scholarly journals Identification of Echinococcus granulosus Genotypes G1 and G3 by SNPs Genotyping Assays

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 125
Author(s):  
Piero Bonelli ◽  
Silvia Dei Giudici ◽  
Angela Peruzzu ◽  
Lorena Mura ◽  
Cinzia Santucciu ◽  
...  
Keyword(s):  
Echinococcus Granulosus ◽  
Animal Species ◽  
Cost Effective ◽  
Sensu Stricto ◽  
Sybr Green ◽  
Hydatid Cysts ◽  
Genotype Identification ◽  
Host Selectivity ◽  
Nad5 Gene ◽  
Echinococcus Granulosus Sensu Lato

Echinococcus granulosus sensu lato (s.l.) is the causative agent of cystic echinococcosis in animals and humans. Different E. granulosuss.l. genotypes exhibit great diversity in their life cycle, host selectivity and pathogenicity. For this reason, the study of genetic variation within Echinococcus species is of importance for their epidemiological implication. We employed two SNP genotyping technologies to distinguish G1 and G3 E. granulosus sensu stricto (s.s.). genotypes. The genotypes of DNA samples (n = 28) extracted from hydatid cysts of different animal species were identified by amplification and sequencing of a fragment of the mitochondrial nad5 gene. Two SYBR green and three TaqMan real time PCR assays were developed for targeting of three nad5 informative positions (SNP758, 1123, and 1380) known to be able to discriminate G1 from G3. Genotyping by SYBR Green PCR based on cycle threshold (Ct) with melting temperature (Tm) analysis and performed on SNP1123 and SNP1380 failed to identify one DNA sample. TaqMan assays for SNP758, 1123 and 1380 effectively confirmed genotype identification obtained by Sanger sequencing. Our results demonstrated that the combination of the three Taqman assays developed in this study represents a valuable and cost effective tool alternative to DNA sequencing for E. granulosus s.s. genotyping.

scholarly journals Species detection within the Echinococcus granulosus sensu lato complex by novel probe-based Real-Time PCRs

2020 ◽  
Author(s):  
Pavlo Maksimov ◽  
Hannes Bergmann ◽  
Marion Wassermann ◽  
Thomas Romig ◽  
Bruno Gottstein ◽  
...  
Keyword(s):  
Real Time ◽  
Echinococcus Granulosus ◽  
Cystic Echinococcosis ◽  
Cost Effective ◽  
Sensu Stricto ◽  
Single Step ◽  
Control Measures ◽  
Chain Reactions ◽  
Disease Epidemiology ◽  
Echinococcus Granulosus Sensu Lato

AbstractInfections with eggs of Echinococcus granulosus sensu lato (s.l.) can cause cystic echinococcosis in intermediate host animals and humans. Upon ingestion of viable eggs, oncospheres hatch from the eggs and subsequently develop into fluid-filled larval cysts, most frequently in the liver or the lungs. The slowly growing cysts progressively interfere with organ function. The risk of infection is determined by the host range of the parasite, its pathogenicity and other epidemiologically relevant parameters, which differ significantly among the five species within the E. granulosus s.l. complex. It is therefore essential to diagnose the correct species within E. granulosus s.l. to help understand specific disease epidemiology and to facilitate effective implementation of control measures. For this purpose, simple, fast and cost-effective typing techniques are needed.We developed quantitative Real-Time Polymerase Chain Reactions (qPCRs) and corresponding sequence-specific hydrolysis DNA probes to target polymorphic regions in the mitochondrial genome of E. granulosus s.l.. In a single-step typing approach, we distinguished E. granulosus s.l. members in four epidemiologically relevant subgroups. These were E. granulosus sensu stricto (G1, G3), E. equinus (G4), E. ortleppi (G5) and the E. canadensis cluster (G6 to G8 and G10). The technique also allowed identification and differentiation of these species from other Echinococcus or Taenia taxa for samples isolated from cysts or faeces.Single-step genotyping techniques for the molecular diagnosis of Echinococcus spp. by qPCRs may not only improve diagnostic performance, but also our knowledge on the epidemiology of the parasites and help controlling the various agents of cystic echinococcosis.

scholarly journals Species Detection within the Echinococcus granulosus sensu lato Complex by Novel Probe-Based Real-Time PCRs

