Integrated PCR-Based Molecular Detection System for the Simultaneous Detection of Four Zoonotic Intestinal Parasites from Multiple Sources

Infectious diseases are critical factors affecting human health, economic development, and social stability. Among them, zoonotic intestinal parasitic diseases have increasingly become a focus of global concern as emerging, re-emerging infectious diseases, particularly Cryptosporidium, Enterocytozoon bieneusi, Blastocystis, and Giardia. Only a few studies were published on the epidemiology of the above parasites, accompanied by issues such as single detection targets, low detection rates, and high missing detection rates. This study aims to establish an integrated molecular detection system based on a combination of PCR tests for simultaneous detection of the above four zoonotic intestinal parasites in samples collected from wild mice, companion pets, and livestock (cattle and sheep), in order to provide an efficient technical support for routine parasite detection in multiple scenarios, including animal-environment interfaces especially in local surveillance facilities. In this study, taking the detection of Enterocytozoon bieneusi in feces from 95 wild mice and 30 cats and dogs (25 from cats and 5 from dogs) as primary test, when two sets of primers were used to amplify the ITS gene, the positive results of each PCR were exactly the same (5.26%), but the combination of 2 sets of polymerase chain reaction (PCR) tests resulted higher positive rate (6.32%) for Enterocytozoon bieneusi. For Giardia, 9 positive samples were detected using the beta-giardin (BG) gene, while no positives were detected using the glutamate dehydrogenase (GDH) and triose phosphate isomerase (TPI) genes. The above results demonstrated that a combination of PCR tests for multiple target genetic segments enhanced the detection capacity for designated pathogens, compared to any single PCR test. Target sequences were confirmed by Sanger sequencing. This system was designed to enhance and facilitate the surveillance of infection status, distribution, and phylogenetic evolution of zoonotic intestinal parasites from multiple sources, providing a basis for accurate and sensitive warnings at an early stage. This aims to build a technical bridge for achieving the One Health goal across human-animal-environment interfaces.