Real time PCR to detect the environmental faecal contamination by Echinococcus multilocularis from red fox stools

Knapp, Jenny; Millon, Laurence; Mouzon, Lorane; Umhang, Gérald; Raoul, Francis; Ali, Zeinaba Said; Combes, Benoît; Comte, Sébastien; Gbaguidi‐Haore, Houssein; Grenouillet, Frédéric; Giraudoux, Patrick

doi:10.1016/j.vetpar.2013.12.023

Cited by 69 publications

(72 citation statements)

References 21 publications

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“…This particular PCR protocol succeeded in increasing the sensitivity of detection, which is of particular interest when focusing on infection with E. multilocularis of dogs where a low prevalence is generally expected. The real-time PCR recently developed by Knapp et al (2014) may constitute a relevant approach in order to overcome the lack of sensitivity of conventional PCR and enable identification of the potential presence of inhibitors by using an internal control.…”

Section: Discussionmentioning

confidence: 99%

First detection of Echinococcus multilocularis in dogs in a highly endemic area of Poland

Karamon¹,

Samorek-Pieróg²,

Kochanowski³

et al. 2016

FOLIA PARASIT

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Abstract:The aim of the investigation was to estimate the epizootic situation concerning infection by the cestode Echinococcus multilocularis Leuckart, 1863 in dogs (Canis lupus familiaris Linnaeus) from a Polish region where this parasite is highly prevalent in red foxes. Faecal samples (n = 148) were collected from rural dogs in Podkarpackie Province. Samples were examined through nested PCR (for E. multilocularis), multiplex PCR (E. multilocularis, species of Taenia Linnaeus, 1758) and PCR [E. granulosus (Batsch, 1786)]. Specific products were sequenced. Faeces were also examined coproscopically. In samples from two dogs (1.4%), there were positive PCR results for E. multilocularis. Taenia-specific PCR products were found in nine dogs (6.1%). Sequencing identified Taenia serialis (Gervais, 1847), T. hydatigena Pallas, 1766, T. pisiformis (Bloch, 1780) and Hydatigera taeniaeformis (Batsch, 1786). One sample (0.7%) was identified as Mesocestoides litteratus (Batsch, 1786). All samples were negative for E. granulosus with PCR. Taking into account coproscopic and PCR results, 28% of dogs were infected with helminths (8% with tapeworms). It should be stressed that one of the infected with E. multilocularis dogs shed eggs of the Taenia type and had a habit of preying on rodents. This investigation revealed the presence of E. multilocularis in dogs for the first time in Poland.

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Section: Discussionmentioning

confidence: 99%

First detection of Echinococcus multilocularis in dogs in a highly endemic area of Poland

Karamon¹,

Samorek-Pieróg²,

Kochanowski³

et al. 2016

FOLIA PARASIT

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“…The qPCR was run on a model 7500 Fast real-time PCR system (Life Technologies, Foster City, CA). The PCR program was the same as that used to detect E. multilocularis in fox stools by qPCR with 45 cycles, designed by Knapp and coworkers (23). All PCRs were performed in duplicate, and results were expressed as quantitative cycle (C q ) numbers.…”

Section: Methodsmentioning

confidence: 99%

“…The previously developed qPCR for E. multilocularis detection and quantification (23) was tested in this study in duplex with the Alea target (Em/Alea qPCR). The marker was designed from part of the mitochondrial rrnL gene.…”

Section: Methodsmentioning

confidence: 99%

“…Thanks to these advanced techniques, zoonotic parasites such as Toxocara spp. (21) and Echinococcus multilocularis (22,23) can be detected and quantified in stools. Real-time nested PCR using fluorescence resonance energy transfer (FRET) probes was designed to detect and discriminate between different host species by melting curve analysis for field studies focusing on Echinococcus multilocularis investigations and the carnivore hosts Vulpes vulpes (red fox), Vulpes ferrilata (Tibetan fox), and Canis lupus familiaris/Canis lupus (domestic dog/wolf) (22).…”

mentioning

confidence: 99%

“…multilocularis DNAs in fox stools were quantified by a qPCR targeting a mitochondrial marker (23). However, to date, very few sensitive tools have been developed to describe environmental contamination by Toxocara spp.…”

mentioning

confidence: 99%

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Development of a Real-Time PCR for a Sensitive One-Step Coprodiagnosis Allowing both the Identification of Carnivore Feces and the Detection of Toxocara spp. and Echinococcus multilocularis

