Molecular Identification of Echinococcus spp. and other Taeniid Tapeworms Using Next Generation Sequence Analysis of PCR Amplified 18s rRNA gene

Cystic echinococcosis (CE) is a prevalent zoonotic disease caused by Echinococcus granulosus, with cosmopolitan distribution. The parasite is transmitted cyclically between canines and numerous intermediate herbivorous livestock animals. Also other taeniid tapeworm could infect domestic dogs and they pose significant veterinary and public health concerns worldwide. This study aimed to develop a sensitive molecular method for detecting Echinococcus spp. DNA in dog fecal samples using next-generation sequencing (NGS). A set of PCR primers targeting conserved regions of Taeniid tapeworms 18s rRNA genes was designed and tested for amplifying genomic DNA from various tapeworm species. The PCR system demonstrated high sensitivity, amplifying DNA from all tested tapeworm species, with differences observed in amplified band sizes. The primers were adapted for NGS analysis by adding forward and reverse adapters, enabling sequencing of amplified DNA fragments. Application of the developed PCR system to dog fecal samples collected from Yatta town, Palestine, revealed the presence of E. granulosus DNA in five out of 50 samples. NGS analysis confirmed the specificity of the amplified DNA fragments, showing 98-99% similarity with the 18s rDNA gene of E. granulosus. This study demonstrates the utility of NGS-based molecular methods for accurate and sensitive detection of Echinococcus spp. in dog fecal samples, providing valuable insights for epidemiological surveillance and control programs of echinococcosis in endemic regions. Author SummaryCystic echinococcosis, or hydatidosis, is a serious and chronic zoonotic disease in humans caused by the dog tapeworm Echinococcus granulosus. The disease is transmitted cyclically between canines and numerous herbivorous livestock animals. Determining E. granulosus infection in dogs is crucial for assessing infection risk and identifying new foci of active infections. The infection rate in dogs is also necessary for evaluating transmission dynamics and assessing the efficacy of control programs. In this study, we present a PCR system based on amplification of the 18S rDNA. New primers were designed following an alignment of various taeniid tapeworms 18S rDNA sequences. The current PCR system was adapted to be used in amplicon sequencing utilizing next-generation sequencing technology. This strategy enables accurate detection of tapeworm DNA extracted from dogs fecal samples and provides quantitative measurement of taeniid infection in dogs.