Duplex quantitative real-time PCR assay for the detection and discrimination of the eggs of <it>Toxocara canis</it> and <it>Toxocara cati</it> (Nematoda, Ascaridoidea) in soil and fecal samples

• Main Authors: Durant Jean-Francois, Irenge Leonid M, Fogt-Wyrwas Renata, Dumont Catherine, Doucet Jean-Pierre, Mignon Bernard, Losson Bertrand, Gala Jean-Luc
• Format: Article
• Language: English
• Published: BMC 2012-12-01
• Series: Parasites & Vectors
• Subjects: Duplex real-time PCR; ITS2; Toxocara; Eggs; Fecal; Soil; Samples
• Online Access: http://www.parasitesandvectors.com/content/5/1/288

<p>Abstract</p> <p>Background</p> <p>Toxocarosis is a zoonotic disease caused by <it>Toxocara canis</it> (<it>T. canis</it>) and/or <it>Toxocara cati</it> (<it>T. cati</it>)<it>,</it> two worldwide distributed roundworms which are parasites of canids and felids, respectively. Infections of humans occur through ingestion of embryonated eggs of <it>T. canis</it> or <it>T. cati</it>, when playing with soils contaminated with dogs or cats feces. Accordingly, the assessment of potential contamination of these areas with these roundworms eggs is paramount.</p> <p>Methods</p> <p>A duplex quantitative real-time PCR (2qPCR) targeting the ribosomal RNA gene internal transcribed spacer (ITS2) has been developed and used for rapid and specific identification of <it>T. canis</it> and <it>T. cati</it> eggs in fecal and soil samples. The assay was set up on DNA samples extracted from 53 adult worms including <it>T. canis</it>, <it>T. cati</it>, <it>T. leonina</it>, <it>Ascaris suum</it> (<it>A. suum</it>) and <it>Parascaris equorum</it> (<it>P. equorum</it>). The assay was used to assess the presence of <it>T. cati</it> eggs in several samples, including 12 clean soil samples spiked with eggs of either <it>T. cati</it> or <it>A. suum</it>, 10 actual soil samples randomly collected from playgrounds in Brussels, and fecal samples from cats, dogs, and other animals. 2qPCR results on dogs and cats fecal samples were compared with results from microscopic examination.</p> <p>Results</p> <p>2qPCR assay allowed specific detection of <it>T. canis</it> and <it>T. cati</it>, whether adult worms, eggs spiked in soil or fecal samples. The 2qPCR limit of detection (LOD) in spiked soil samples was 2 eggs per g of soil for a turnaround time of 3 hours. A perfect concordance was observed between 2qPCR assay and microscopic examination on dogs and cats feces.</p> <p>Conclusion</p> <p>The newly developed 2qPCR assay can be useful for high throughput prospective or retrospective detection of <it>T.canis</it> and/or <it>T. cati</it> eggs in fecal samples as well as in soil samples from playgrounds, parks and sandpits.</p>