Understanding the role of pigs in the transmission of zoonotic Echinococcus ortleppi in Haryana, India.

The etiological agents of zoonotic cystic echinococcosis comprise the Echinococcus granulosus sensu lato (s.l.) species complex. The present study was aimed at investigating the zoonotic genotypes of Echinococcus granulosus s.l. circulating in the pig population of Haryana, India. Out of 253 slaughtered pigs screened, 5 showed the presence of hydatid cysts. The amplification of the partial mitochondrial NADH dehydrogenase subunit 1 (nad1) gene for the molecular confirmation and phylogenetics of the retrieved metacestodes (n = 2) revealed the presence of E. ortleppi. The sequences generated herein exhibited 99.80% homology to the GenBank archived E. ortleppi sequences. Cladistics targeting genetic diversity and haplotype network analysis involved 37 E. granulosus s.l. GenBank archived sequences from India corresponding to different hosts (large and small ruminants and humans) along with the sequences (n = 2) generated in the present study. Overall, 14 haplotypes with high haplotype (0.780 ± 0.059) and low nucleotide (0.033 ± 0.010) diversities were recorded for the overall data set, which evinced a population expansion. The median-joining haplotype network revealed a stellate shape of E. granulosus sensu stricto (s.s.) sequences, which was indicative of rapid population expansion. High genetic differentiation (FST = 0.840 - 0.983) and low gene flow (Nm = 0.003 - 0.047) were recorded between the pig intermediate hosts infected with E. ortleppi and other hosts infected with E. granulosus s.s. The findings are of paramount significance for the formulation of effective control strategies considering the public health and economic impact of cystic echinococcosis.

A pilot study of microRNAs expression profile in plasma of patients with hydatid disease: potential immunomodulation of hydatid disease.

Echinococcosis is a zoonotic disease, which seriously endangers human health. The immune game between parasite and host is not fully understood. Exosomes are thought to be one of the ways of information communication between parasite and host. In this study, we attempted to explore the communication between Echinococcus granulosus and its host through the medium of exosomes. We collected plasma from E. granulosus patients (CE-EXO) and healthy donors (HD-EXO) and extracted exosomes from the plasma. The expression profile of miRNA in plasma was determined by second generation sequencing. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to annotate the function of target genes of differential miRNAs. Meanwhile, we co-cultured plasma exosomes from healthy donors and plasma exosomes from E. granulosus patients with Jurkat T cells with or without phytohaemagglutinin (PHA) stimulation. The expression of CD69 on Jurkat T cells was detected by flow cytometry. The results showed that the miRNA of exosomes between healthy donors and E. granulosus patients was significantly different. GO and KEGG were used to annotate the function of target genes of differential miRNAs. The results indicate that many important pathways are involved in inflammation, metabolism, and immune response after parasite infection, such as p53 signaling pathway, PI3K-Akt signaling pathway, and glycolysis/gluconeogenesis. Flow cytometry showed that CE-EXO reduced the expression of CD69 + on Jurkat T cells. Our present results suggest that these differentially expressed miRNAs may be important regulators of parasite-host interactions. Meanwhile, functional prediction of its target genes provides valuable information for understanding the mechanism of host-parasite interactions. These results provide clues for future studies on E. granulosus escape from host immune attack, which could help control E. granulosus infection.

Deciphering the role of miR-71 and let-7 in the fertility of cystic echinococcosis cysts: a preliminary assessment.

Cystic echinococcosis (CE) is a neglected helminthic zoonosis in many parts of the world. Some CE cysts in the intermediate host are non-fertile. Considering the function of microRNAs in many biological processes such as embryonic development, cell proliferation, and apoptosis, this study investigated the function and comparison of miR-71 and let-7 in fertile and non-fertile CE cysts. Here, we determined the expression level of the miRNAs for 33 animal cysts and 16 human cysts (Echinococcus granulosus sensu stricto (G1). The quantitative real-time PCR method was conducted for the expression evaluation of miR-71 and let-7. The expression of both miRNAs in all samples was determined using the following formula: [ΔCT = CT (target) - CT (internal control)]. A comparison of Δct of miR-71 and let-7 in fertile and non-fertile cysts did not show a significant difference (P = 0.911 and 0.354). In cattle, sheep, and humans, Δct of miR-71, and let-7 were higher, respectively. Therefore, the mean expression of miR-71 and let-7 indicates an increase in humans compared to other intermediate hosts. Also, statistical results show a significant difference in the expression of these miRNAs in sheep, cattle, and human cysts (P = 0.025 and 0.01). The lower expression of these miRNAs in cattle cysts and their common infertility might be associated with the hypothesis and function of miRNAs in the fertility of CE cysts. So we should not ignore the function and role of miRNAs in this subject due to the importance of infertility in E. granulosus epidemiology.

