Molecular Identification and Differentiation of Fasciola Isolates Using PCR- RFLP Method Based on Internal Transcribed Spacer (ITS1, 5.8S rDNA, ITS2)

Background: In this study, we used both ITS1 and ITS2 for molecular identification of Fasciola species.Methods: The region between 18S and 28S of ribosomal DNA was used in PCR-RFLP method for molecular identification of Fasciola species. Ninety trematodes of Fasciola were collected during abattoir inspection from livers of naturally infected sheep and cattle from Khorasan, East Azerbaijan, and Fars provinces in Iran. After DNA extraction, PCR was performed to amplify region ITS1, 5.8S rDNA, ITS2. To select a suitable restriction enzyme, we sequenced and ana-lyzed the PCR products of F. hepatica and F. gigantica samples from sheep and cattle. Tsp509I fast digest restriction enzyme was selected for RFLP method that caused the separation specifi-cally of Fasciola species. Results: The fragment approximately 1000bp in all of the Fasciola samples was amplified and then digested with the Tsp509I restriction endonuclease. Seventy F. hepatica and 20 F. gigantica were identified of total 90 Fasciola isolates.Conclusion: The new PCR-RFLP assay using Tsp509I restriction enzyme provides a simple, practical, fast, low cost, and reliable method for identification and differentiation of Fasciola isolates

Prevalence and severity of animal Fasciolosis in six provinces of Iran

Background: Fasciolosis is one of the most important parasitic disease common among both humans and livestock. Considering the health and economic importance of the disease, an understanding of the epidemiology of Fasciolosis is highly crucial. This study aimed to evaluate the prevalence and severity of Fasciola infection in animals from different geographical regions of Iran during 2009-10. Materials and Methods: In this descriptive study, 11100 livers taken from slaughtered sheep and cattle were carefully examined for Fasciola parasites at the six industrial slaughterhouses of East Azerbaijan, Khorasan-Razavi, Khuzestan, Fars, Mazandaran and Markazi provinces. All Fasciola parasites isolated from the livers of infected animals were transferred to the laboratory, and then the parasite species were identified and counted. Finally, the frequency distribution and the severity of infection were analyzed. Results: In this study, 1.10 of the total sheep and cattle slaughtered in six industrial slaughterhouses were found positive for Fasciolosis. The severity of Fasciola in sheep and cattle livers was 7.77±0.42 and 15.24±1.78, respectively. Khorasan Razavi and Fars provinces had the highest (14.54±3.16) and lowest (7.75±0.79) severity of infection, respectively. Conclusion: Results of the study show a reduction in the prevalence and severity of Fasciolosis in sheep and cattle. But considering the importance of the disease and its endemicity, the preventive measures should be taken against the animal and human Fasciolosis in Iran

miR-143 acts as an inhibitor of migration and proliferation as well as an inducer of apoptosis in melanoma cancer cells in vitro

Melanoma is a serious form of skin cancers begins in the melanocyte. Micro-RNAs are small noncoding RNA with 19 to 25 nucleotides in length involves in the regulation of a wide range of biological processes. MicroRNAs are affected by an aberrant epigenetic alteration in the tumors that may lead to their dysregulation and formation of cancer. Recently, dysregulation of numerous microRNAs has been reported in different types of cancer. The present study focused on the role of miR-143 in carcinogenesis of melanoma cancer. Here, we evaluated the expression level of miR-143 in three melanoma cell lines in comparison with the normal human epidermal melanocyte cell line. Then, miR-143 gene plasmid transfected into the WM115 cell line, for having the lowest expression of miR-143. In addition, the effect of miR-143 transfection on mRNA and protein levels of metastasis-related genes was performed along with MTT assay, wound healing assay, and flow cytometry. The results showed that mRNA and protein expression levels of metastasis-related genes including MMP-9, E-cadherin, Vimentin, and CXCR4 have been reduced following transfection of miR-143. Moreover, the results of the scratch test showed that miR-143 re-expression inhibited cell migration. Also, the role of miR-143 in the induction of apoptosis and inhibition of proliferation by flow cytometry and MTT was confirmed. As a result, the present study showed that miR-143 was involved in metastatic and apoptotic pathways, suggesting that miR-143 acts as a tumor-suppressor microRNA in melanoma cancer. © 2020 International Union of Biochemistry and Molecular Biolog