Pathogens ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 791
Author(s):  
Pavlo Maksimov ◽  
Hannes Bergmann ◽  
Marion Wassermann ◽  
Thomas Romig ◽  
Bruno Gottstein ◽  
...  
Keyword(s):  
Real Time ◽  
Echinococcus Granulosus ◽  
Cost Effective ◽  
Sensu Stricto ◽  
Single Step ◽  
Control Measures ◽  
Chain Reactions ◽  
Disease Epidemiology ◽  
Polymerase Chain Reactions ◽  
Echinococcus Granulosus Sensu Lato

Infections with eggs of Echinococcus granulosus sensu lato (s.l.) can cause cystic echinococcosis in intermediate host animals and humans. Upon ingestion of viable eggs, oncospheres hatch from the eggs and subsequently develop into fluid-filled larval cysts, most frequently in the liver or the lungs. The slowly growing cysts progressively interfere with organ function. The risk of infection is determined by the host range of the parasite, its pathogenicity and other epidemiologically relevant parameters, which differ significantly among the five species within the E. granulosus s.l. complex. It is therefore essential to diagnose the correct species within E. granulosus s.l. to help understand specific disease epidemiology and to facilitate effective implementation of control measures. For this purpose, simple, fast and cost-effective typing techniques are needed. We developed quantitative real-time polymerase chain reactions (qPCRs) to target polymorphic regions in the mitochondrial genome of E. granulosus s.l. In a single-step typing approach, we distinguished E. granulosus s.l. members in four epidemiologically relevant subgroups. These were E. granulosus sensu stricto, E. equinus, E. ortleppi and the E. canadensis cluster. The technique also allowed identification and differentiation of these species from other Echinococcus or Taenia taxa for samples isolated from cysts or faeces.

scholarly journals Echinococcus granulosus sensu stricto: silencing of thioredoxin peroxidase impairs the differentiation of protoscoleces into metacestodes

Parasite ◽  
2018 ◽  
Vol 25 ◽  
pp. 57 ◽  
Author(s):  
Hui Wang ◽  
Jun Li ◽  
Chuanshan Zhang ◽  
Baoping Guo ◽  
Qin Wei ◽  
...  
Keyword(s):  
Echinococcus Granulosus ◽  
Sensu Stricto ◽  
Antioxidant Defence ◽  
Culture Studies ◽  
Antioxidant Defence System ◽  
Thioredoxin Peroxidase ◽  
Survival And Growth ◽  
Echinococcus Granulosus Sensu Lato

Cystic echinococcosis (CE) is a cosmopolitan parasitic disease caused by infection with the larval stage of Echinococcus granulosus sensu lato. Thioredoxin peroxidase (TPx) may play an essential role in the antioxidant defence system of E. granulosus s.l. as neither catalase nor glutathione peroxidase activities have been detected in the parasite. However, it is not known whether TPx affects the survival and growth of E. granulosus s.l. during development. In this study, three fragments of siRNA specific for EgTPx (siRNA-1/2/3) were designed and transfected into protoscoleces of E. granulosus sensu stricto by electroporation. Quantitative real-time PCR and Western blotting analysis showed that siRNA-3 significantly reduced the expression of EgTPx. Coincidentally, knockdown of EgTPx expression in protoscoleces with siRNA-3 significantly reduced the viability of the parasite under oxidative stress induced by 0.6 mM H2O2. In vitro culture studies showed that protoscoleces treated with siRNA-3 reduced pre-microcyst formation. In vivo experiments showed that injecting mice intraperitoneally with protoscoleces treated with siRNA-3 resulted in a significant reduction in the number, size and weight of CE cysts compared with those of control animals. Silencing of EgTPx led to the impairment of growth of E. granulosus s.s. both in vitro and in vivo, indicating that EgTPx is an important factor for protoscoleces survival and plays an important role in the antioxidant defence against the host during development.

scholarly journals Cystic Echinococcosis: Clinical, Immunological, and Biomolecular Evaluation of Patients from Sardinia (Italy)

Pathogens ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 907
Author(s):  
Cinzia Santucciu ◽  
Piero Bonelli ◽  
Angela Peruzzu ◽  
Alessandro Fancellu ◽  
Vincenzo Marras ◽  
...  
Keyword(s):  
Molecular Biology ◽  
Echinococcus Granulosus ◽  
Cystic Echinococcosis ◽  
Public Health Problem ◽  
Sensu Stricto ◽  
Surgical Removal ◽  
Neighbor Joining ◽  
Intermediate Hosts ◽  
High Prevalence ◽  
Echinococcus Granulosus Sensu Lato