Transcriptome analysis uncovers the key pathways and candidate genes related to the treatment of Echinococcus granulosus protoscoleces with the repurposed drug pyronaridine.

BACKGROUND: Cystic echinococcosis (CE) is a life-threatening zoonosis caused by the larval form of Echinococcus granulosus tapeworm. Our previous study showed that an approved drug pyronaridine (PND) is highly effective against CE, both in vitro and in an animal model. To identify possible target genes, transcriptome analysis was performed with E. granulosus sensu stricto protoscoleces treated with PND. RESULTS: A total of 1,321 genes were differentially expressed in protoscoleces treated with PND, including 541 upregulated and 780 downregulated genes. Gene ontology and KEGG analyses revealed that the spliceosome, mitogen-activated protein kinase (MAPK) pathway and ATP-binding cassette (ABC) transporters were the top three enriched pathways. Western blot analysis showed that PND treatment resulted in a dose-dependent increase in protein expression levels of EgMKK1 (MKK3/6-like) and EgMKK2 (MEK1/2-like), two members of MAPK cascades. Interestingly, several heat shock protein (HSP) genes were greatly downregulated including stress-inducible HSPs and their constitutive cognates, and some of them belong to Echinococcus-specific expansion of HSP70. CONCLUSIONS: PND has a great impact on the spliceosome, MAPK pathway and ABC transporters, which may underline the mechanisms by which PND kills E. granulosus protoscoleces. In addition, PND downregulates HSPs expression, suggesting a close relationship between the drug and HSPs.

MicroRNA profile in immune response of alveolar and cystic echinococcosis patients.

It is known that miRNAs are effective in immune response in the diagnosis and treatment of many infectious diseases. However, the miRNAs profile is unknown in Alveolar and Cystic Echinococcosis which can be fatal if left untreated. The miRNAs profile that activates the T and B cells forming the immune system in Alveolar and Cystic Echinococcosis patients was investigated in this study. A total of 50 liver tissue samples were obtained from Alveolar and Cystic Echinococcosis patients in Kilis State Hospital Pathology Laboratory in southeast of Turkey. The circulating cell-free miRNAs were evaluated by a quantitative real-time polymerase chain reaction, statistically calculated within ΔΔCt values and fold changes were evaluated by Welch T test, in which P < .05 was considered to be significant. Twenty-five microRNAs, including let-7a-5p, let-7c, let-7e-5p, miR-15b-5p, miR16, miR-17-5p, miR-23a-5p, miR-24-3p, miR-25-3p, miR-26a-3p, miR-26b-3p, miR-29b-3p, miR-29c-3p, miR-30a-5p, miR-30b-5p, miR-30c-5p, miR-30d-5p, miR-30e-5p, miR-98-5p, miR-101-3p, miR-106b-5p, miR-125b-5p, miR-142-5p, miR-222-3p and miR-223-3p, were found as down-regulated in Alveolar and Cystic Echinococcosis patients than control groups. Twelve miRNAs, including miR-15a-5p, miR-21-5p, miR-27a-3p, miR-29a-3p, miR-146a-5p, miR-181a-5p, miR-181b-5p, miR-181d, miR-181c-5p, miR-195-5p, miR-214-3p and miR-365-3p, were found as up-regulated in Alveolar and Cystic Echinococcosis patients than healthy person. It has been shown that T- and B-cell activities are related in the progressive of both Alveolar and Cystic Echinococcosis in this study. The miRNA panel activated by T and B cells may be important for exploring the mechanisms underlying early development in Alveolar and Cystic Echinococcosis providing novel information that may be used to discover new therapeutics for these diseases.

microRNA-125b-5p is a promising novel plasma biomarker for alveolar echinococcosis in patients from the southern province of Qinghai.

