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Background: Natural products have been important resources for potential drug development. Among the many classes of natural products, alkaloids have the most therapeutic applications. Treatment of leishmaniasis by chemical drugs remains a challenge because of toxic side effects, limited efficacy, and drug resistance. This review focuses to embrace all researches on leishmanicidal alkaloids over a specific range of time, with special consideration the molecular mechanism of action, and structure-related activity. Methods: All publications (in English) from Web of Science, PubMed, Science Direct, Scopus, and Google Scholar from 2000-2021 using a variety of keywords such as natural alkaloids, herbal alkaloids, marine alkaloids along with leishmaniasis were included in the present study. In this summary, the focus is mainly on natural alkaloids of plant, mineral, marine origin, etc., which have empirically demonstrated the antileishmanial effect. Results: Fourteen categories of alkaloids with anti-leishmaniasis activity were extracted from the articles. The highest number of alkaloids belonged to isoquinoline, indole, and quinoline alkaloids (21.78%, 20.67%, and 16.48% respectively). This review indicated that the mentioned alkaloids are able to inhibit the proliferation of parasites, the respiratory chain and protein synthesis, arrest the cell cycle, disrupt the mitochondrial membrane integrity, inhibit leishmanial topoisomerase, induce mitochondrial dysfunction, and changes in the parasite morphology. Discussion: The present study highlighted antileishmanial alkaloids that are active against different species of Leishmania in vitro and some of them are also active in visceral and cutaneous leishmaniasis models. However, more clinical studies are needed to clarify the anti-leishmanial activity of alkaloids against leishmania in detail. Conclusion: Among the reported compounds, two main classes of alkaloids including isoquinoline and indole alkaloids cover a wider range of anti-parasitic compounds, and structure-activity relationships (SAR) studies of these molecular skeletons may be good lead compounds and afford the structural clues to develop novel medicines with more selective therapeutic profiles.

Multiple sclerosis (MS) is characterized by the destruction of the blood-brain barrier, loss of myelin sheath, and contribution of inflammatory interleukins such as TNF-alpha, interleukin-17, and interleukin-6.The current study investigated the effect of antigen B of hydatid cyst fluid on the reduction of anti-inflammatory cytokines and nerve conduction velocity in rats with experimental autoimmune encephalomyelitis (EAE)-induced MS. After isolation of antigen B from sterile cyst fluid, the rats were randomly divided into four groups: saline, EAE, EAE + teriflunomide (EAE + TF), and EAE + antigen B (EAE + AngB). The EAE model was induced using cow spinal cord homogenization, in combination with Freund's complete adjuvant. The serum concentration of cytokines including IL-1B and IL-17, IL-10, IL-6, and TNF-X was measured by the ELISA method, and real-time PCR was performed to study gene expression. Electrophysiological, behavioral, and neuropathological tests were also conducted.Nerve conduction velocity and IL-10 concentration were increased in the antigen B group. The results of this study showed that antigen B reduced the inflammatory component of the EAE MS animal model by modulating the immune system compared to teriflunomide, which eventually led to a reduction in symptoms at the behavioral and electrophysiological level.It seems that antigen B plays a critical role in regulating immunity and it can be used as a possible therapeutic agent to modulate the immune system in MS patients. It might be rational to consider hydatid cyst fluid antigen as a modifier in MS.

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm harbored in the intestine of canines. There is an urgent need for applied genetic research to understand the mechanisms of pathogenesis and disease control and prevention. However, the lack of an effective gene evaluation system impedes direct interpretation of the functional genetics of cestode parasites, including the Echinococcus species. The present study demonstrates the potential of lentiviral gene transient transduction in the metacestode and strobilated forms of E. granulosus. Protoscoleces (PSCs) were isolated from hydatid cysts and transferred to specific biphasic culture media to develop into strobilated worms. The worms were transfected with harvested third-generation lentivirus, along with HEK293T cells as a transduction process control. A pronounced fluorescence was detected in the strobilated worms over 24 h and 48 h, indicating transient lentiviral transduction in E. granulosus. This work presents the first attempt at lentivirus-based transient transduction in tapeworms and demonstrates the promising outcomes with potential implications in experimental studies on flatworm biology.

