Revealing novel cytb and nad 5 genes-based population diversity and benzimidazole resistance in Echinococcus granulosus of bovine origin

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

doi:10.3389/fvets.2023.1191271

Revealing novel cytb and nad 5 genes-based population diversity and benzimidazole resistance in Echinococcus granulosus of bovine origin

Front Vet Sci. 2023 Jun 16:10:1191271. doi: 10.3389/fvets.2023.1191271. eCollection 2023.

Authors

Mughees Aizaz Alvi^{1

2}, Ayed Alshammari³, Rana Muhammad Athar Ali², Shahbaz Ul Haq⁴, Rizwan Bashir⁵, Li Li¹, Muhammad Saqib², Muhammad Sohail Sajid⁶, Muzafar Ghafoor², Muhammad Imran⁷, Muhammad Umar Ijaz⁸, Bao-Quan Fu¹, Mohd Saeed⁹, Irfan Ahmad¹⁰, You-Yu Liu¹, Hong-Bin Yan¹, Wan-Zhong Jia^{1

11}

Affiliations

¹ State Key Laboratory for Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University, National Para-Reference Laboratory for Animal Echinococcosis, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China.
² Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad, Pakistan.
³ Department of Biology, College of Science, University of Hafr Al Batin, Hafr Al Batin, Saudi Arabia.
⁴ Key Laboratory of Veterinary Pharmaceutical Development, Ministry of Agriculture and Rural Affairs, Lanzhou Institute of Husbandry and Pharmaceutical Sciences, Chinese Academy of Agricultural Sciences, Lanzhou, China.
⁵ Veterinary Disease Diagnostic Laboratory Sialkot, Livestock and Dairy Development Department, Government of Punjab, Lahore, Pakistan.
⁶ Department of Parasitology, University of Agriculture, Faisalabad, Pakistan.
⁷ Department of Pathology, University of Agriculture, Faisalabad, Pakistan.
⁸ Department of Zoology, Wildlife and Fisheries, University of Agriculture, Faisalabad, Pakistan.
⁹ Department of Biology, College of Science, University of Hail, Hail, Saudi Arabia.
¹⁰ Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, Abha, Saudi Arabia.
¹¹ Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Disease, Yangzhou, China.

Abstract

Cystic echinococcosis (CE) is a neglected zoonotic disease caused by Echinococcus granulosus (sensu stricto). The parasite affects a wide range of livestock and wild animals. In this study, the population diversity of the Echinococcus species was investigated based on mitochondrial cytochrome b (cytb) and NADH dehydrogenase subunit 5 (nad5) genes. In addition to this, β-tubulin gene isoforms of Echinococcus granulosus were amplified to determine the resistance against benzimidazoles. For this purpose, 40 cyst samples from cattle (n = 20) and buffaloes (n = 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 E. granulosus. BLAST-based searches of sequences of each isolate for each gene (nad5 and cytb) confirmed their maximum similarity with the G1 genotype. Overall, high haplotype diversity (Hd nad5 = 1.00; Hd cytb = 0.833) and low nucleotide diversity (π nad5 = 0.00560; π = cytb = 0.00763) was identified based on diversity indices. For both the genes, non-significant values of Tajima's D (nad5 = -0.81734; cytb = -0.80861) and Fu's Fs (nad5 = -1.012; cytb = 0.731) indicate recent population expansion. Bayesian phylogeny-based results of nad5 and cytb sequences confirmed their genotypic status as distinct from other Echinococcus species. This study shed light on the status of benzimidazole resistance in Echinococcus granulosus for the very first time from Pakistan. The findings of this study will significantly add in the information available on genetic diversity of Echinoccous granulosus based on cytb and nad5 genes sequences.

Keywords: Echinococcus granulosus; benzimidazole resistance; cytb; genetic diversity; nad5; phylogeny.