AUTHOR=Alvi Mughees Aizaz , Alshammari Ayed , Ali Rana Muhammad Athar , Ul Haq Shahbaz , Bashir Rizwan , Li Li , Saqib Muhammad , Sajid Muhammad Sohail , Ghafoor Muzafar , Imran Muhammad , Ijaz Muhammad Umar , Fu Bao-Quan , Saeed Mohd , Ahmad Irfan , Liu You-Yu , Yan Hong-Bin , Jia Wan-Zhong 

TITLE=Revealing novel cytb and nad5 genes-based population diversity and benzimidazole resistance in Echinococcus granulosus of bovine origin

JOURNAL=Frontiers in Veterinary Science

VOLUME=10

YEAR=2023

URL=https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2023.1191271

DOI=10.3389/fvets.2023.1191271

ISSN=2297-1769

ABSTRACT=<p>Cystic echinococcosis (CE) is a neglected zoonotic disease caused by <italic>Echinococcus granulosus</italic> (<italic>sensu stricto</italic>). The parasite affects a wide range of livestock and wild animals. In this study, the population diversity of the <italic>Echinococcus</italic> species was investigated based on mitochondrial cytochrome b (<italic>cyt</italic>b) and NADH dehydrogenase subunit 5 (<italic>nad</italic>5) genes. In addition to this, β-tubulin gene isoforms of <italic>Echinococcus granulosus</italic> were amplified to determine the resistance against benzimidazoles. For this purpose, 40 cyst samples from cattle (<italic>n</italic> = 20) and buffaloes (<italic>n</italic> = 20) were collected from the main abattoir of Sialkot. DNA extraction was performed using Qiagen Blood and Tissue Kits. Amplification was performed through PCR. Each amplicon was confirmed by GelRed™ stained agarose gel (2%). Samples were sequenced in a DNA analyzer and viewed for any misread nucleotide by using MEGA (v.11). Corrections in nucleotide sequence and multiple sequence alignment were made through the same software. NCBI-BLAST was used for sample specific sequences to identify them as belonging to a particular species. Diversity indices were estimated using DnaSP (v.6) while phylogenetic analysis was inferred using the Bayesian method using MrBayes (v.1.1). β-tubulin gene isoforms sequence analysis was performed to find out the candidate gene causing benzimidazole resistance. All 40 isolates were found positive for <italic>E. granulosus.</italic> BLAST-based searches of sequences of each isolate for each gene (<italic>nad</italic>5 and <italic>cyt</italic>b) confirmed their maximum similarity with the G1 genotype. Overall, high haplotype diversity (Hd <italic>nad5</italic> = 1.00; Hd <italic>cytb</italic> = 0.833) and low nucleotide diversity (<italic>π nad5</italic> = 0.00560; <italic>π = cytb</italic> = 0.00763) was identified based on diversity indices. For both the genes, non-significant values of Tajima’s D (<italic>nad</italic>5 = −0.81734; <italic>cyt</italic>b = −0.80861) and Fu’s Fs (<italic>nad</italic>5 = −1.012; <italic>cyt</italic>b = 0.731) indicate recent population expansion. Bayesian phylogeny-based results of <italic>nad5</italic> and <italic>cytb</italic> sequences confirmed their genotypic status as distinct from other <italic>Echinococcus</italic> species. This study shed light on the status of benzimidazole resistance in <italic>Echinococcus granulosus</italic> for the very first time from Pakistan. The findings of this study will significantly add in the information available on genetic diversity of <italic>Echinoccous granulosus</italic> based on <italic>cytb</italic> and <italic>nad5</italic> genes sequences.</p>