MicroRNAs are critical gene regulators at the post-transcriptional level and play essential roles in numerous developmental processes in metazoan parasites including the causative agent of cystic echinococcosis, Echinococcus granulosus. The molecular basis of different patterns of E. granulosus development in the canine definitive host and in in vitro culture systems is poorly understood. In the present study, miRNA transcriptomes of the strobilated worms derived from experimental infection in the definitive host were compared with those from diphasic culture system after 60-day protoscoleces cultivation. Total RNA was extracted from in vivo- and in vitro-derived strobilated worms. Small RNA libraries were constructed, and deep sequencing was performed. Subsequently, differential miRNA expressions and target predictions were obtained, and pathway analysis was performed by gene ontology and KEGG. Seven miRNAs were differentially expressed between the in vivo- and in vitro-derived worms. In addition, we reported 13 novel miRNA candidates and 42 conserved miRNAs. Four out of five top miRNAs with the highest read counts were shared between the in vivo and in vitro-derived worms, i.e., egr-miR-10a-5p, egr-let-7-5p, egr-bantam-3p, and egr-miR-71-5p. Target prediction of the differential miRNAs between the two systems showed significant differences in the membrane-enclosed lumen, membrane part, and an intrinsic component of the membrane. Findings of KEGG analysis indicated that differentially expressed miRNAs were involved in hippo, MAPK, and WNT signaling pathways. The study demonstrated a significant difference in miRNA transcriptomes and related signaling pathways between the two systems, suggesting the importance of host-parasite interplay in the fate of protoscoleces development in in vivo and in vitro systems.

Cystic Echinococcosis (CE) is a parasitic infection caused by the larval stage of Echinococcus granulosus. Exploring safe and effective scolicidal agents for the surgery is an urgent need for the successful treatment of CE. This study aimed to determine scolicidal activity of the synthesized chitosan nanoparticles. Physicochemical properties of synthesized nanoparticles were determined by using DLS, FTIR, and SEM. Different concentrations of chitosan nanoparticles from 125 to 1000 μg/ml were examined at different incubation times (10, 60, 120, and 180 min). Scolicidal and cytotoxic activity of chitosan nanoparticles were confirmed by eosin exclusion and hemolysis activity tests. FTIR spectra, zeta potential (+42 ± 2.08) and PDI (0.388 ± 0.034) value revealed that the chitosan nanoparticles were synthesized. Significant differences among the scolicidal effects of chitosan nanoparticles were observed in comparison to the control treatments and highest scolicidal activity was observed at 1000 μg/ml after 180 min exposure time. Hemolytic activity was not significant at all concentrations of chitosan nanoparticles. Our findings support the hypothesis that Chitosan nanoparticles have the potential to be a safe and efficient scolicidal agent candidate at very low concentrations and in a wide range of exposure time. Further in vivo studies are recommended to evaluate chitosan nanoparticle efficacy before clinical application.

The article's abstract is not available.

Designing and implementing Cystic Echinococcosis control programs require quantitative information about the worm load and the intensity of infection in dog populations in endemic areas. So far no “probe-based” molecular quantification tool has been available for E. granulosus. This study was conducted in order to develop and evaluate a qPCR technique for measuring worm load of E. granulosus in the final host. A species-specific TaqMan probe was designed based on the available sequences in GenBank. The study was conducted in two stages. First, stool samples from an experimentally infected dog were collected in days 7, 14, 21, 28, 35 and 49, and were analyzed by real-time qPCR assay. In the second stage, 600 mg negative stool specimens were manually spiked with 1, 5, 10, 20 and 40 eggs and the specimens were analyzed using real-time qPCR. According to the standard curve analysis, 93% efficiency and coefficients of correlation (Rsq) > 0.991 were documented. Quantitative PCR assays showed an increasing signal of infection during the 7-week course of infection. As revealed by the qPCR results from week 5 onward, signals indicative of egg excretion began and reached maximum on week 7. No qPCR signal from the samples containing 1, 10 and 20 eggs was recorded, however the samples containing 5 and 40 eggs produced signals proportional to the primary DNA. The study presents a molecular tool to quantify the burden of E. granulosus infection in dogs. This tool could be applied for measuring the burden of infection in the definitive hosts in surveillance and control programs.

Background and Aim: Cystic echinococcosis (CE), as a major zoonotic helminth infection, imposes remarkable socioeconomic burden on many endemic countries across the world, including Iran. Due to the high importance of free-roaming dogs in the transmission of CE, epidemiological and molecular studies in this type of hosts are required in the endemic regions. This study aimed to investigate the epidemiology and genotyping of Echinococcus granulosus isolated from stray dogs in Kerman, Southeast Iran. Materials and Methods: Eighty-four samples were isolated from stray dogs in the city and suburbs of Kerman in coordination with the health authorities and the municipality office for rabies control and dog population management. Dog demographic data, including age and sex were collected. The worm was isolated by necropsy and genomic DNA was extracted and partial cytochrome c oxidase subunit 1 gene was amplified using specific primers. Phylogenetic and Templeton-Crandall-Sing (TCS) network analyses were carried out on the sequence data. Results: The overall prevalence of CE in the surveyed dogs was 10.7% (9/84 cases). Out of 84 stray dogs, 33 (39.3%) and 51 (60.7%) cases were male and female, respectively. There was not a statistically significant difference between the infection and gender of dogs. However, infection is shown more in dogs under one year of age with a statistically significant difference (p

