Transcriptomic landscape of hepatic lymph nodes, peripheral blood lymphocytes and spleen of swamp buffaloes infected with the tropical liver fluke Fasciola gigantica.

The tropical liver fluke Fasciola gigantica is a parasitic helminth that has been frequently reported to infect mammals, typically involving water buffaloes. In this study, we characterized the tissue transcriptional landscape of buffaloes following infection by F. gigantica. RNAs were isolated from hepatic lymph nodes (hLNs), peripheral blood lymphocytes (pBLs), and spleen at 3-, 42- and 70-days post-infection (dpi), and all samples were subjected to RNA sequencing analyses. At 3 dpi, 2603, 460, and 162 differentially expressed transcripts (DETs) were detected in hLNs, pBLs, and spleen, respectively. At 42 dpi, 322, 937, and 196 DETs were detected in hLNs, pBLs, and spleen, respectively. At 70 dpi, 376, 334, and 165 DETs were detected in hLNs, pBLs, and spleen, respectively. Functional enrichment analysis identified upregulated immune-related pathways in the infected tissues involved in innate and adaptive immune responses, especially in hLNs at 42 and 70 dpi, and pBLs at 3 and 42 dpi. The upregulated transcripts in spleen were not enriched in any immune-related pathway. Co-expression network analysis further identified transcriptional changes associated with immune response to F. gigantica infection. Receiver operating characteristic (ROC) curve analysis showed that 107 genes in hLNs, 32 genes in pBLs, and 36 genes in spleen correlated with F. gigantica load. These findings provide new insight into molecular mechanisms and signaling pathways associated with F. gigantica infection in buffaloes.

RAA-Cas12a-Tg: A Nucleic Acid Detection System for Toxoplasma gondii Based on CRISPR-Cas12a Combined with Recombinase-Aided Amplification (RAA).

Toxoplasmosis, caused by the intracellular protozoon Toxoplasma gondii, is a significant parasitic zoonosis with a world-wide distribution. As a main transmission route, human infection can be acquired by the ingestion of T. gondii oocysts from the environment (e.g., soil, water, fruits and vegetables). Regarding the detection of T. gondii oocysts in environmental samples, the development of a time-saving, cost-effective and highly sensitive technique is crucial for the surveillance, prevention and control of toxoplasmosis. In this study, we developed a new method by combining recombinase-aided amplification (RAA) with CRISPR-Cas12a, designated as the RAA-Cas12a-Tg system. Here, we compared this system targeting the 529 bp repeat element (529 bp-RE) with the routine PCR targeting both 529 bp-RE and ITS-1 gene, respectively, to assess its ability to detect T. gondii oocysts in soil samples. Our results indicated that the 529 bp RE-based RAA-Cas12a-Tg system was able to detect T. gondii successfully in nearly an hour at body temperature and was more sensitive than the routine PCR assay. The sensitivity of this system reached as low as 1 fM with high specificity. Thus, RAA-Cas12a-Tg system provided a rapid, sensitive and easily operable method for point-of-care detection of T. gondii oocysts in soil, which will facilitate the control of T. gondii infection in humans and animals.

Differential expression of microRNAs and tRNA fragments mediate the adaptation of the liver fluke Fasciola gigantica to its intermediate snail and definitive mammalian hosts.

The tropical liver fluke Fasciola gigantica affects livestock and humans in many Asian countries, large parts of Africa, and parts of Europe. Despite the public health and economic impacts of F. gigantica, understanding of F. gigantica biology and how the complex lifecycle of this liver fluke is transcriptionally regulated remain unknown. Here, we tested the hypothesis that the regulatory small non-coding RNAs (sncRNAs), microRNAs (miRNAs) and tRNA-derived fragments (tRFs) play roles in the adaptation of F. gigantica to its intermediate and definitive hosts. We sequenced sncRNAs of eight lifecycle stages of F. gigantica. In total, 56 miRNAs from 33 conserved families and four Fasciola-specific miRNAs were identified. Expression analysis of miRNAs suggested clear stage-related patterns. By leveraging the existing transcriptomic data, we predicted a miRNA-based regulation of metabolism, transport, growth and developmental processes. Also, by comparing miRNA complement of F. gigantica with that of Fasciola hepatica, we detected a high level of conservation and identified differences in some miRNAs, which can be used to distinguish the two species. Moreover, we found that tRFs at each lifecycle stage were predominantly derived by tRNA-Lys and tRNA-Gly at 5' half sites, but relatively high expression was related to the buffalo-infecting stages. Taken together, we provided a comprehensive overview of the dynamic transcriptional changes of small RNAs that occur during the developmental stages of F. gigantica. This global analysis of F. gigantica lifecycle stages revealed new roles of miRNAs and tRFs in parasite development and will facilitate future research into understanding of fasciolosis pathobiology.

Proteomic Profiling of the Liver, Hepatic Lymph Nodes, and Spleen of Buffaloes Infected with Fasciola gigantica.

In the present study, we used an isobaric tag for relative and absolute quantitation (iTRAQ) proteomics technology to characterize the differentially expressed proteins (DEPs) in the liver, hepatic lymph nodes (hLNs), and spleen of buffaloes infected with Fasciola gigantica (F. gigantica). We also used the parallel reaction monitoring (PRM) method to verify the expression levels of the DEPs in the three infected tissues. At three days post-infection (dpi), 225, 1821, and 364 DEPs were detected in the liver, hLNs, and spleen, respectively. At 42 dpi, 384, 252, and 214 DEPs were detected in the liver, hLNs, and spleen, respectively. At 70 dpi, 125, 829, and 247 DEPs were detected in the liver, hLNs, and spleen, respectively. Downregulation of metabolism was prominent in infected livers at all time points, and upregulation of immune responses was marked in the hLNs during early infection (three dpi); however, no changes in the immune response were detected at the late stages of infection (42 and 70 dpi). Compared to the hLNs, there was no significant upregulation in the levels of immune responses in the infected spleen. All the identified DEPs were used to predict the subcellular localization of the proteins, which were related to extracellular space and membrane and were involved in host immune responses. Further PRM analysis confirmed the expression of 18 proteins. These data provide the first simultaneous proteomic profiles of multiple organs of buffaloes experimentally infected with F. gigantica.

Transcriptomic Profiling of Mouse Brain During Acute and Chronic Infections by Toxoplasma gondii Oocysts.

Infection by the protozoan Toxoplasma gondii can have a devastating impact on the structure and function of the brain of the infected individuals, particularly immunocompromised patients. A systems biology view of the brain transcriptome can identify key molecular targets and pathways that mediate the neuropathogenesis of cerebral toxoplasmosis. Here, we performed transcriptomic analysis of the brain of mice infected by T. gondii Pru strain oocysts at 11 and 33 days post-infection (dpi) compared to uninfected (control) mice using RNA sequencing (RNA-seq). T. gondii altered the expression of 936 and 2,081 transcripts at 11 and 33 dpi, respectively, and most of these were upregulated in the infected brains. Gene Ontology (GO) enrichment and pathway analysis showed that immune response, such as interferon-gamma (IFN-γ) responsive genes were strongly affected at 11dpi. Likewise, differentially expressed transcripts (DETs) related to T cell activation, cytokine production and immune cell proliferation were significantly altered at 33 dpi. Host-parasite interactome analysis showed that some DETs were involved in immune signaling, metabolism, biosynthesis-related processes and interspecies interaction. These findings should increase knowledge of the mouse brain transcriptome and the changes in transcriptional regulation and downstream signaling pathways during acute and chronic T. gondii infections.