BACKGROUND: Alveolar echinococcosis (AE) is caused by parasitic infection by Echinococcus multilocularis. Its diagnosis is usually based on clinical symptoms, ultrasound, and other imaging methods. MicroRNAs (miRNAs) play important roles in disease processes and can exist in a highly stable cell-free form in body fluids. It is important to identify specific, sensitive diagnostic markers for early diagnosis and evaluation of AE. In this study, we examined hsa-miR-125b-5p as a potential plasma biomarker of E. multilocularis infection. METHODS: Plasma samples from patients with AE and healthy individuals were screened for the presence of five miRNAs using miRNA chips. We used quantitative polymerase chain reaction to measure miRNA expression levels in plasma and liver tissue samples from patients with AE. RESULTS: hsa-miR-125b-5p was stably upregulated in the plasma and liver tissue samples from patients with AE. CONCLUSIONS: The results suggest that hsa-miR-125b-5p may be a promising biomarker for early, non-invasive diagnosis of AE.

Expression profile of circular RNAs in cystic echinococcosis pericystic tissue.

OBJECTIVE: The aim of the present study was to investigate the expression profiles of circular RNAs (circRNAs) in the pericystic tissue of patients with cystic echinococcosis (CE). PATIENTS AND METHODS: CircRNA expression profiles were obtained by circRNA microarray of four matched pairs of pericystic tissues affected by CE and adjacent normal liver tissues. qRT-PCR was used to validate the differential expression of some circRNAs identified by the microarray analysis. The potential functions of the differentially expressed circRNAs in the CE pericystic tissues were predicted by bioinformatic analysis. RESULTS: Compared with the adjacent normal liver tissues, 177 circRNAs were upregulated and 166 circRNAs were downregulated in CE pericystic tissues based on a ≥2.0-fold change. The top 10 upregulated circRNAs were hsa_circRNA_001654,hsa_circRNA_103361,hsa_circRNA_001490,hsa_circRNA_104310,hsa_circRNA_100395,hsa_circRNA_102485,hsa_circRNA_001459,hsa_circRNA_104193,hsa_circRNA_400043, and hsa_circRNA_006773; The top 10 downregulated circRNAs were hsa_circRNA_400633,hsa_circRNA_404974,hsa_circRNA_068482 ,hsa_circRNA_100974,hsa_circRNA_049637,hsa_circRNA_404798,hsa_circRNA_400064,hsa_circRNA_004045,hsa_circRNA_101379, and hsa_circRNA_016771;The circRNA-seq results for 15 selected differentially expressed circRNAs were validated by qRT-PCR. The qRT-PCR analysis showed that hsa_circRNA_006773, hsa_circRNA_049637, hsa_circRNA_104349, and hsa_circRNA_406281 were differentially expressed in CE pericystic tissues when compared with their expression in the adjacent normal liver tissues. Interestingly, 319 miRNAs and 52 mRNAs were predicted to be adsorbed by these four differentially expressed circRNAs. Gene Ontology analysis revealed that these circRNAs may be involved in the response to organic cyclic compounds and endogenous stimuli and in cellular organismal processes. CONCLUSION: Differential expression of circRNAs may be associated with the development and progression of CE, and these circRNAs might be useful as biomarkers for prognosis prediction and as treatment targets.

MicroRNA expression profile in patients with cystic echinococcosis and identification of possible cellular pathways.