Background: Human echinococcosis is a neglected zoonotic disease distributed worldwide. It comprises cystic and alveolar forms, the former being the more prevalent disease. Imaging techniques are the first choice for diagnosis of cystic echinococcosis and serology is used as an additional diagnostic technique in doubtful cases or as the sole test in low-resource settings. Rapid diagnostic tests are useful and convenient for immunodiagnosis of cystic echinococcosis in endemic areas, where medical facilities often struggle with limited resources. Methods: Recently, we have developed Hyd Rapid™, an IgG4 lateral flow dipstick test using recombinant antigen B1 for detection of cystic echinococcosis. This study was performed between 2016 until 2018 at the Institute for Research in Molecular Medicine, Universiti Sains Malaysia. The diagnostic performance of Hyd Rapid™ was tested in-house and at two international laboratories in Switzerland and Iran. Results: The overall diagnostic sensitivity for detection of cystic and alveolar echinococcosis was 95% (56/59). Meanwhile, the diagnostic specificity, with and without exclusion of cysticercosis and fascioliasis, was 100% (n=48) and 88% (63/72), respectively. Conclusion: Hyd Rapid™ detected cystic echinococcosis as well as probable cases of alveolar echinococcosis. Therefore, Hyd Rapid™ showed good potential as a serological tool for echinococcosis, and merits further evaluation.

Alveolar echinococcosis caused by Echinococcus multilocularis is an important zoonotic disease, a great threat to human health due to limited interventions. microRNAs are a type of small non-coding RNA that plays a key role in many diseases and is considered as a potential therapeutic target for control of parasitic diseases. However, naked miRNAs are difficult to enter into cells and are easily degraded in both external and internal environments. Chitosan (CS) has recently been used as a promising vehicle for delivery of nucleic acids. Therefore, we prepared miRNA-bearing CS nanoparticles and investigated the physicochemical properties as well as the delivery efficiency. We found that CS nanoparticles was relatively stable, offered miRNA strong protection from degradation and had low cytotoxicity with no significant effects on cell proliferation and apoptosis. CS nanoparticles were shown to be easily absorbed by cells and have remarkable liver tropism. Furthermore, CS nanoparticles were used to efficiently deliver E. multilocularis miR-4989 in vitro and in vivo and caused a significant reduction in the expression of UBE2N in the liver, a potential target of emu-miR-4989, at both mRNA and protein levels. Our data demonstrate that CS nanoparticles can act as a vehicle for efficient liver-targeted delivery of miRNAs and for development of miRNA-based therapeutics against E. multilocularis infection.

Human echinococcoses are the infection caused by the larval stages of different species of the genus Echinococcus, mostly E. granulosus and E. multilocularis. There is no aggregated information on the nature and characteristics echinococcosis in patients with immunodeficiency. This study presents a systematic review of the current literature published on the status of echinococcosis in immunocompromised individuals.An electronic search of related articles in four major databases (PubMed, Scopus, Web of Science and Google Scholar) was performed up to November 2021. All related studies meeting the inclusion criteria were assessed for qualitative analysis. Data available on different characteristics of the diseases were extracted. The data were subsequently categorized into two subgroups: Cystic Echinococcosis (CE) and Alveolar Echinococcosis (AE).Twenty-eight articles related to the existence of echinococcosis in immunocompromised hosts were included. HIV/AIDS was found as the most frequent condition in immunocompromised CE patients. Most of the CE cases with immunodeficiency were female (66.4%). The dominant stages of the cysts were CE2 and CE3. Surgery was performed for 76.2% of the patients. A high mortality rate of 23.8% was recorded in CE patients. Malignancies was the dominant condition in AE patients.Findings of the present study can potentially improve our understanding of the impact of immunodeficiency syndromes on echinococcoses and contribute to an improved diagnosis, treatment and quality of care in immunocompromised patients suffering from cystic and alveolar echinococcosis.

The larvae of

Sumac has been traditionally used by people as a medicinal plant for the treatment of different disorders. Cystic echinococcosis (CE) is one of the major zoonotic diseases of human with a worldwide distribution. Long term albendazole therapy is usually associated with side effects including impaired liver function and leukopenia. The present study investigated the efficacy of the methanolic extract of Sumac, Rhus coriaria, on the secondary hydatid cyst development in mice and evaluated sumac effects on the expression of a profile of genes with a potential role in parasite development. Thirty-six mice were intraperitoneally inoculated with of with 3000 protoscoleces and six months after induction of infection were divided into three groups that received either oral sumac extract, albendazole or distilled water. The mice were necropsied 45 days later and the volume and weight of cysts were measured. The expression level of five target genes were analyzed using RT-qPCR. The volume and weight of the cysts were significantly lower in the sumac group compared to the controls. Decreased expressions were found in four out of the five genes following sumac administration. While significantly lower expressions in the sumac group were found for the cdk6, b-raf, fgfr and ras genes, no significant difference was found in cdk2 expression as compared with the control groups. Findings of the present study indicate high efficacy of sumac on the size and volume of secondary hydatid cysts in a murine CE model. Further studies are required to explore the most active and effective ingredients of this natural product.

