Project description:Giardia lamblia is one of most common agents causing persistent abdominal symptoms in developed and developing countries. There are several diagnostic methods for Giardia infection, but none are optimal. In this study our aim was to find a new method based on Giardia microRNA (miRNA) that would contribute to the currently available diagnostic methods of giardiasis. Profiling Giardia small RNAs by deep sequencing revealed that the previously reported putative miR5 and miR6 are expressed in several G. lamblia isolates. These miRNAs were later tested by PCR in duodenal biopsies from 8 patients with positive pathology for giardiasis, while gastric biopsies served as matched negative controls. Additionally, these miRNAs were evaluated in stool samples of patients with proven giardiasis. All 8 duodenal samples of patients with histologically proven G. lamblia infection were positive for Giardia miR5 with a mean Ct of 23.7. These results were superior to Ct levels of G. lamblia DNA, which were 26.3 (p=0.004). The miR6 results were close to negative. All 10 gastric biopsies were negative for miR5. Stool studies showed 90% specificity but only 50% sensitivity in diagnosing giardiasis using miR6. The results of miR5 in stool were even less accurate. In conclusion, miR5 testing for Giardia infection in duodenal biopsies, may be a breakthrough method for diagnosis of giardiasis. It seems to be superior to G. lamblia DNA in duodenal biopsies. It would be important to investigate the contribution of routine Giardia miRNA testing in duodenal biopsies and duodenal aspirates from patients with persistent abdominal symptoms.
Project description:Giardia gene expression is being examined using Serial Analysis of Gene Expression (SAGE) to monitor genome-wide levels of messenger RNA (mRNA) expression throughout Giardia's life cycle. Examination of genome-wide gene expression patterns will provide a coherent picture of activation and inactivation of biological pathways. This research will provide a comprehensive understanding of changes in giardial gene expression in response to important host physiological signals and will serve as a valuable model for study of other parasites and complex eukaryotes, such as yeast and animals. It will provide a dynamic framework, in the context of the life cycle, to the annotation of the Giardia genome, including the detection of unpredicted genes via detection of their tags. Keywords: Giardia, SAGE, trophozoites, encystation, excystation, cysts
Project description:To investigate the transcriptional responses of intestinal epithelial cells and Giardia intestinalis, assemblage A isolate WB-C6, trophozoites during infection, we infected human enteroids with preconditioned trophozoites for 1h and 3h. Giardia intestinalis trophozoites were preconditioned before the infection with either DMEM/F-12 or DMEM/F-12 supplemented with 10% FBS to modify the trophozoites’ fitness.
Project description:To investigate the magnitude of the transcriptional changes occurring during the life cycle of Giardia lamblia we compared the transcriptome of trophozoites and cysts. Cysts were found to possess a much smaller transcriptome, both in terms of mRNA diversity and abundance. Genes encoding proteins related to ribosomal functions are highly over-represented. In cysts which have lost infectivity the transcriptome is further depleted. In contrast, exposure of cysts to conditions which promote excystation induced transcription. Six cysts and three trophozoites life cycle stages were analyzed.
Project description:Despite of Giardia duodenalis being one of the most commonly found intestinal pathogens in humans and animals, little is known of the host-parasite interactions in natural hosts. Therefore, the objective of this study was to investigate the intestinal response in calves following a G. duodenalis infection, using a bovine high-density oligo microarray to analyze global gene expression in the small intestine. The resulting microarray data suggested a decrease in inflammation, immune response and immune cell migration in infected animals, which was examined in more detail by quantitative real-time PCR on a panel of cytokines combined with histological analyses. The cytokine transcription levels showed a trend of down regulated expression in infected animals compared to the negative controls, best seen in jejunum for IL-6 and IL-8 and statistically significant for IL-17, IL-13 and IFN-?. No increased immune cell recruitment could be seen after infection, as well as no intestinal pathologies, such as villus shortening or increased levels of apoptosis. Key regulators in this intestinal response seem to be the nuclear peroxisome proliferator-activated receptors alpha (PPARA) and gamma (PPARG), for which an up-regulated expression was seen on microarray and qRT-PCR data. The activation of PPARs can exert an anti-inflammatory effect with inhibition of pro-inflammatory cytokines and a decrease in cell recruitment. . How the PPARs are activated during a Giardia infection still needs to be further elucidated. Eight male Holstein calves aged two to four weeks old were used for the trial. Prior to arrival, all animals were screened for the presence of Giardia cysts in their faecal samples. After confirming their negative status for all these pathogens, four of the animals were randomly chosen and placed in a G. duodenalis contaminated environment, whereas the four remaining animals were kept as negative controls in separate G. duodenalis-free stables. All calves in the study received the same commercial milk replacer. After three weeks, the presence or absence of a G. duodenalis infection was confirmed by IFA on faecal samples after which the animals were euthanized. Changes in gene expression profiles induced by Giardia duodenalis infection were compared using a high-density 60mer bovine oligo microarray.
Project description:To elucidate IL-17A-dependent immune mechanims involved in regulating host defense, we employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential of regulating protective immunity that failed to be upregulated in the absence of IL-17RA signaling. Whole small intestine tissue collected from IL-17RA-deficient and C57BL/6 (wild-type) mice was collected 2 weeks after Giardia muris infection and from uninfected controls. Expression levels of several genes that may have anti-parasitic potential (Defb1, Retnlb, Saa1, Saa2) and may be involved in parasite clearance were, on average, lower or unchanged in IL-17RA deficient compared to wild-type mice after infection. Two weeks after Giardia muris challenge, total RNA was extracted and analyzed from tissue of the small intestines of infected IL-17RA-deficient and wild-type mice, and compared to uninfected controls. Each sample contained equal amounts of total RNA from 6 female mice which were pooled and used in the experiment.
Project description:Research shows that children who are reared in households with low socioeconomic status are more vulnerable to heart disease, respiratory infection, and some cancers when they reach adulthood. This study conducted transcriptional profiling of PBMC in healthy adults who were low vs. high in early-life SES to explore the long-lasting genomic effects of early experience. Keywords: life stress, gene expression, inflammation, socioeconomic status Samples from 30 adults with low early-life SES and 30 adults with high early-life SES
Project description:We have performed strand-specific RNA-seq of trophozoites from four different Giardia intestinalis strains (A=WB and AS175, B=GS, E=P15). Comparison of mRNA levels in four different Giardia intestinalis strains.