30th Annual Meeting of the German Society for Parasitology

Prof. Dr.

Asst Prof., Dr.

Prof.

Elucidation of Dihydroquinine Mechanism(s) of Action against T. gondii

Abstract text Daniel A. Abugri1, 2, 3*, Joseph A. Ayariga1, Melissa Boersma4, Audrey Napier1, Boakai K Robertson1,2 1Department of Biological Sciences, 2Microbiology PhD Program, 3Laboratory of Ethnomedicine, Parasitology, and Drug Discovery, College of Science, Technology, Engineering and Mathematics, Alabama State University, Montgomery, AL, 36104, 4Department of Chemistry and Biochemistry, Auburn University, Auburn, AL, 36849 * Corresponding Author: Daniel A. Abugri, Ph.D. Email address: dabugri@alasu.edu;Tel:334-604-8158 Abstract Dihydroquinine (DHQ) also known as hydroquinine, is a quinine-derived compound. DHQ has a history of inhibiting Plasmodium falciparum, and Plasmodium berghei, and possesses anti-arrhythmia properties. Though, previous studies in Plasmodium spp have shown its target to be nucleic acids and protein synthesis. Our team recently showed that it disrupts mitochondria membrane potential, upregulates ROS production, and depletes ATP production in T. gondii. To unify these observations with previously identified targets, we tested for the first time the effect of DHQ on T. gondii tachyzoites metabolites and lipid production in a concentration dependent manner. Interestingly, the multi-omics (metabolomics and lipidomics) studies showed that DHQ down-regulates certain lipid classes, nucleic acid precursors, and amino acid synthesis in a concentration-dependent manner. Also, for the first time, in silico analysis showed that DHQ binds strongly to DNA gyrase, Calcium Dependent Protein Kinase 1 (CDPK 1), and prolyl tRNA synthetase and thus could affect DNA replication and translational activities in T. gondii. In summary, our findings indicate that DHQ will be an effective anti-T. gondii agent and could be further developed for clinical use. Keywords: Dihydroquinine, metabolomics, lipidomic, DNA Gyrase, tRNA Synthetase, Calcium Dependent Protein Kinases, T. gondii

Drug Development/Target Identification

Molecular Parasitology

Talk

Dr. Muhammad Afzal

In-vivo anticoccidial efficacy of green synthesized iron-oxide nanoparticles using leaf extract of Ficus racemosa Linn. (Moraceae) against Emeria tenella infection in broiler chickens

Abstract text Introduction Green synthesis of iron oxide nanoparticles (IONPs) using botanical extracts has recently fascinated substantial attention due to their environmental protection advantages, their low-cost, and their easy fabrication with the targeted applications. Objectives &amp; Methods In the present study, IONPs were synthesized using the aqueous leaf extract of Ficus racemosa as a reducing and capping agent. These IONPs were evaluated for their impacts on the growth performance, biochemistry, blood profile, and histology in the coccidiodized broiler chicken with Emeria tenella under in vivo conditions. The confirmation of characteristics features of green synthesized IONPs was first obtained using UV-Vis spectroscopy, Fourier transforms infrared (FTIR), X-Ray diffraction (XRD), energy dispersive X-ray absorption (EDX), scanning electron microscopy (SEM), zeta potential and zeta size. Results The results indicated that IONPs at the moderate dose of 15 mg/kg significantly (p &lt; 0.05) reduced the coccidial impacts by eliminating oocyst shedding per gram feces (up to 91%) and reducing clinical symptoms (lesions (LS = 0), bloody diarrhea (No), and mortality (0%)) in chicken at day 10 of treatment as compared to the negative control group-B (infected &amp; non-treated). A dose-dependent and time-dependent trend were observed during treatments (10, 15, and 20 mg/kg) of 1-3 weeks using IONPs against the coccidial impacts on the growth parameters (body weight gain, mean feed consumption, feed conversion ratio) and biochemistry (plasma glucose, total protein, uric acid, ALT, AST, and ALP) in chickens. Additionally, F. racemosa IONPs at a dose of 15 and 20 mg/kg significantly recovered the parasitized and highly damaged hepatocytes, liver tissues, and ceca tissues after 1-3 weeks of treatment in broiler chickens. Our findings revealed that green synthesized IONPs at the concentration of 15 mg/kg play an important role in the recovery and growth enhancement in coccidiodized broiler chickens. Conclusion Therefore, the green synthesis of IONPs using leaf extract of F. racemosa could be a potential and safe anticoccidial agent with targeted implications in the poultry industry.

Veterinary Parasitology

Prof., Dr.

Differential affinity chromatography coupled to mass spectrometry: a 3R-relevant tool to identify common binding proteins of a broad-range antimicrobial peptide and potentially other compounds active against Toxoplasma gondii