The article's abstract is not available.

Our knowledge on the susceptibility of humans to different genotypes of the zoonotic tapeworm Echinococcus granulosus and the genetic make-up of the cysts in multi-organ involvements is li...

Albendazole, as the main anti-echinococcal benzimidazole, has demonstrated safe and effective therapeutic outcomes in the treatment of echinococcosis. The emergence of resistance or reduced response to albendazole sulfoxide (ABZ_SOX) and other benzimidazoles have been demonstrated in several parasitic helminths of medical and veterinary importance. As the genetic makeup and miRNA profile of helminths affects their response to albendazole sulfoxide, the present study was conducted to investigate the expression of miRNAs in different developmental stages of Echinococcus granulosus exposed to albendazole sulfoxide in vitro. Different developmental stages of the helminth were obtained from in vitro cultured E. granulosus in monophasic and diphasic media. In both ABZ-SOX-treated and control parasites miRNAs were extracted from microcysts, intact protoscoleces and strobilated worms with one and three segments. Expression of two miRNAs, let-7 and miR-61 was evaluated using RT-qPCR for each stage. Results of the present study revealed significant differential expression of both let-7 and miR-61 at different drug concentrations. A significant difference of let-7 expression was observed between the strobilated and metacestode stages of E. granulosus exposed to ABZ-SOX. In the treated protoscoleces, let-7 expression was significantly reduced in the presence of ABZ-SOX at 1000 μg/ml concentration. In contrast higher expression levels were documented in the segmented worms. In the microcysts exposed to different drug concentrations a significant decline of miR-61 expression was demonstrated. Also, a significant increase in expression of miR-61 was observed in one proglottid worms as well as the protoscoleces. Under high drug concentration or long-term exposure of the protoscoleces to ABZ-SOX significantly higher miR-61 expression was observed compared to the controls. Our findings suggested that under in vitro benzimidazole exposure the expression of two E. granulosus miRNAs were significantly affected in the microcyst stage. This study presents the first evidence of the nature of benzimidazole effects on miRNA expression in platyhelminths.

Background: Cystic echinococcosis, a major public health and economic concern, is a zoonotic helminth infection with worldwide distribution. This study was conducted to investigate the genetic characteristics of hydatid cysts isolated from human and livestock in Hamadan region, western Iran during 2016-2017. Methods: Ten human hydatid cysts and 40 animal hydatid cysts including 32 sheep, 5 cattle and 3 goats were genotyped by PCR amplification of two mitochondrial genes, cox1 and nad1. Genetic identification of the isolates was performed by using bioinformatics software and mtDNA nucleotide sequences of the parasite, available in GenBank database. Results: The PCR amplification was successfully carried out on 50 hydatid cyst isolates and then the nucleotide sequencing was conducted. The sequence analysis of the samples found that the isolates belonged to E. granulosus sensu stricto including G1 (42/50, 84), G2 (4/50, 8) and G3 (4/50, 8) genotype. The G1 genotype was detected in human (8/10, 80), sheep (26/32, 81), cattle (5/5, 100) and goat (3/3, 100) hydatid cysts. The G2 and G3 genotypes were found only in sheep and human isolates. Alignment analysis of the cox1 and nad1 gene sequences revealed thirteen and ten sequence types, respectively. Conclusion: G1 was the prevailing genotype of E. granulosus in the area and dogsheep transmission cycle should be considered when implementing hydatidosis control programs. In addition, high genetic diversity was detected among the hydatid cyst isolates. © 2019, Tehran University of Medical Sciences (TUMS). All rights reserved.

Cysticercus pisiformis, the larval stage of Taenia pisiformis, causes serious illness in rabbits that severely impacts the rabbit breeding industry. An inhibitive Th2 immune response can be induced by let-7-enriched exosomes derived from T. pisiformis cysticercus. However, the underlying molecular mechanisms are not completely understood. Here, we report that exosomal miR-let-7-5p released by T. pisiformis cysticercus played a critical role in the activation of M2 macrophages. We found that overexpression of let-7-5p in M1 macrophages decreased M1 phenotype expression while promoting polarization to the M2 phenotype, which is consistent with experimental data in exosome-treated macrophages alone. In contrast, knockdown of let-7-5p in exosome-like vesicles promoted M1 polarization and decreased M2 phenotype expression. Furthermore, down-regulation of transcription factor CCAAT/enhancer-binding protein (C/EBP)-δ resulted in the decrease of M1 phenotype markers and increase of M2 phenotype markers. These results suggested that let-7 enriched in exosome-like vesicles from T. pisiformis metacestodes can induce M2 macrophage polarization via targeting C/EBP δ, which may be involved in macrophage polarization induced by T. pisiformis metacestodes. The finding helps to expand our knowledge of the molecular mechanism of immunosuppression and Th2 immune response induced by metacestodes.