Cystic echinococcosis (CE) is a neglected tropical disease, caused by metacestode (larval) form of the Echinococcus granulosus sensu lato (sl) in humans. MicroRNAs (miRNAs) are small, stable, tissue-specific RNA molecules encoded by the genome that are not translated into proteins. Circulating miRNA expression profiles vary in health and disease. The aim of this study is to determine the altered cellular pathways in CE by comparing the miRNA profiles of controls and CE patients with active or inactive cysts. Following abdominal ultrasonography (US) examination, 20 patients diagnosed with active CE (CE1, CE2, CE3a and CE3b) or inactive CE (CE4 and CE5) and three healthy controls were included in the study. The expression profiles of 372 biologically relevant human miRNAs were investigated in serum samples from CE patients and healthy controls with miScript miRNA HC PCR Array. Compared with the control group, expression of 6 miRNAs (hsa-miR-4659a-5p, hsa-miR-4518, hsa-miR-3977, hsa-miR-4692, hsa-miR-181b-3p, hsa-miR-4491) and one miRNA (hsa-miR-4687-5p) were found to be downregulated in CE patients with active and inactive cysts, respectively (p < 0.05). For downregulated miRNAs in this study, predicted targets were found to be associated mainly with cell proliferation, apoptosis, cell-cell interactions and cell cycle regulation. Further studies in this direction may elucidate the pathogenesis of human CE and the relationship between CE and other pathologies.

Gene expression in human liver fibrosis associated with Echinococcus granulosus sensu lato.

Liver fibrosis is a dynamic process that occurs in response to chronic liver disease resulting from factors such as chronic infections, autoimmune reactions, allergic responses, toxins, radiation, and infectious agents. Among the infectious agents, multicellular parasites cause chronic inflammation and fibrosis. Twenty-five patients with different stages of cystic echinococcosis (CE) were enrolled in the study. The expression of ACTA2, COL3A1, IFN-γ, MMP2, MMP9, TGF-ß1, and TNF-α genes was determined by qRT-PCR in healthy and fibrotic liver tissue of the CE patients. TGF-ß1 expression was evaluated by immunohistochemistry, and histology was conducted to assess the development of liver fibrosis. Expression of MMP9, ACTA2, COL3A1, and MMP2 was found significantly higher in the fibrotic tissue compared to healthy tissue. We observed a significant correlation between TGF-ß1 and TNF-α gene expressions and liver fibrosis. The mRNA level of IFN-γ was lower in the fibrotic than in the healthy hepatic tissue. Immunohistochemistry analysis revealed TGF-ß1 upregulation in the fibrotic tissue. Histology showed inflammation and fibrosis to be significantly higher in the fibrotic tissue. The findings of this study suggest that Echinococcus granulosussensu lato can promotes fibrosis through the overexpression of TGF-ß1, MMP9, ACTA2, COL3A1, and MMP2. The downregulation of IFN-γ mRNA in fibrotic samples is probably due to the increased production of TGF-ß1 and the suppression of potential anti-fibrotic role of IFN-γ during advanced liver injury caused by E. granulosussensu lato.

Phylogenetic Characteristics of Echinococcus granulosus Sensu Lato in Uzbekistan.

Echinococcosis occurs mainly in areas with heavy livestock farming, such as Central Asia, America, and Australia. Echinococcus granulosus sensu lato (s.l.) infection causes echinococcosis in intermediate hosts, such as sheep, cattle, goats, camels, and horses. Numerous cases of echinococcosis occur in Uzbekistan as stock farming is a primary industry. Epidemiological and genetic studies of E. granulosus s.l. are very important for mitigating its impact on public health and the economy; however, there are no such studies on E. granulosus s.l. in Uzbekistan. In the present study, to determine which genotypes exist and are transmitted, we isolated Echinococcus sp. from definitive hosts (one isolate each from jackal and dog) and intermediate hosts (52 isolates from humans and 6 isolates from sheep) in Uzbekistan and analyzed the isolates by sequencing 2 mitochondrial DNA components (cox1 and nad1). The results showed that all of isolates except one belonged to the E. granulosus sensu stricto (s.s.) G1 and G3 genotypes. Phylogenetic analysis based on cox1 sequences showed that 42 isolates from humans, 6 isolates from sheep, and one isolate from jackal were the G1 genotype, whereas the remaining 8 isolates from human and the one isolate from dog were the G3 genotype. These results suggest that the G1 and G3 genotypes of E. granulosus s.s. are predominant in Uzbekistan, and both wild animals and domestic animals are important for maintaining their life cycle. Only one isolate from human sample was confirmed to be E. eqiinus (G4 genotype), which is known to be for the first time.