Cystic echinococcosis (CE), a zoonotic disease caused by the larval stage of the tapeworm Echinococcus granulosus sensu lato (s.l.), is a worldwide public health problem. Echinococcus granulosus sensu stricto (s.s.), associated with G1 and G3 genotypes, is endemic with high prevalence in the Mediterranean basin. The parasite’s life cycle comprises definitive hosts (canids) and intermediate hosts (ruminants) and can occasionally involve humans. The main aim of this research was to confirm the diagnosis of 13 patients suspected of CE who presented different complications and needed the surgical removal of the cysts. We also wanted to understand and clarify more the diagnosis of echinococcosis in humans. For this purpose, the patients first underwent cyst evaluation by ultrasound (US), immunological analysis, and then total pericystectomy, followed by parasitological, histopathological, and molecular biology examinations of the cysts. US stadiated one CE1, one CE2, eight CE3b, one CE4, and two CE5; immunology evidenced nine positives; histopathology confirmed 11 CE cysts, of which 8 fertile presenting protoscoleces were identified as E. granulosus s.s. by molecular biology, genotyped as three G1 and four G3 by neighbor-joining (NJ) phylogenetic tree. In conclusion, the results showed that 11 patients were affected by E. granulosus s.s. G1 orG3, and 2 cystic neoformations were of non-parasitic origin.

scholarly journals Molecular Characterization of Echinococcus granulosus sensu lato from Humans in Slovenia

Pathogens ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 562
Author(s):  
Barbara Šoba ◽  
Špela Gašperšič ◽  
Darja Keše ◽  
Tadeja Kotar
Keyword(s):  
Molecular Characterization ◽  
Echinococcus Granulosus ◽  
Nadh Dehydrogenase ◽  
Sensu Stricto ◽  
Intermediate Hosts ◽  
Western Balkans ◽  
Nadh Dehydrogenase Subunit ◽  
Important Intermediate ◽  
Echinococcus Granulosus Sensu Lato

The larval form of tapeworms of the Echinococcus granulosus sensu lato species cluster cause an important zoonotic infection, cystic echinococcosis (CE). Molecular characterization of the cluster’s isolates from different hosts greatly contributes to a better understanding of its transmission dynamics. To date, no genetic information is available on CE in Slovenia. In this work, we characterized isolates from human CE cases. Parasite samples from 18 patients were collected, together with the patients’ demographic and clinical data. Genomic DNA was analyzed by conventional PCR and sequencing at four mitochondrial loci (cytochrome c oxidase subunit 1, cox1; NADH dehydrogenase subunit 1, nad1; NADH dehydrogenase subunit 5, nad5; and small ribosomal RNA, rrnS). Thirteen isolates were successfully amplified and sequenced. Seven (58.8%) patients were infected with E. granulosus sensu stricto (s.s.) G1, five (38.5%) with E. canadensis G7 and one (7.7%) with E. granulosus s.s. G3. Echinococcus canadensis G7, the pig genotype, was identified exclusively in autochthonous Slovenes, while the patients originating from the Western Balkans were all infected with E. granulosus s.s. Our findings suggest that pigs are important intermediate hosts for human CE in Slovenia.

Genetic characterization of human hydatid cysts shows coinfection by Echinococcus canadensis G7 and Echinococcus granulosus sensu stricto G1 in Argentina

2017 ◽  
Vol 116 (9) ◽  
pp. 2599-2604 ◽  
Author(s):  
María Florencia Debiaggi ◽  
Silvia Viviana Soriano ◽  
Nora Beatriz Pierangeli ◽  
Lorena Evelina Lazzarini ◽  
Luis Alfredo Pianciola ◽  
...  
Keyword(s):  
Echinococcus Granulosus ◽  
Genetic Characterization ◽  
Sensu Stricto ◽  
Hydatid Cysts ◽  
Echinococcus Canadensis ◽  
Echinococcus Granulosus Sensu Stricto

scholarly journals Species detection within the Echinococcus granulosus sensu lato complex by novel probe-based Real-Time PCRs