Echinococcosis is considered a cosmopolitan zoonosis caused by different species of small taeniid tapeworms of the genus Echinococcus and is regarded as a neglected zoonosis. Cystic and alveolar echinococcoses are endemic diseases of Tibetan, Pamir, and Iranian plateaus. All of the countries within the Iranian plateau are affected by echinococcosis. Pakistan, Turkey, and Iran are the three most populous countries of the region, in which echinococcosis is highly endemic. The three neighboring countries share strong cultural and socioeconomic ties. The present study aimed to provide a broad review of the status of cystic and alveolar echinococcosis, summarizing the current knowledge about geographical distribution, molecular epidemiology, and transmission dynamics of Echinococcus granulosus sensu lato and Echinococcus multilocularis in this region. Additionally, we aimed to understand disease burden and risk factors as basic requirements for establishing a surveillance system and planning prevention and control programs. A considerable body of information is available on different aspects of echinococcosis in this region; however, several information and research gaps need to be filled before planning control programs. None of the countries in the region have an elaborate echinococcosis control program. Effective control programs require multi/intersectoral coordination within a One Health approach with a long-term political and administrative commitment and enhanced international collaboration among the three countries.

Understanding dynamics of free-roaming dog (FRD) population is critical for planning and implementation of dog population management programs. FRD population size estimation as well as dynamic modeling of dog population under different female dog neutering interventions were investigated in order to determine the most appropriate animal birth control approach. We performed population size estimate of dogs using sight-resight surveys by photography in a randomly selected 25 blocks of the city and all the suburbs of greater Kerman area. Main demographic features were characterized and the dog density distribution was mapped. A dynamic model was developed to predict free-roaming dog population variations after 5 and 10 years. Different scenarios based on 10, 30, 50, 60 and 70% female dog sterilization were considered to predict the effects of animal birth control measures. Free roaming dog population was estimated at 6781 dogs (65.3% males) in Kerman and suburbs with several major population hotspots. Analysis of the dog locations within the city showed that the largest proportion of the dogs were observed in the vacant lots (46.2%). Modeling predictions indicated that, in the absence of management, the free-roaming dog population could increase from a baseline of 6781 to 13,665 dogs (2.02 fold increase) in 5 years and to 19,376 dogs in 10 years (2.86 fold increase). Using a population dynamics model, we simulated five neutering coverages to explore the impact of female neutering on free-roaming dog population size. The 5-year projections of the model have shown that 50% annual female dog sterilization significantly reduced free-roaming dog population by 0.44 comparing to the baseline population. Findings of the present study improve our knowledge on the nature and extent of dog population dynamics in Iran. Effective population control and selection of the most appropriate neutering interventions require a comprehensive knowledge of the characteristics and dynamics of FRD population.

Background The complex life cycle of Echinococcus granulosus involves numerous environmental conditions within different intermediate and definitive hosts. This requires adaptation at different levels of transcript regulation. Alternative splicing (AS) and the related cellular functions as one of the major fields of post-genomics has been poorly studied in tapeworms. In the present study, we investigated AS events and their potential biological effects in E. granulosus. Methods Whole transcriptome sequencing data of four groups of protoscoleces were prepared for RNA-seq library construction. Fresh protoscoleces were either used as non-induced controls (NT group) or incubated for 15 min with pepsin (PEP group) and cultivated in a biphasic medium for 12 and 24 h (12 and 24 h groups). The frequency and different types of AS events were identified using rMATS software. Functional annotations and gene ontology of differential AS (DAS) genes were performed using Blast2GO software. AS events were experimentally validated by PCR on the protoscolex cDNAs using specific primers for each gene. Results At least one AS event was found in 38.1% of the genes (3904 out of 10,245) in the protoscoleces during early strobilar development. The genes were associated primarily with cellular and metabolic processes and binding and catalytic activity. KEGG pathway analysis of DAS events revealed a number of genes belonging to different components of the spliceosome complex. These genes tended to belong to common SR proteins, U1-related factors, U2-related factors, complex A-specific factors and other splicing-related proteins. Conclusions The high number of AS events in the transcriptome regulatory mechanisms indicates the essential rapid molecular processes required by the parasite for adaptation in different environments. Graphical Abstract