Knapp

Umhang

Poulle

et al. 2016

Appl Environ Microbiol

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dStudying the environmental occurrence of parasites of concern for humans and animals based on coprosamples is an expanding field of work in epidemiology and the ecology of health. Detecting and quantifying Toxocara spp. and Echinococcus multilocularis, two predominant zoonotic helminths circulating in European carnivores, in feces may help to better target measures for prevention. A rapid, sensitive, and one-step quantitative PCR (qPCR) allowing detection of E. multilocularis and Toxocara spp. was developed in the present study, combined with a host fecal test based on the identification of three carnivores (red fox, dog, and cat) involved in the life cycles of these parasites. A total of 68 coprosamples were collected from identified specimens from Vulpes vulpes, Canis lupus familiaris, Canis lupus, Felis silvestris catus, Meles meles, Martes foina, and Martes martes. With DNA coprosamples, real-time PCR was performed in duplex with a qPCR inhibitor control specifically designed for this study. All the coprosample host identifications were confirmed by qPCR combined with sequencing, and parasites were detected and confirmed (E. multilocularis in red foxes and Toxocara cati in cats; 16% of samples presented inhibition). By combining parasite detection and quantification, the host fecal test, and a new qPCR inhibitor control, we created a technique with a high sensitivity that may considerably improve environmental studies of pathogens. For detection of infectious agents in the environment, which involves both wild and domestic animals, coprosamples are noninvasive and valuable isolates, as opposed to necropsy, which is time-consuming, logistically onerous, and often considered unethical by the public. In this field of work, Echinococcus multilocularis is the causative agent of alveolar echinococcosis, one of the most worrying zoonoses in the Northern Hemisphere (1), and is in constant progression (2-4). Toxocara spp. are the causative agents of toxocariasis, a widespread disease that is still described in known areas of endemicity despite relevant anthelminthic programs (5). Both E. multilocularis and Toxocara spp. are zoonotic agents involved in a fecal-oral transmission cycle from carnivores to humans, with no vectors or intermediate actors (5). These helminth parasites are both found in at least one wild carnivore (red fox) and two domestic carnivore (dog and cat) host species. While E. multilocularis is a common tapeworm of the red fox, low parasite prevalences have often been described for European domestic host species (cat and dog) (6-8). E. multilocularis eggs are highly resistant to environments with low temperatures and high percentages of relative humidity. Under controlled laboratory conditions, the eggs were found to survive in water for 478 days at 4°C. Under these experimental conditions, eggs remained infectious for rodents (9). Carnivores become infected after predation of contaminated rodents and release eggs into the environment via their feces. Alveolar echinococcosis in humans remains a let...

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2015

EFSA Journal

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The European Food Safety Authority (EFSA) was required to support the European Commission in preparing the review of Regulation (EU) No 1152/2011. In Europe, red fox (Vulpes vulpes) is the main definitive host of the Echinococcus multilocularis (EM) lifecycle. There is no evidence that any other carnivore species can maintain the lifecycle in the absence of red fox, and this makes it to most relevant target species for surveillance. Movement of infected definitive hosts is an important introduction pathway. The knowledge on the geographical distribution of the environmental factors for the persistence of the lifecycle is scarce. In areas where no suitable autochthonous wild canid hosts and no highly suitable intermediate hosts are present, e.g. Malta, establishment of the EM cycle is considered close to impossible. Such countries do not need to carry out surveillance on domestic dogs to substantiate absence of EM in the relevant animal population. Reconsideration of some aspects of the current legislation regarding surveillance activities might be relevant; for example the identification of epidemiologically relevant units should be independent from political borders. Studies to improve the knowledge on epidemiological risk factors should be encouraged to enable risk-based sampling. Echinococcus notification should always be done at species level in order to discriminate between the more severe alveolar echinococcosis and the cystic echinococcosis. Praziquantel is the substance of choice for the treatment of dogs. However, the treatment window should be reconsidered to reduce the risk of re-infection: a general rule is to treat as close as possible to entry into a non-infected country. There is a lack of standardization of the diagnostic methods between laboratories. The diagnostic sensitivity of the tests should be established in accordance to the World Organisation for Animal Health (OIE) standards for validation. For the time being, the diagnostic sensitivity can be set conservatively to 78%.

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Real time PCR to detect the environmental faecal contamination by Echinococcus multilocularis from red fox stools

Cited by 69 publications

References 21 publications

First detection of Echinococcus multilocularis in dogs in a highly endemic area of Poland

First detection of Echinococcus multilocularis in dogs in a highly endemic area of Poland

Development of a Real-Time PCR for a Sensitive One-Step Coprodiagnosis Allowing both the Identification of Carnivore Feces and the Detection of Toxocara spp. and Echinococcus multilocularis

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