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

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, ß-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). ß-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.

Enhancing activity of ß-lactam and fluoroquinolones antibiotics by artemisinin and its derivatives against MDR Escherichia coli.

Artemisinin and its derivatives had played a biocidal role in biomedical remedies, while they were expected to enhance the activity of antibiotics against multiple drug-resistant (MDR) bacteria. The current study evaluated the interaction of artemisinin (ART), dihydroartemisinin (DHA), artesunate (AS), and artemisinic acid (AA) with ß-lactam and fluoroquinolones antibiotics against Escherichia coli. Antibiotic strip test (E-test), Kirby Bauer's disc test (KB method), and broth microdilution method were adopted for susceptibility analysis, while the checkerboard method was applied to assess synergisms. ART, DHA, AS, and AA showed significantly enhanced antibacterial effects of ß-lactam antibiotics against different strains of E. coli. The study showed ciprofloxacin to be most effective by presenting the least MIC (0.017125 ± 0.0022 µg/ml), while oxacillin was least effective (MIC 256 µg/ml) against E. coli. Synergism between AA and penicillin G (75%), ampicillin (25%), and oxacillin (50%) was observed in all isolates tested. AA and AS significantly decreased the MIC of ampicillin (-0.912 ± 0.908 µg/ml) and ciprofloxacin (-0.901 ± 0.893 g/ml), respectively. Artemisinin and its derivatives increased antibiotic accumulation within E. coli in a dose-dependent manner. The time-kill assay significantly reduced the bacterial number within 24 h of incubation. The study thus concludes greater room for improvement in enhancing the efficacy of antibiotics if used with artemisinin and its derivatives.

Toxicological evaluation of the ultrasonic extract from Dichroae radix in mice and wistar rats.

This study was aimed at evaluating the acute and subchronic toxicity of ultrasonic extract of Dichroae radix (UEDR) in mice and rats. High performance liquid chromatography (HPLC) and thin layer chromatogrephy (TLC) were used to detect ß-dichroine and α-dichroine in UEDR for quality control. The levels of ß-dichroine and α-dichroine in UEDR were 1.46 and 1.53 mg/g, respectively. An oral LD50 of 2.43 g/kg BW was observed in acute toxicity test. After 28-day repeated oral administration, compared with the control group, treatment-related changes in body weight (BW) and body weight gain (BWG), lymphocyte counts and ratios, as well as in the relative organ weights (ROWs) of liver, kidney, lung, and heart, were detected in the middle- and high-dose groups (P < 0.05, P < 0.01), no differences were noted in the serum biochemical parameters and necropsy examinations in both sexes at all doses. Histopathological examinations exhibited UEDR-associated signs of toxicity or abnormalities. After 14 days withdrawal, no statistically significant or toxicologically relevant differences were observed in any of the UEDR-treated groups, and the hispathological lesions in the high-dose group were alleviated. Findings showed that long-course and high-dose of UEDR administration was toxic, and showed dose-dependence, the toxic damage was reversible.