Background Cystic echinococcosis (CE) is a disease caused by the larval stage of Echinococcus granulosus sensu lato (s.l.). The treatment of CE mainly relies on the use of benzimidazoles, which can commonly cause adverse side effects. Therefore, more efficient treatment options are needed. Drug repurposing is a useful approach for advancing drug development. We have evaluated the in vitro protoscolicidal effects of tropisetron and granisetron in E. granulosus sensu stricto (s.s.) and assessed the expression of the calcineurin (CaN) and calmodulin (CaM) genes, both of which have been linked to cellular signaling activities and thus are potentially promising targets for the development of drugs. Methods Protoscoleces (PSC) of E. granulosus (s.s.) (genotype G1) obtained from sheep hepatic hydatid cysts were exposed to tropisetron and granisetron at concentrations of 50, 150 and 250 µM for various periods of time up to 10 days. Cyclosporine A (CsA) and albendazole sulfoxide were used for comparison. Changes in the morphology of PSC were investigated by light microscopy and scanning electron microscopy. Gene expression was assessed using real-time PCR at the mRNA level for E. granulosus calcineurin subunit A (Eg-CaN-A), calcineurin subunit B (Eg-CaN-B) and calmodulin (Eg-CaM) after a 24-h exposure at 50 and 250 µM, respectively. Results At 150 and 250 µM, tropisetron had the highest protoscolicidal effect, whereas CsA was most effective at 50 µM. Granisetron, however, was less effective than tropisetron at all three concentrations. Examination of morphological alterations revealed that the rate at which PSC were killed increased with increasing rate of PSC evagination, as observed in PSC exposed to tropisetron. Gene expression analysis revealed that tropisetron at 50 μM significantly upregulated Eg-CaN-B and Eg-CaM expression while at 250 μM it significantly downregulated both Eg-CaN-B and Eg-CaM expressions; in comparison, granisetron decreased the expression of all three genes at both concentrations. Conclusions Tropisetron exhibited a higher efficacy than granisetron against E. granulosus (s.s.) PSC, which is probably due to the different mechanisms of action of the two drugs. The concentration-dependent effect of tropisetron on calcineurin gene expression might reflect its dual functions, which should stimulate future research into its mechanism of action and evaluation of its potential therapeutical effect in the treatment of CE. Graphical Abstract

MicroRNAs are critical gene regulators at the post-transcriptional level and play essential roles in numerous developmental processes in metazoan parasites including the causative agent of cystic echinococcosis, Echinococcus granulosus. The molecular basis of different patterns of E. granulosus development in the canine definitive host and in in vitro culture systems is poorly understood. In the present study, miRNA transcriptomes of the strobilated worms derived from experimental infection in the definitive host were compared with those from diphasic culture system after 60-day protoscoleces cultivation. Total RNA was extracted from in vivo- and in vitro-derived strobilated worms. Small RNA libraries were constructed, and deep sequencing was performed. Subsequently, differential miRNA expressions and target predictions were obtained, and pathway analysis was performed by gene ontology and KEGG. Seven miRNAs were differentially expressed between the in vivo- and in vitro-derived worms. In addition, we reported 13 novel miRNA candidates and 42 conserved miRNAs. Four out of five top miRNAs with the highest read counts were shared between the in vivo and in vitro-derived worms, i.e., egr-miR-10a-5p, egr-let-7-5p, egr-bantam-3p, and egr-miR-71-5p. Target prediction of the differential miRNAs between the two systems showed significant differences in the membrane-enclosed lumen, membrane part, and an intrinsic component of the membrane. Findings of KEGG analysis indicated that differentially expressed miRNAs were involved in hippo, MAPK, and WNT signaling pathways. The study demonstrated a significant difference in miRNA transcriptomes and related signaling pathways between the two systems, suggesting the importance of host-parasite interplay in the fate of protoscoleces development in in vivo and in vitro systems.

Background: In recent decades platyhelminths have been used as model organisms to address some of the fundamental questions related to the growth and development of animal organisms. Epidermal Growth Factor Receptors (EGFR) and Transforming Growth Factor beta (TGF-beta) have a regulatory role in the growth and development of Echinococcus species. This study determined the effect of alpha-tocopherol on the expression of EGFR and TGF-beta genes in three in vitro developmental stages of E. granulosus. Methods: E. granulosus protoscoleces were cultured in diphasic medium containing bovine serum and CMRL 1066. Three developmental stages of E. granulosus, i.e. invaginated protoscoleces, evaginated protoscoleces and three-proglottid worms, were treated by alphatocopherol (250 μg/ml for 36 h) and the expression of EGFR and TGF-beta genes were evaluated by using qPCR analysis. Results: Intact protoscoleces were successfully developed to the segmented worms in diphasic culture media. Higher levels of both EGFR and TGF-beta gene expression were observed in the invaginated protoscoleces as well as the segmented worms in comparison to the non-treated controls. Conclusion: Administration of alpha-tocopherol to different developmental stages of E. granulosus significantly enhanced EGFR and TGF-beta expression in the parasite. Both oxidant and non-oxidant activities of alpha-tocopherol could explain the study findings. Overexpression of the genes could in turn enhance growth factor effects and facilitates the viability of the parasite.