Validation of a one-step PCR assay for the molecular identification of Echinococcus granulosus sensu stricto G1-G3 genotype.

The Italian National Reference Center for Echinococcosis (CeNRE, Sassari, Italy) set up a diagnostic protocol of "one-step-PCR" useful for the detection of E. granulosus sensu stricto (E.g.s.s.) and the identification of its genotype (G1-G3). The purpose of this work was to perform the validation of the "PCR E.g.s.s." method. The procedures were performed employing the criteria of the World Organization for Animal Health as well as of the Italian Accreditation Body (ACCREDIA) based on the Regulation UNI CEI EN ISO/IEC 17025. Positive DNA samples belonging to E. granulosus, E. ortleppi, E. multilocularis, E. canadensis species were used for the experiments. Analytical specificity evidenced primer pairs Cal (Calreticulin l gene of 1001 bp) with an specificity higher respect to Ef1 (Elongation-Factor 1 Alpha gene of 706 bp) and NAD (Dehydrogenase-subunit 1 gene of 219 bp). The analytical sensitivity presented the capability to detect a very low amount of parasite DNA corresponding to a concentration of 12.5 pg/µl; accuracy and precision related to the operator performance, along with repeatability and reproducibility, evidenced high concordance among results and demonstrated an excellent κ values of Cohen. According to the good performance related to the evaluated parameters, the method "PCR E.g.s.s." was suitable for the validation procedure, and consequently, to be undergone to the accreditation process. In conclusion, the results demonstrated an elevated robustness and reliable features of the "PCR E.g.s.s." able to perform a rapid diagnosis of E. granulosus in only "one step", hence, it is likely to avoid the sequencing step.

Proteomic Analysis on Exosomes Derived from Patients' Sera Infected with Echinococcus granulosus.

Cystic echinococcosis (CE), a zoonotic disease caused by Echinococcus granulosus at the larval stage, predominantly develops in the liver and lungs of intermediate hosts and eventually results in organ malfunction or even death. The interaction between E. granulosus and human body is incompletely understood. Exosomes are nanosized particles ubiquitously present in human body fluids. Exosomes carry biomolecules that facilitate communication between cells. To the best of our knowledge, the role of exosomes in patients with CE is not reported. Here, we isolated exosomes from the sera of patients with CE (CE-exo) and healthy donors and subjected them to liquid chromatography-tandem mass spectrometry analysis. Proteomic analysis identified 49 proteins specifically expressed in CE-exo, including 4 proteins of parasitic origin. The most valuable parasitic proteins included tubulin alpha-1C chain and histone H4. And 8 proteins were differentially regulated in CE-exo (fold change>1.5), as analyzed with bioinformatic methods such as annotation and functional enrichment analyses. These findings may improve our understanding about the interaction between E. granulosus and human body, and may contribute to the diagnosis and prevention of CE.

Pro-Angiogenic Activity of Monocytic-Type Myeloid-Derived Suppressor Cells from Balb/C Mice Infected with Echinococcus Granulosus and the Regulatory Role of miRNAs.

BACKGROUND/AIMS: This study aims to predict the pro-angiogenic functions of monocytic-type myeloid-derived suppressor cells (M-MDSCs) derived from mice infected with Echinococcus granulosus. METHODS: M-MDSCs were collected from Balb/c mice infected with E. granulosus and normal mice (control) and cultured in vitro. Human umbilical vein endothelial cells (HUVECs) were stimulated with the cell supernatant, and angiogenesis was investigated and analysed by the Angiogenesis module of the software NIH Image J. RNA was extracted from fresh isolated M-MDSCs and analysed with miRNA microarray; differentially expressed miRNAs and their potential functions were analysed through several bioinformatics tools. Finally, quantitative PCR was used to confirm the results of microarray analysis. RESULTS: M-MDSCs from mice infected with E. granulosus could promote the formation of tubes from HUVECs in vitro. Moreover, vascular endothelial growth factor (VEGF) showed significantly high expression, whereas soluble fms-like tyrosine kinase-1 (sFlt-1) showed low expression at the transcriptional level in M-MDSCs from mice infected with E. granulosus. Microarray analysis of miRNAs showed that 28 miRNAs were differentially expressed in M-MDSCs from the two experimental mice groups, and 272 target genes were predicted using the microRNA databases TargetScan, PITA and microRNAorg. These target genes were mainly involved in the biological processes of intracellular protein transport, protein targeting to the lysosome and protein transport, and mainly located in the cytoplasm, neuronal cell body and membrane. Moreover, they were mainly involved in the molecular functions of protein binding, metal ion binding and SH3 domain binding. Further, the differentially expressed miRNAs were mainly enriched in the endocytosis, Wnt and axon guidance pathways, as well as the MAPK, focal adhesion, PI3K-Akt, cAMP, mTOR and TGF-ß signalling pathways, which are linked to immunoregulation and angiogenesis based on the results of bioinformatics analysis with DIANA-miRPath 3.0. In addition, the expression of eight miRNAs was randomly verified by quantitative PCR independently in three mice infected with E. granulosus and three normal mice. CONCLUSION: M-MDSCs have a potential angiogenic role during E. granulosus infection, and miRNAs may play a role in the immune response and angiogenesis functions of M-MDSCs through regulation of the identified signalling pathways.

