity-related cytokines and immune-related C-type lectins. Conclusion: Our results not only showed that host miRNA expression is altered by T. gondii but also revealed differences in the regulation of key biological processes and pathways involved in host responses to acute versus chronic T. gondii infection. This will aid future research into miRNA-target interactions during T. gondii infection in pigs and the development of novel therapies against T. gondii. [ABSTRACT FROM AUTHOR]

Background: Eimeria is a common genus of apicomplexan parasites that infect diverse vertebrates, most notably poultry, causing serious disease and economic losses. Eimeria species have complex life-cycles consisting of three developmental stages. However, the molecular basis of the Eimeria reproductive mode switch remains an enigma., Methods: Total RNA extracted from second- (MZ-2) and third-generation merozoites (MZ-3) of Eimeria necatrix was subjected to transcriptome analysis using RNA sequencing (RNA-seq) followed by qRT-PCR validation., Results: A total of 6977 and 6901 unigenes were obtained from MZ-2 and MZ-3, respectively. Approximately 2053 genes were differentially expressed genes (DEGs) between MZ-2 and MZ-3. Compared with MZ-2, 837 genes were upregulated and 1216 genes were downregulated in MZ-3. Approximately 95 genes in MZ-2 and 48 genes in MZ-3 were further identified to have stage-specific expression. Gene ontology category and KEGG analysis suggested that 216 upregulated genes in MZ-2 were annotated by 70 GO assignments, 242 upregulated genes were associated with 188 signal pathways, while 321 upregulated genes in MZ-3 were annotated by 56 GO assignments, 322 upregulated genes were associated with 168 signal pathways. The molecular functions of upregulated genes in MZ-2 were mainly enriched for protein degradation and amino acid metabolism. The molecular functions of upregulated genes in MZ-3 were mainly enriched for transcriptional activity, cell proliferation and cell differentiation., Conclusions: To the best of our knowledge, this is the first RNA-seq data study of the MZ-2 and MZ-3 stages of E. necatrix; it demonstrates a high number of differentially expressed genes between the MZ-2 and MZ-3 of E. necatrix. This study forms a basis for deciphering the molecular mechanisms underlying the shift from the second to third generation schizogony in Eimeria. It also provides valuable resources for future studies on Eimeria, and provides insight into the understanding of reproductive mode plasticity in different Eimeria species.

Background: Gametocyte proteins of Eimeria (E.) spp. are important components of the oocyst wall and some have been used to develop transmission-blocking vaccines against avian coccidiosis., Methods: Total RNA isolated from E. necatrix gametocytes was utilized as templates for RT-PCR amplification and sequencing of cDNA encoding a gametocyte protein using gene-specific primers. The cDNA was cloned into the bacterial expression vector pET28a(+) and expressed in E. coli BL21 cells. The antigenicity of the recombinant gametocyte protein and its localization in different E. necatrix life-cycle stages were determined by western blot and indirect immunofluorescence analyses, respectively., Results: A 731-nucleotide sequence of cDNA [GenBank: KF649255] of E. necatrix had 97.7% identity to that of Etgam22 of E. tenella. The cDNA ORF encoded a 186-amino acid protein containing a histidine-proline-rich region. The recombinant gametocyte protein (rEnGAM22) was predominately expressed in the insoluble inclusion body and recognized by antiserum from chickens immunized with oocysts of E. necatrix, E. maxima and E. tenella. A specific antibody to the rEnGAM22 protein recognized the wall-forming bodies in macrogametocytes and the walls of oocysts and sporocysts., Conclusions: The gene cloned from E. necatrix gametocytes is an ortholog to Etgam22 of E. tenella and presents a potential target for future recombinant subunit vaccines against coccidiosis.