Designing and implementing Cystic Echinococcosis control programs require quantitative information about the worm load and the intensity of infection in dog populations in endemic areas. So far no “probe-based” molecular quantification tool has been available for E. granulosus. This study was conducted in order to develop and evaluate a qPCR technique for measuring worm load of E. granulosus in the final host. A species-specific TaqMan probe was designed based on the available sequences in GenBank. The study was conducted in two stages. First, stool samples from an experimentally infected dog were collected in days 7, 14, 21, 28, 35 and 49, and were analyzed by real-time qPCR assay. In the second stage, 600 mg negative stool specimens were manually spiked with 1, 5, 10, 20 and 40 eggs and the specimens were analyzed using real-time qPCR. According to the standard curve analysis, 93% efficiency and coefficients of correlation (Rsq) > 0.991 were documented. Quantitative PCR assays showed an increasing signal of infection during the 7-week course of infection. As revealed by the qPCR results from week 5 onward, signals indicative of egg excretion began and reached maximum on week 7. No qPCR signal from the samples containing 1, 10 and 20 eggs was recorded, however the samples containing 5 and 40 eggs produced signals proportional to the primary DNA. The study presents a molecular tool to quantify the burden of E. granulosus infection in dogs. This tool could be applied for measuring the burden of infection in the definitive hosts in surveillance and control programs.

Cystic echinococcosis (CE) is distributed worldwide, extending from China to the Middle East and from Mediterranean countries to the sub-Saharan Africa and South America. According to WHO, one million people around the world are suffering from CE with an estimated burden of 183,573 DALYs. The annual monetary burden of the disease due to treatment costs and CE-related livestock losses has been estimated at US$ 3 billion. CE is endemic in all countries within the WHO Eastern Mediterranean Regional Office (EMRO). The region, which includes most of the Middle East and North Africa, is one of the most ancient foci of the domestic cycle of CE and is recognized as one of the major hotspots of CE. There are 22 countries in the EMRO, where about 688 million people are living at risk of CE. In many EMRO countries, little is known about CE epidemiology and transmission. WHO included echinococcosis in a list of 17 neglected tropical diseases (NTDs) and 12 neglected zoonotic diseases (NZDs). Accordingly, different regional offices of WHO organized several initiatives for CE control and prevention. WHO's Western Pacific regional office considered echinococcosis as one of the region's major health topics, and several preventive measures have been implemented in the American region with the support of Pan American Health Organization (PAHO) in Argentina, Peru, Uruguay, and Chile. Although CE is endemic in all 22 EMRO countries, surprisingly, CE is absent from the health topics list of diseases and conditions in this region. Therefore, CE clearly requires further attention in the WHO EMRO agenda, and the need for elaboration of specific measures for CE control is becoming apparent in EMRO countries, where substantial collaborations among the member states and WHO EMRO is of paramount importance. Major topics of collaborative activities include training programs and health communication on different aspects of CE control, analysis of CE burden, national and international surveillance and disease registry systems, technical support to promote epidemiological studies for collecting baseline data, cost-benefit analysis of control interventions, and intersectoral cooperation among the agriculture, veterinary, medical, and health sectors.

Background Cystic echinococcosis, caused by the cestode Echinococcus granulosus, is a neglected tropical disease with remarkable morbidity in humans and a problem of worldwide economic importance in livestock industry. Understanding the molecular basis of the parasite growth and development is essential for the disease diagnosis, management and control. The tetraspanin (TSP) family of proteins are transmembrane proteins with a role in many physiological processes of eukaryotic organisms. TSPs present in the tegumental surface of platyhelminths play pivotal roles in host-parasite interaction. However, little is known about the role of TSPs in growth and development in the Platyhelminthes. To understand the role of TSP1 in the growth and development of E. granulosus we investigated the effect of EgTSP1-specific long dsRNA in different in vitro stages of the parasite. Methods Different stages of E. granulosus, protoscoleces and strobilated worms, were cultivated In vitro in di-phasic media. Using long dsRNA and two delivery methods, i.e. electroporation and electro-soaking, EgTSP1 silencing was performed with an EgTSP1-specific dsRNA. The TSP1 expression profile was assessed as well as the biological and ultrastructural properties of the parasites. Results After three days of dsRNA treatment, EgTSP1 expression was significantly reduced in both stages of E. granulosus as compared to irrelevant/unrelated dsRNA and untreated controls. Silencing expression of EgTSP1 in different stages of E. granulosus resulted in reduced viability and body contractions, inhibition of protoscoleces evagination and distinctive tegumental changes. Ultrastructural morphology of the strobilated worms treated with EgTSP1-specific dsRNA was indicative of the microtriches impairments and vacuolated tegument compared to the control helminths. Conclusions Results of the present study suggest that EgTSP1 plays important structural roles in tegument configuration in E. granulosus. EgTSP1 is proved to be a potential target for the development of vaccines and RNAi-based drugs.

