Abstract

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.

Highlights

  • Infection with Echinococcus granulosus sensu lato (E. granulosus s.l.) [1] can cause cystic echinococcosis (CE) in animals and humans [2,3,4,5].Previously, E. granulosus was considered to be a single species, subdivided into “strains”and genotypes (G1 to G8, G10 and the “lion strain”) [4], most of them associated with specificPathogens 2020, 9, 791; doi:10.3390/pathogens9100791 www.mdpi.com/journal/pathogensPathogens 2020, 9, 791 definitive–intermediate hosts and geographic distribution patterns [6]

  • To test the performance of the TaqMan® quantitative real-time polymerase chain reactions (qPCRs) assays when used with faecal samples, fox faeces were spiked with serially diluted reference DNA to simulate the testing of faeces from definitive hosts infected with members of E. granulosus s.l

  • We developed four sequence-specific, DNA-probe-based qPCR assays that allow differentiation of E. granulosus s.l. species detected in DNA samples derived from cyst material or spiked faecal matter, namely E. granulosus s.s. (G1–G3), E. equinus (G4), E. ortleppi (G5) and E. canadensis (G6–8, G10)

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Summary

Introduction

Infection with Echinococcus granulosus sensu lato (E. granulosus s.l.) [1] can cause cystic echinococcosis (CE) in animals and humans [2,3,4,5]. Identifying the individual species within E. granulosus s.l. is necessary for a detailed epidemiological understanding of the disease in its geographic distribution, for surveillance purposes, and for implementing effective prevention and control measures This is important in endemic areas, where several Echinococcus species and other taeniid cestodes coexist. Diagnostic DNA probe qPCR tests were successfully applied, together with an internal control in duplex or triplex format to identify the single species derived from cystic material or faecal matter We anticipate that this tool will make diagnosis easier and faster, but will be of use for epidemiological studies, effective prevention planning and control programs [13,14]

Results
Detection of Echinococcus
Discussion
Primer and DNA Probe Design for qPCRs
Reference DNA Samples and Faecal Spiking
Conventional PCR
Sequencing of qPCR Products
Full Text
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