2020 ◽  
Author(s):  
Pavlo Maksimov ◽  
Hannes Bergmann ◽  
Marion Wassermann ◽  
Thomas Romig ◽  
Bruno Gottstein ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Real Time ◽  
Echinococcus Granulosus ◽  
Cystic Echinococcosis ◽  
Sensu Stricto ◽  
Single Step ◽  
Sybr Green ◽  
Control Measures ◽  
Analytical Sensitivity ◽  
Chain Reactions

Abstract Background: Infections with eggs of Echinococcus granulosus sensu lato (s.l.) can cause cystic echinococcosis in intermediate host animals and humans. Upon ingestion of viable eggs, oncospheres hatch from the eggs and subsequently develop into fluid-filled larval cysts, most frequently in the liver or the lungs. The slowly growing cysts progressively interfere with organ function. The risk of infection is determined by the host range of the parasite, its pathogenicity and other epidemiologically relevant parameters, which differ significantly among the five species within the E. granulosus s.l. complex. It is therefore essential to diagnose the correct species within E. granulosus s.l. to help understand specific disease epidemiology and to facilitate effective implementation of control measures. For this purpose, simple, fast and cost-effective typing techniques are needed.Methods: Mitochondrial genome sequences from the respective members of E. granulosus s.l. complex were used to identify target regions for the detection and genotyping. The selected primer pairs were first tested in a SYBR-green assay. In the next step, primers discriminating between the genotypes (according to post-amplification melting curves) were tested together with the respective probes in a TaqMan® Real-Time Polymerase Chain Reactions (qPCRs). For the analysis of analytical sensitivity and specificity of the real-time PCRs (SYBR-green and TaqMan), a panel of reference DNAs from various Echinococcus spp. was used.Results: We developed quantitative qPCRs and corresponding sequence-specific hydrolysis DNA probes to target polymorphic regions in the mitochondrial genome of E. granulosus s.l.. In a single-step typing approach, we distinguished E. granulosus s.l. members in four epidemiologically relevant subgroups. These were E. granulosus sensu stricto (G1, G3), E. equinus (G4), E. ortleppi (G5) and the E. canadensis cluster (G6 to G8 and G10). The technique also allowed identification and differentiation of these species from other Echinococcus or Taenia taxa for samples isolated from cysts or faeces. Conclusions: Single-step genotyping techniques for the molecular diagnosis of Echinococcus spp. by qPCRs may not only improve diagnostic performance, but also our knowledge on the epidemiology of the parasites and help controlling the various agents of cystic echinococcosis.

National survey and molecular diagnosis of Echinococcus granulosus sensu lato in livestock in France, 2012

Parasitology ◽  
2020 ◽  
Vol 147 (6) ◽  
pp. 667-672 ◽  
Author(s):  
Gérald Umhang ◽  
Céline Richomme ◽  
Vanessa Bastid ◽  
Jean-Marc Boucher ◽  
Carine Peytavin de Garam ◽  
...  
Keyword(s):  
Echinococcus Granulosus ◽  
National Survey ◽  
Parasite Species ◽  
Hydatid Cysts ◽  
Livestock Species ◽  
Zoonotic Risk ◽  
Causative Agents ◽  
National Prevalence ◽  
Systematic Control ◽  
Echinococcus Granulosus Sensu Lato

AbstractThe parasitic species of the Echinococcus granulosus sensu lato (sl) complex are the causative agents of cystic echinococcosis in humans. The lifecycle of E. granulosus sl is essentially domestic, and is based on the consumption by dogs of hydatid cysts in viscera of livestock species. The aim of this study was to survey E. granulosus sensu lato in livestock in France. A 1-year national survey of E. granulosus sl in livestock at the slaughterhouse was organized in 2012 in France, with systematic molecular confirmation. The prevalence of E. granulosus ss nationally was 0.002% in sheep, mainly focused in the Alpine area, and 0.001% in cattle, with the distribution of cases throughout the country. Echinococcus canadensis G6/7 was observed only in Corsica in pigs, with a prevalence of nearly 1% in the island. A national prevalence of 0.0002% was estimated for E. ortleppi in cattle, due to seven cases distributed in two foci. The results of this survey are of particular interest because of the zoonotic risk associated with the presence of these parasite species, for which systematic control at the slaughterhouse should enable their elimination.

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