Antigen B from Echinococcus granulosus is a novel ligand for C-reactive protein.

Antigen B (EgAgB) is a phosphatidylcholine (PC)-rich lipoprotein of Echinococcus granulosus s.l. larva, potentially capable of modulating the activation of various myeloid cells, including macrophages. As C-reactive protein (CRP) can act as an innate receptor with ability to bind the phosphocholine moiety of PC in lipoproteins, we investigated whether EgAgB and CRP could interact during cystic echinococcosis infection (CE), and how CRP binding could affect the modulation activities exerted by EgAgB on macrophages. To that end, we firstly investigated the occurrence of CRP induction during human CE. We found that 61% of CE patients, but none of healthy donors, exhibited serum CRP levels higher than 10 mg/mL, suggesting that CRP can be induced during the chronic phase of CE. Furthermore, human CRP was capable of binding specifically to EgAgB with high affinity (0.6 ± 0.1 nM); this binding was Ca2+ -dependent and involved the phosphocholine moiety of PC, but not EgAgB8/1, EgAgB8/2 or EgAgB8/3 apolipoproteins. Finally, CRP presence altered the modulation exerted by EgAgB on the cytokine response of LPS-activated macrophages. Overall, our results suggest that CRP presence during CE may contribute to a complex scenario of interactions between EgAgB and myeloid cells, influencing the cytokine response induced during macrophage activation.

Immunotherapy of alveolar echinococcosis via PD-1/PD-L1 immune checkpoint blockade in mice.

The growth potential of the tumour-like Echinococcus multilocularis metacestode (causing alveolar echinococcosis, AE) is directly dependent upon the nature/function of the periparasitic adaptive host immune-mediated processes. PD-1/PD-L1 pathway (programmed cell death 1), which inhibits lymphocytic proliferation in tumour development, is over-expressed at the chronic stage of AE. We tested the impact of a PD-1/PD-L1 pathway blockade on the outcome of both chronic AE (intraperitoneal metacestode inoculation, secondary AE and SAE) and acute AE (peroral egg infection, primary AE and PAE). To assess the parasite proliferation potential, we measured parasite mass weight for SAE and liver lesion number for PAE. In both models, the parasite load was significantly decreased in response to anti-PD-L1 antibody treatment. In SAE, anti-PDL1 administration was associated with increased Th1 response parameters and decreased Treg responses, while in PAE anti-PDL1 administration was associated with fewer lesions in the liver and decreased Treg/Th2 responses. Our findings highly suggested that a PD-1/PD-L1 pathway blockade triggered the host immune responses in favour of an immune-mediated control of E. multilocularis proliferation. Based on this, future studies that combine PD-1/PD-L1 blockade with a parasitostatic albendazole medication may yield in a putatively curative therapeutic approach to control alveolar echinococcosis.

First molecular characterization of Echinococcus granulosus (sensu stricto) genotype 1 among cattle in Sudan.