Sero-molecular detection, multi-locus genotyping, and clinical manifestations of ocular toxoplasmosis in patients in northwest Iran

Background Our goal was to use molecular techniques to verify and characterise clinical diagnoses of ocular toxoplasmosis. Clinical cases were evaluated against IgM and IgG Toxoplasma antibodies, and IgG avidity was tested. B1 gene was assessed for molecular detection, and multi-locus genotyping were conducted to type Toxoplasma infections. Methods A cross-sectional study was conducted in 33 patients with suspected active ocular toxoplasmosis. Patients were examined by an ophthalmologist and clinical manifestations were recorded. Toxoplasma gondii IgG and IgM from serum samples were analysed by chemiluminescence immunoassay and ELISA. Acute vs chronic infection was evaluated by IgG avidity testing. Molecular diagnosis of T. gondii infection targeted the B1 gene, and the T. gondii genotype was determined by amplification of the GRA6, SAG2, SAG3, BTUB and APICO loci. The correlation of age, gender, occupation, education, contact with cats or soil, and the consumption of undercooked meat with the incidence of ocular toxoplasmosis was evaluated. Results Twenty-eight patients (84.8) were seropositive, two (6) were both IgG and IgM positive, while one (3) showed IgG avidity <40. Molecular testing confirmed toxoplasmosis in 27 patients (81.8). Chorioretinal scarring (p=0.014) and posterior uveitis (p=0.004) was significantly associated with ocular toxoplasmosis patients. Multi-locus genotyping showed genotype I. Ocular toxoplasmosis showed no significant correlation with gender, age, behaviours, occupation or education. Conclusion Clinical manifestations, serological and molecular detection of Toxoplasma were highly correlated in the diagnosis of ocular toxoplasmosis. Genotype I was predominant in ocular toxoplasmosis in northwest Iran. A larger comparative study should be conducted to provide a broader view of the molecular epidemiology of T. gondii genotypes and its role in toxoplasmosis. © 2018 The Author(s)

Transcriptomic insights into the early host-pathogen interaction of cat intestine with Toxoplasma gondii

Abstract Background Although sexual reproduction of the parasite Toxoplasma gondii exclusively occurs in the cat intestine, knowledge about the alteration of gene expression in the intestine of cats infected with T. gondii is still limited. Here, we investigated the temporal transcriptional changes that occur in the cat intestine during T. gondii infection. Methods Cats were infected with 100 T. gondii cysts and their intestines were collected at 6, 12, 18, 24, 72 and 96 hours post-infection (hpi). RNA sequencing (RNA-Seq) Illumina technology was used to gain insight into the spectrum of genes that are differentially expressed due to infection. Quantitative RT-PCR (qRT-PCR) was also used to validate the level of expression of a set of differentially expressed genes (DEGs) obtained by sequencing. Results Our transcriptome analysis revealed 2363 DEGs that were clustered into six unique patterns of gene expression across all the time points after infection. Our analysis revealed 56, 184, 404, 508, 400 and 811 DEGs in infected intestines compared to uninfected controls at 6, 12, 18, 24, 72 and 96 hpi, respectively. RNA-Seq results were confirmed by qRT-PCR. DEGs were mainly enriched in catalytic activity and metabolic process based on gene ontology enrichment analysis. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that transcriptional changes in the intestine of infected cats evolve over the course of infection, and the largest difference in the enriched pathways was observed at 96 hpi. The anti-T. gondii defense response of the feline host was mediated by Major Histocompatibility Complex class I, proteasomes, heat-shock proteins and fatty acid binding proteins. Conclusions This study revealed novel host factors, which may be critical for the successful establishment of an intracellular niche during T. gondii infection in the definitive feline host