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.

Comparative anthelmintic efficacy of Arundo donax, Areca catechu, and Ferula assa-foetida against Haemonchus contortus / Eficácia anthelmíntica comparativa de Arundo donax, Areca catechu e Ferula assa-foetida contra Haemonchus contortus

Abstract In the present study, anthelmintic activities of Arundo (A.) donax L., Areca (Ar.) catechu L., and Ferula (F.) assa-foetida L. were determined. Leaves of A. donax L., latex of F. assa-foetida L. and seeds of Ar. catechu L. in different solvent fractions were subjected to in vitro (egg hatch assay; EHA, and adult motility assay; AMA) and in vivo (faecal egg count reduction test; FECRT) tests of anthelmintic activity using Haemonchus contortus model. In the AMA, crude aqueous methanol extracts (CAME) and ethyl acetate fractions of F. assa-foetida at 10 hr post-treatment showed maximum mortality of H. contortus at 12.5-50 mg mL-1. In the EHA, CAME of F. assa-foetida was identified as a potent ovicide based on its low LC50 (16.9 µg mL-1), followed in order by Ar. catechu and A. donax. Results from the FECRT also showed the extract of F. assa-foetida L. to be more effective than those of Ar. catechu L. and A. donax L., against the gastrointestinal parasitic nematodes. Chloroform and ethyl acetate fractions showed better anthelmintic activities against the adult worms in vitro, while CAME of these plants were better than their crude powders in vivo. It is recommended to document and investigate indigenous knowledge of possible medicinal plants to plan scientific trials that may justify their endorsement.

Resumo No presente estudo, as atividades anti-helmínticas de Arundo (A.) donax L., Areca (Ar.) Catechu L. e Ferula (F.) assa-foetida L. foram determinadas. Folhas de A. donax L., látex de F. assa-foetida L. e sementes de Ar. catechu L. em diferentes frações de solvente foram submetidos a testes in vitro (teste de eclosão de ovos, EHA e ensaio de motilidade em adultos, AMA); e in vivo (teste de redução da contagem de ovos fecais, FECRT) de atividade anti-helmíntica, usando-se Haemonchus contortus. Na AMA, extratos aquosos brutos de metanol (CAME) e frações de acetato de etila de F. assa-foetida. Dez horas pós-tratamento, apresentaram mortalidade máxima de H. contortus em 12,5-50 mg mL-1. No EHA, CAME de F. assa-foetida foi identificado como um ovicida potente baseado em seu baixo LC50 (16,9 µg mL-1), seguido em ordem por Ar. catechu e A. donax. Os resultados do FECRT também mostraram que o extrato de F. assa-foetida L. é mais eficaz do que o de Ar. catechu L. e A. donax L., contra nematoides parasitas gastrointestinais. As frações clorofórmio e acetato de etila mostraram melhores atividades anti-helmínticas contra vermes adultos in vitro, enquanto o CAME dessas plantas foi melhor do que o pó bruto in vivo. Recomenda-se documentar e investigar o conhecimento indígena de possíveis plantas medicinais para planejar ensaios científicos que possam justificar seu endosso.

Identification of two novel HSP90 proteins in Babesia orientalis: molecular characterization, and computational analyses of their structure, function, antigenicity and inhibitor interaction.

BACKGROUND: HSP90 protects the cells from heat stress and facilitates protein maturation and stability. The full genome sequences of piroplasms contain two putative HSP90 proteins, which are yet uncharacterized. To this end, the two putative HSP90 proteins of Babesia orientalis were identified and characterized by molecular and in silico methods. METHODS: The two putative proteins in B. orientalis genome showing homology with putative HSP90 of other piroplasms were cloned and sequenced. A computational analysis was carried out to predict the antigenic determinants, structure and function of these proteins. The interactions of two HSP90 isoforms with respective inhibitors were also examined through docking analysis. RESULTS: The length of BoHSP90-A gene (amplified from gDNA) was 2706 bp with one intron from position 997 to 1299 bp. This gene amplified from cDNA corresponded to full length CDS with an open reading frame (ORF) of 2403 bp encoding a 800 amino acid (AA) polypeptide with a predicted size of 91.02 kDa. The HSP90-B gene was intronless with an ORF of 2349 bp, and predicted polypeptide comprised of 797 AA with a size of 90.59 kDa. The AA sequences of these two proteins of B. orientalis were the most identical to those of B. bovis. The BoHSP90-A and BoHSP90-B were recognized as 90 kDa in the parasite lysate by the rabbit antisera raised against the recombinant BoHSP90 proteins. The anti-B. orientalis buffalo serum reacted with the rBoHSP90s expressed in E. coli, indicating that these proteins might be secreted by the parasite before entry into host cells. The overall structure and functional analyses showed several domains involved in ATPase activity, client protein binding and HSP90 dimerization. Likewise, several HSP90 inhibitors showed binding to ATP binding pockets of BoHSP90-A and BoHSP90-B, as observed through protein structure-ligand interaction analysis. CONCLUSIONS: The two putative HSP90 proteins in B. orientalis were recognized as 90 kDa. The rBoHSP90-A and rBoHSP90-B were reacted with the B. orientalis infected buffalo serum. The computational structure and functional analyses revealed that these two proteins may have chaperonic activity. The protein structure-ligand interaction analyses indicated that these two proteins had many drug target sites.

Effect of ivermectin on the cellular and humoral immune responses of rabbits.

The objective of this paper is to determine the effect of ivermectin administration on cell mediated (CMI) and humoral immunity (HI) of rabbits. CMI against dinitrochlorobenzene (DNCB) and sheep red blood cells (SRBC) in rabbits was determined by delayed-type hypersensitivity and macrophage engulfment assay (MEA), respectively; whereas, HI to Pasteurella multocida B2 vaccine and SRBC was determined by indirect haemagglutination assay (IHA) and Jerne hemolytic plaque formation assay (JHPFA), respectively. The rabbits were divided into four major groups (A through D) each subdivided into four sub-groups (1 through 4). Rabbits of group A served as vehicle control while those of groups B, C and D were treated with ivermectin at the dose rates of 200 microg/kg, 400 microg/kg and 600 microg/kg b.w., respectively. Cellular immunity was determined in sub-groups 1 and 2 through DNCB and MEA, respectively while HI was determined in sub-groups 3 and 4 through IHA and JHPFA, respectively. The skin sensitivity to DNCB at 24 and 48 h and macrophage engulfment of SRBC were highest (P>0.05) in rabbits administered with 600 microg/kg b.w. The highest geometric mean titers (14.00+/-0.31) and number of plaque forming units (1860+/-0.75) were found in rabbits that received ivermectin at a dose of 600 microg/kg b.w. followed, in order by the groups that received 400 microg/kg, 200 microg/kg b.w. and controls. Leukocyte counts were significantly higher in ivermectin-treated groups (C and D) than group A (vehicle control) and B (ivermectin at the rate of 200 microg/kg). A graded dose immune response suggested an immunopotentiating effect of ivermectin at higher doses.