Standardization of parasitic and molecular techniques for the detection and characterization of toxocara spp. from feces and soil

Title : Standardization of parasitic and molecular techniques for the detection and characterization of toxocara spp. from feces and soil

Abstract:

Zoonotic parasites present in dogs and cats, such as Toxocara canis and Toxocara cati, pose a risk, especially in recreational areas where people live with their pets. These helminths are responsible for visceral larva migrans syndrome. The objective of this study was to standardize parasitological and molecular techniques for the detection and differentiation of Toxocara spp. species in fecal and soil samples. Diagnostic sensitivity was evaluated by inoculating parasite-free fecal (1 g) and soil (10 g) samples with T. canis and T. cati eggs at concentrations ranging from 1 × 104 to 1 egg obtained from a female T. canis. The parasitological method was performed by concentration and flotation with a saturated sugar solution [A/C]. DNA extraction was performed using the GENEJET kit, with prior mechanical lysis and quality verification by electrophoresis and A260/A280 spectrophotometry. A PCR assay specific to the genus Toxocara, targeting the IT1–IT2 intergenic region of the 5S ribosomal subunit, was standardized with different primer sets that produce 1000 and 500 bp amplicons. Differentiation between species was achieved by RFLP analysis interpreted with Total1D software. The parasitological method recovered between 60 and 80% of the inoculated eggs and had a sensitivity of 1 egg for both the inoculum alone and with fecal and soil matrices inoculated with eggs. The 1000 and 500 bp PCRs detected 10 fg and 1 Toxocara egg (Te), and 10 pg of DNA and 10 Te, respectively. The sensitivity of PCR for detecting Te decreased to 0 eggs in fecal or soil matrices. The addition of 50 µg of BSA improved amplification in inhibited samples, allowing the detection of between 1 × 10² Te in feces using the 500 bp PCR. For both matrices, amplification was possible by combining parasitological and molecular methods, achieving a sensitivity of 1 × 10⁰ Te. The polymorphism obtained from the 500 bp amplicon allowed differentiation between T. canis and T. cati. These results support the use of [A/C] as the parasitological method for Te detection and the 500 bp PCR for the detection and molecular characterization of Toxocara spp. in fecal and soil matrices with high sensitivity, strengthening epidemiological studies in areas shared by humans and pets.

Biography:

Maria Alejandra Vethencourt Ysea is a microbiologist who graduated from the University of the Andes in Venezuela (1991) and holds a Ph.D. in Science with a concentration in Immunology (2014) from the Venezuelan Institute for Scientific Research (IVIC), as well as a Master’s degree in Molecular Biology from the European Center for Master’s and Postgraduate Studies (2023). She had taught at the Central University of Venezuela (10 years), the University of Medical Sciences (Costa Rica) for 5 years, and currently, since 2025, at the Latin University of Costa Rica. Her current line of research is related to One Health, specifically the molecular detection of parasites and bacteria with zoonotic potential. She has more than 20 publications in this field.