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

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.

Species and genotypes belonging to Echinococcus granulosus sensu lato complex causing human cystic echinococcosis in Europe (2000–2021): a systematic review

“…species have largely been generated on the basis of mitochondrial cox1 gene sequencing [ 58 ]. Recently, a much more sensitive real-time PCR assay [ 78 ] and a validated method not requiring sequencing [ 79 ] to differentiate E. granulosus s.l. species have been made available in literature, as well as new molecular tools for genotype differentiation (G1 versus G3 and G6 versus G7) within this species complex [ 80 , 81 ].…”

The First Record of Echinococcus ortleppi (G5) Tapeworms in Grey Wolf (Canis lupus)

“…Therefore, recently, there is an interest in developing a universal method for detecting E. granulosus s.l. infections, e.g., a recently published study presenting a set of qPCR methods covering all species of the E. granulosus complex [51].…”

Wild Carnivore Survey of Echinococcus Species in Slovenia

“…Echinococcus multilocularis was detected using a protocol by Knapp et al [ 22 ] with a detection limit of 5 × 10 −5 ng/µL, corresponding to one EM egg. For EGss, a protocol by Maksimov et al [ 23 ] was used, while EC was screened using a protocol published by Grech-Angelini et al [ 24 ]. All qPCRs were performed in a 96-well plate format using ABI 7500 Fast (Applied Biosystems ® , Waltham, MA, USA) with the same thermal cycling conditions consisting of a preheating step at 50 °C for 2 min, followed by 95 °C for 10 min and 45 cycles of denaturation at 95 °C for 15 s with annealing and extension at 60 °C for 1 min.…”