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

Abstract

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...

Support for targeted sampling of red fox (Vulpes vulpes) feces in Sweden: a method to improve the probability of finding Echinococcus multilocularis

“…Cats may also be infected; however, cats are considered poor hosts due to low infection intensity and few infective eggs produced [33]. Recent studies have used molecular methods to confirm species identification of feces [35, 36], but these methods were not used here as background environmental contamination from feces occurs regardless of the definitive host. Although misidentified feces may have led to an underestimation of the positive proportions, this underestimation would not change the conclusions drawn from the results.…”

A flotation/sieving method to detectEchinococcus multilocularisandToxocaraspp. eggs in soil by real-time PCR

“…The qPCRs were performed in duplicate and run on a RotorGene thermocycler (Qiagen) with a program that consists of 10 min at 95 °C and 45 cycles of 15 s at 95 °C and 60 s at 60 °C. All E. multilocularis -positive copro-samples obtained by real-time PCR were confirmed by sequencing the PCR products of a second real-time PCR, performed on the same gene, as proposed by Knapp et al [15]. Briefly, the same qPCR was performed but using a new reverse primer to obtain a longer fragment.…”

Space invaders: Searching for invasive Smallmouth Bass (Micropterus dolomieu) in a renowned Atlantic Salmon (Salmo salar) river

“…Many factors will influence the degradation of eDNA (Shogren et al, ; Strickler et al, ) and which is complicated by the river's discharge (Jane et al, ; Wilcox et al, ). While we are learning more about these factors and interpreting/misinterpreting eDNA in lotic environments, we have minimized the potential for false negatives in our study by collecting field blanks to identify possible containments (Carim et al, ), tested similar species (Wilcox et al, ), and conducted the Sanger sequence to cross‐validated qPCR results (Knapp, Umhang, Poulle, & Millon, ). There remain pathways that may produce false positives (Roussel, Paillisson, Tréguier, & Petit, ; Wilcox et al, ).…”

“…positive amplifications", number of positive amplification; "C T values" are the associated C T values, and "No. molecules" are the number of molecules to identify possible containments , tested similar species (Wilcox et al, 2013), and conducted the Sanger sequence to cross-validated qPCR results (Knapp, Umhang, Poulle, & Millon, 2016). There remain pathways that may produce false positives (Roussel, Paillisson, Tréguier, & Petit, 2015;Wilcox et al, 2013).…”