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

Summary

Here, we present a protocol to detect four zoonotic intestinal parasites from multiple sources simultaneously based on a combination of polymerase chain reaction (PCR) based integrated molecular detection system.

Abstract

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.

Introduction

The Cryptosporidium, Enterocytozoon bieneusi, Blastocystis, and Giardia are common and easily neglected protozoan pathogens with global prevalence. In recent years, due to factors such as reclamation and intrusion into the ecological niches of natural foci, the popularity of companion animal breeding, and the development of livestock and poultry breeding, the prevalence of multi-source zoonotic intestinal parasitic diseases in rodents, companion animals (cats and dogs), and livestock (cattle and sheep) has increasingly become a focus of emerging, outbreak, and re-emerging infectious diseases at domestic and rural areas, seriously en....

Protocol

The study was approved and conducted by the animal ethics committee of Jiangsu Provincial Center for Disease Control and Prevention. This study was conducted in accordance with the 2020 Edition of the American Veterinary Medical Association Guidelines on Animal Euthanasia, the guiding principles of the Regulations on the Administration of Experimental Animals (Order No. 2 of the State Science and Technology Commission of the People's Republic of China, 1988), and the Guiding Principles for the Humane Treatment of Animals issued by the Ministry of Science and Technology of the People's Republic of China.

1. Preparation of fresh....

Results

Mice Cryptosporidium, Blastocystis SSU rRNA gene, Enterocytozoon bieneusi ITS gene, and Giardia BG gene PCR testing results
All 95 rectal fecal samples from mice were tested for the ITS gene of Enterocytozoon bieneusi using two sets of primers, resulting 5 positive samples for each method, resulting 6 positive samples in t.......

Discussion

The Cryptosporidium, Enterocytozoon bieneusi, Blastocystis, and Giardia are common zoonotic intestinal parasites; the first two are classified as Category B biological pathogens by the U.S. National Institutes of Health (NIH), and all four are listed by the World Health Organization (WHO) as 24 of the most important food-borne parasites worldwide. They can potentially infect humans, animals, and contaminate the environment, which may cause disease outbreaks and significantly affect pub.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose	Qingke Tech	TSJ001
Qiaquick gel extraction kit	Qiagen	28704	DNA gel extraction kit
100 bp Plus DNA Ladder	TransGen Biotech	BM311	DNA Ladder
4S Green Plus Nucleic Acid Stain	BBI	A616696-0500	DNA stain
10×TBE Buffer	Solarbio	T1051	electrophoreisis buffer
Qubit 4.0	Thermo	https://www.thermofisher.com/in/en/home /industrial/spectroscopy-elemental -isotope-analysis/molecular-spectroscopy/ fluorometers/qubit/models/qubit-4.html? ef_id=:G:s&s_kwcid=AL!3652!10!77515 785702067!!!!77516061528018!!380468 453!1240249321530142&cid=bid_pca_ aqb_r01_co_cp1359_pjt0000_bid 00000_0se_bng_bt_pur_con	Fluorescence quantitative instrument
MINI Space 1000	Tanon	http://en.biotanon.com/PRODUCT/ PRODUCTLIST?ID=3040& TYPE=DETAIL	Gel imager
Fast DNA Stool Mini Kit for DNA extraction	QIAGEN	51604	Nucleic acid extraction kit
ProFlex Base	Thermo	https://www.thermofisher.com/ in/en/home/life-science/pcr/ thermal-cyclers-realtime- instruments/thermal-cyclers /proflex-pcr-system.html	PCR
Dream Taq Green PCR Mix	Thermo	K1082	Pcr amplification