Quantifying the load of Echinococcus granulosus eggs in experimental dog infection using probe-based copro-qPCR analysis
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ORIGINAL ARTICLE
Quantifying the load of Echinococcus granulosus eggs in experimental dog infection using probe-based copro-qPCR analysis Maliheh Riahi1 • Mohammad Ali Mohammadi1 • Ali Afgar1 • Hossein Kamyabi2 • Saeid Nasibi1 Majid Fasihi Harandi1
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Received: 3 May 2020 / Accepted: 25 August 2020 Ó Indian Society for Parasitology 2020
Abstract Designing and implementing Cystic Echinococcosis control programs require quantitative information about the worm load and the intensity of infection in dog populations in endemic areas. So far no ‘‘probe-based’’ molecular quantification tool has been available for E. granulosus. This study was conducted in order to develop and evaluate a qPCR technique for measuring worm load of E. granulosus in the final host. A species-specific TaqMan probe was designed based on the available sequences in GenBank. The study was conducted in two stages. First, stool samples from an experimentally infected dog were collected in days 7, 14, 21, 28, 35 and 49, and were analyzed by real-time qPCR assay. In the second stage, 600 mg negative stool specimens were manually spiked with 1, 5, 10, 20 and 40 eggs and the specimens were analyzed using real-time qPCR. According to the standard
& Majid Fasihi Harandi [email protected]; [email protected] Maliheh Riahi [email protected] Mohammad Ali Mohammadi [email protected] Ali Afgar [email protected] Hossein Kamyabi [email protected] Saeid Nasibi [email protected] 1
Research Center for Hydatid Disease in Iran, Kerman University of Medical Sciences, 76169114115 Kerman, Iran
2
Department of Medical Parasitology, School of Medicine, Kerman University of Medical Sciences, 7616914115 Kerman, Iran
curve analysis, 93% efficiency and coefficients of correlation (Rsq) [ 0.991 were documented. Quantitative PCR assays showed an increasing signal of infection during the 7-week course of infection. As revealed by the qPCR results from week 5 onward, signals indicative of egg excretion began and reached maximum on week 7. No qPCR signal from the samples containing 1, 10 and 20 eggs was recorded, however the samples containing 5 and 40 eggs produced signals proportional to the primary DNA. The study presents a molecular tool to quantify the burden of E. granulosus infection in dogs. This tool could be applied for measuring the burden of infection in the definitive hosts in surveillance and control programs. Keywords Canine echinococcosis TaqMan probe Quantitative PCR Diagnosis
Introduction Cystic echinococcosis (CE) is caused by a small tapeworm known as Echinococcus granulosus. The final host of the parasite are dogs and the intermediate hosts are livestock. Human infection known as hydatid disease usually form space-occupying cysts inside the internal organs especially liver and lungs (Thompson 2017). Increasing dog population is one of the main determinants of the parasite transmission and the spread of disease in human societies. The prevalence of E. granulosus infection in dogs varies from 5 to 70% in different cou
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