Cystic echinococcosis (CE) is an important public health challenge in Iran that requires a One Health approach to control. Filling information gaps and research needs on different aspects of the disease, including parasite biology, disease ecology, human/animal infection, and administrative issues, is essential to address challenges faced by CE control programs.

Environmental contamination of vegetables and fruits with intestinal protozoan trophozoites, cysts and oocysts is a means of transmitting parasitic agents of public health importance. The purpose of this systematic review and meta-analysis was to determine the global prevalence of intestinal protozoan parasite contamination in vegetables and fruits. Several databases (Web of Science, PubMed, Scopus, ProQuest and Google Scholar) were searched for literature published up to August 2021. Pooled prevalence was determined using the meta-package in R (version 3.6.1). Out of 90,404 publications, 189 articles (202 datasets) met the inclusion criteria. Among these, 183 investigations documented protozoan contamination in vegetables and 20 in fruits. The pooled prevalence (95% confidence interval) was 20% (16%–24%) for vegetables and 13% (7%–21%) for fruits. The highest pooled prevalence was found in South-East Asian WHO region 37% (6%–76%). The most prevalent protozoan parasite in vegetables was Cryptosporidium spp. (11%, 7%–15%). As well, Entamoeba histolytica was the most common agent found in fruits (9%, 4%–14%). Furthermore, the unwashed samples had the highest pooled prevalence of contamination (22%, 3%–49%). Our data suggest a possible risk of protozoan infection in humans via unwashed vegetables and fruits. Accidental ingestion of protozoa occurs through consumption of contaminated vegetables and fruits that have been improperly washed and prepared under poor sanitation. Using sanitary irrigation water, consuming properly cleaned and cooked vegetables, and practicing good hygiene can all assist to reduce the risk of protozoa infection.

Little is known about the genetic and morphological characters ofTaenia ovis. The purpose of the present study was to characterize sheep isolates ofT. ovisusing rostellar hook morphometry as well as mitochondrial genes sequence analysis. Ninety sheep specimens ofCysticercus oviswere collected from 18 slaughterhouses in Iran. The mean ±s.d.for total length of large and small hooks were 174.1 ± 6.4 and 116.7 ± 5.4µm, respectively. CO1 and 12S rRNA sequence analysis showed 11 and nine haplotypes, respectively. The level of pairwise nucleotide variations between individual haplotypes of CO1 and 12S rRNA genes were 0.3–1.1 and 0.2–1.0%, respectively. Level of nucleotide variation in CO1 and 12S rRNA betweenT. ovishaplotypes from present study and eight otherTaeniaspecies was found to be 11.3–17.8 and 5.3–16.3%, respectively. Phylogenetic analysis clustered allT. ovisisolates into a single clade comprised of the all CO1 and 12S rRNA haplotypes. CO1 nucleotide difference betweenT. ovis ovisandT. asiaticawas 13.6% that is lesser than the corresponding difference betweenT. ovis ovisandT. ovis krabbei, warranting the designation of two separate species asT. ovisandT. krabbei. Interclass correlation coefficients showed that there was no significant association between rostellar hook length variation and the variability of the mitochondrial genes.

Synanthropic fly species can be potential mechanical vectors of many infectious agents. The potential of the flies to carry Echinococcus granulosus eggs is not fully documented. The purpose of the present study was to determine the possible role of non-biting flies to carry taeniid eggs. A total of 210 flies were collected from seven selected sites in areas of Kerman city, southeastern Iran from November 2016 to May 2017. Adult flies were live-caught using sweeping nets. Flies were placed individually in small glass bottles and transported to the laboratory. All the flies were killed by deep freezing and then identified to the species level using both morphological and molecular methods. The flies were homogenized in test tubes and genomic DNA was extracted and amplified by PCR. PCR protocols were used both to identify the live-caught flies to the species level, and for the detection of E. granulosus. The laboratory reared second generation flies were experimentally exposed to dog feces manually spiked by Echinococcus eggs. Two runs of experiments with 1–3 h of exposure were designed. For each experiment 20 flies were selected from the stock colony and were starved for three days. After each experiment, the flies were frozen for further molecular studies. The dominant fly species were Musca domestica and Lucilia sericata. No eggs were found on the body surface and/or guts of live-caught flies. After the first hour of exposure, 60%, of the flies of both species were found to harbor Echinococcus eggs. However, in the case of L. sericata 50% of the flies harbored Echinococcus eggs after 3 h of exposure. Results of the present study indicate the probable role of synanthropic flies in harboring Echinococcus eggs and mechanical transmission of cystic echinococcosis. When the helminth eggs are susceptible to desiccation grooming flies can remove many of eggs from exterior surfaces of them. Despite this result the role of synanthropic flies in the transmission of certain helminthiases should not be discounted because of their vagility and feeding mechanisms.