BACKGROUND: Echinococcus granulosus sensu lato (s.l.) is the causative agent of cystic echinococcosis (CE), which is a cosmopolitan zoonotic parasitic disease infecting humans and a wide range of mammalian species including cattle. Currently, little information is available on the genetic diversity of Echinococcus species among livestock in Sudan. In the present study, fifty (n = 50) hydatid cysts were collected from cattle carcasses (one cyst sample per animal) at Al-kadarou slaughterhouse, Khartoum North, Sudan. DNA was extracted from protoscolices and the germinal layer of each cyst and subsequently amplified by PCR targeting the mitochondrial NADH dehydrogenase subunit 1 (NADH-1) gene. The amplified PCR products were purified and subjected to direct sequencing for subsequent construction of phylogenetic tree and net work analysis. RESULTS: The phylogenetic tree revealed the presence of Echinococcus canadenesis genotype 6 (G6) in 44 cysts (88.0%), Echinococcus ortleppi genotype 5 (G5) in 4 cysts (8.0%) and Echinococcus granulosus sensu stricto (s.s) genotype 1 (G1) in 2 cysts (4.0%). The phylogenetic network analysis revealed genetic variation among the different haplotypes/genotypes. This report has provided, for the first time, an insight of the role of cattle in the transmission of the zoonotic G1 echinococosis. CONCLUSIONS: The results of the study illustrate that Sudanese breeds of cattle may play an important role in the transmission dynamics and the epidemiology of cystic echinococcosis in Sudan. This study reports the first molecular identification of E. granulosus s.s. in cattle in Central Sudan.

The Echinococcus canadensis (G7) genome: a key knowledge of parasitic platyhelminth human diseases.

BACKGROUND: The parasite Echinococcus canadensis (G7) (phylum Platyhelminthes, class Cestoda) is one of the causative agents of echinococcosis. Echinococcosis is a worldwide chronic zoonosis affecting humans as well as domestic and wild mammals, which has been reported as a prioritized neglected disease by the World Health Organisation. No genomic data, comparative genomic analyses or efficient therapeutic and diagnostic tools are available for this severe disease. The information presented in this study will help to understand the peculiar biological characters and to design species-specific control tools. RESULTS: We sequenced, assembled and annotated the 115-Mb genome of E. canadensis (G7). Comparative genomic analyses using whole genome data of three Echinococcus species not only confirmed the status of E. canadensis (G7) as a separate species but also demonstrated a high nucleotide sequences divergence in relation to E. granulosus (G1). The E. canadensis (G7) genome contains 11,449 genes with a core set of 881 orthologs shared among five cestode species. Comparative genomics revealed that there are more single nucleotide polymorphisms (SNPs) between E. canadensis (G7) and E. granulosus (G1) than between E. canadensis (G7) and E. multilocularis. This result was unexpected since E. canadensis (G7) and E. granulosus (G1) were considered to belong to the species complex E. granulosus sensu lato. We described SNPs in known drug targets and metabolism genes in the E. canadensis (G7) genome. Regarding gene regulation, we analysed three particular features: CpG island distribution along the three Echinococcus genomes, DNA methylation system and small RNA pathway. The results suggest the occurrence of yet unknown gene regulation mechanisms in Echinococcus. CONCLUSIONS: This is the first work that addresses Echinococcus comparative genomics. The resources presented here will promote the study of mechanisms of parasite development as well as new tools for drug discovery. The availability of a high-quality genome assembly is critical for fully exploring the biology of a pathogenic organism. The E. canadensis (G7) genome presented in this study provides a unique opportunity to address the genetic diversity among the genus Echinococcus and its particular developmental features. At present, there is no unequivocal taxonomic classification of Echinococcus species; however, the genome-wide SNPs analysis performed here revealed the phylogenetic distance among these three Echinococcus species. Additional cestode genomes need to be sequenced to be able to resolve their phylogeny.

Scolicidal and apoptotic activities of albendazole sulfoxide and albendazole sulfoxide-loaded PLGA-PEG as a novel nanopolymeric particle against Echinococcus granulosus protoscoleces.

Treatment failures of human cystic echinococcosis (CE) with albendazole (ABZ) have attributed to its low solubility and poor drug absorption rate, resulting in low drug level in plasma. The scolicidal effects of ABZ-loaded liposome nanoparticles have recently evaluated; however, these particles have several challenges due to their low encapsulated load. This investigation was designed to evaluate and compare in vitro apoptotic activities of ABZ sulfoxide (ABZs) and ABZs-loaded poly(lactic-co-glycolic acid) (PLGA)-PEG against protoscoleces (PSCs). ABZs-loaded PLGA-PEG was prepared by a double-emulsion method (W1/O/W2). Various concentrations of ABZs and ABZs-loaded PLGA-PEG (50, 100, 150, and 200 µg/ml) were experimentally tested against PSC of CE at different exposure times (5, 10, 20, 30, and 60 min). ABZs-loaded PLGA-PEG at concentrations of 150 and 200 µg/ml was able to act at a 100 % scolicidal rate in all exposure times (5 to 60 min), while ABZs at a concentration of 200 µg/ml demonstrated 94, 100, and 100 % mortality rates following 20, 30, and 60 min of exposure times, respectively. The messenger RNA (mRNA) expression of caspase-3 was assessed by semi-quantitative RT-PCR after 15 h of exposure. Caspase-3 mRNA expression was higher in both PSC treated with ABZs and PSC treated with ABZs-loaded PLGA-PEG than that in control groups (P < 0.05). No significant difference was observed between the apoptotic intensity of PSC treated with ABZs and that of PSC treated with ABZs-loaded PLGA-PEG (P > 0.05). DNA fragmentation assay and ultrastructural changes revealed that ABZs and ABZs-loaded PLGA-PEG induced the apoptosis of PSC by activation of caspase-3. The higher permeability and scolicidal rate of ABZs-loaded PLGA-PEG can be addressed as an effectual alternative strategy to improve the treatment of human CE.

[Study of the genes correlated with cyst calcification in patients with cystic echinococcosis].

OBJECTIVE: To explore the genes (biomarkers) correlated with cyst calcification in patients with cystic echinococcosis (CE), and to provide the evidence for the judgment on the patients' prognosis at molecular level. METHODS: The liver tissues from 32 patients with liver CE (10 cases for mRNA microarray and 22 cases for real-time PCR analysis) and 11 patients with hepatic cystadenoma were collected from three hospitals in Ningxia from June, 2013 to December, 2014. A comparison of the different gene-expressions between five patients with calcified lesions and five cases with no calcification was carried out using Significant Analysis of Microarrays (SAM) to select a subset of differentially expressed genes (DEGs) . Fold-change analysis was used to assess the changes of the expression quantity in the same genes between two groups. The verification was conducted among the liver tissues from 22 patients with liver CE (11 in the group of calcified or 11 in that of non-calcified) by real-time quantitative PCR (RT-qPCR). With GAPDH as a reference-gene and the liver tissues from 11 cases with hepatic cystadenoma as standardized control groups, the relative expressions of galecitin-4 (LGALS4) and acid ceramidase (ASAH1) in patients with calcified and non-calcified were calculated, respectively. The differences between two groups were compared using t'-test. RESULTS: Five screened genes presented siginificantly different expressions all had showed the low-regulated expressions in the calcified group, with the most distinct low-regulation of LGALS4 and ASAH1 whose fold changes were 0.008 8, and 0.020 3, respectively. The verification by RT-qPCR illustrated that the relative expression of LGALS4 was showed at level of 0.49±0.27 amongst patients with calcified, and at level of 2.70±2.61 amongst non-calcified individuals,,indicating significant differences between two groups (t=-2.59, P=0.026); while the ASAH1 was relatively expressed at levels of 1.36±0.33 and of 1.68±0.67 amongst patients with calcified and non-calcified, respectively, showing insignificant changes statistically (t=-1.44, P=0.167). In the non-calcified group, both LGALS4 and ASAH1 genes expression quantities had a small fluctuation range, but with positively correlated trend (r=0.91, P=0.001), which indicated that a patient with the low LGALS4 expression quantity also had a relative low level of ASAH1 expression quantity. CONCLUSIONS: Low expression quantity of LGALS4 and ASAH1 genes in patients with CE in the calcification might be potential biomarker for an indication of the disease self-healing.