Abstract text Introduction: Leucinostatins and derivatives are antimicrobial peptides (AMPs) with broad-range of activities against cancer cells and infectious agents. One of these, ZHAWOC_6027 (AMP6027), was previously reported as a promising treatment option for African Sleeping Sickness (Brand et al. Angew Chem Int Ed Engl 2021, 60) Objective: To investigate the efficacy of AMP6027 against Toxoplasma gondii in vitro and in vivo, and to identify potential drug targets. Materials &amp; methods: In vitro efficacy (EC50) and ultrastructural studies were done using T. gondiitachyzoites grown in human foreskin fibroblasts (HFF). For in vivo studies, AMP6027 (3 mg/kg/day for 5 days) was applied s. c. in mice experimentally infected with T. gondii oocysts, and cerebral parasite load was determined by real time PCR. The effects of peptide 6027 on B and T cells were investigated in murine splenocytes. To identify peptide 6027-binding proteins in Toxoplasma and splenocytes, differential affinity chromatography coupled to mass spectrometry and proteomics (DAC-MS-proteomics) was performed with cell-free extracts of T. gondii tachyzoites and mouse spleens using AMP6027 or an ineffective analogue. Results: AMP6027 was highly efficacious in vitro (EC50 = 2 nM), and induced distinct mitochondrial changes as seen by TEM. HFF EC50 was 1000 x higher. In vivo, however, AMP6027 exacerbated the infection, caused mild clinical signs and elevated cerebral parasite load. AMP6027 also impaired the proliferation and viability of LPS-stimulated B cells in vitro. Following DAC, MS and proteomics of T. gondii extracts, 269 proteins were identified binding specifically to AMP6027, while in eluates from mouse spleen extracts 645 proteins specifically binding to this peptide were detected. Both datasets contained proteins involved mitochondrial energy metabolism and in protein processing and secretion, and in T. gondii, the cytochrome oxidase (TgApiCox25) complex was identified as the main molecular target among others These results suggest that AMP6027 interacts with common targets involved in essential pathways. Conclusions: Since this methodology can be applied to various compounds as well as target cell lines or organs, DAC-MS-proteomics should be considered a smart and 3R-relevant way to (i) define putative drug targets in pathogens, and (ii) eliminate compounds that are prone to potential side effects before performing tedious and costly safety and efficacy assessments in animals or humans.

Parasite-Host Interaction

Univ.-Prof. Dr. rer. nat.

Quantification of the metabolic activity of Ascaris suum L3 using resazurin reduction

Abstract text Helminth infections are a major health burden of humans and animals alike. Due to rising resistances against anthelmintics, there is an urgent need for new drugs. Current approaches for anthelmintic drug screening are considered subjective, laborious, and low in throughput since their endpoint readouts have to be evaluated microscopically. Here, we aimed to establish and optimize a fluorometric-based assay using resazurin and evaluated the assay by studying the metabolic activity of Ascaris suum (A. suum) larvae (L3), a highly prevalent parasite in swine. A. suum L3 were assessed for their potential to reduce the non-fluorescent resazurin to the highly fluorescent resorufin using up to 1000 L3 and resazurin concentrations up to 15µg/ml. The assay was carried out in 96-microwell plates and fluorescence intensities were measured 24h post-incubation with the dye. Additionally, fluorescence microscopy was used to assess the resazurin reduction site within larvae. Finally, the assay was evaluated with larvae that have been exposed to different metabolic stressors or anthelmintics. Our data show that resazurin is reduced in vital A. suum L3 to resorufin and released into the media. An intact L2 sheath around the L3 of A. suum completely prevented the uptake of resazurin while in unsheathed L3 a fluorescence signal was detected along the intestine. L3 which were metabolically affected by methanol or heat exposure showed a gradually decreased resazurin reduction activity. Exposure to ivermectin at 0.625µM, mebendazole at 5µM and thiabendazole from 10µM to 100µM for 24h significantly decreased larval resazurin reduction activity by 55%, 73% and 70% to 89%, respectively. Together, our data show that metabolic stressors or anthelmintic drugs significantly reduce the resazurin reduction activity of A. suum L3, making the proposed assay a sensitive and easy to use high-throughput method to measure larval metabolic activity in vitro.

Role of intrinsic factors in a long-term evolutionary ivermectin resistance study in the parasite model Caenorhabditis elegans

Abstract text Introduction: Free-living nematodes such as Caenorhabditis elegans (C. elegans) are an appealing tool for experimental evolution, given the easiness of working with them in cultured laboratory conditions. C. elegans is also a critical model for parasitic nematodes working with antiparasitic drugs, from understanding current and new drugs' molecular mechanisms to uncovering more drug targets. This is critical to counter the increase in antiparasitic drug resistance. However, the research of antiparasitic drugs with C. elegans has not been investigated from an evolutionary aspect. Objective: We present an evolution experiment detailing the impact of intrinsic factors such as population size and genetic diversity on the rate of ivermectin resistance evolution. Methods: We used a step-wise ivermectin introduction experiment to evolve resistant strains, which a mathematical model accompanied. The starting ancestor and final ivermectin-resistant populations were subjected to transcriptomic analysis by RNASeq and larval development assays with various anthelmintics. Results: We found that having a genetically diverse population shortened the time it took for the worms to evolve resistance to ivermectin. Different population sizes directly correlated to the rate of ivermectin resistance during the evolutionary experiment. This correlation was also predicted by mathematical modeling. Our RNASeq analysis is ongoing, yet we have many differences in gene expression between the ancestor and resistant worm populations. Lastly, our ivermectin-resistant populations were still sensitive to monepantel and albendazole but resistant to moxidectin and unexpectedly also to emodepside. Conclusion: The work done here is the first of many examples using evolution experiment guidelines to find answers for understanding which factors could influence the occurrence of anthelmintic resistance. We found that intrinsic factors such as genetic diversity and population size play a definitive role in the rate of ivermectin resistance formation in our evolutionary experiment. Our post-evolutionary experiments found that our ivermectin-resistant populations are also resistant to other macrocyclic lactones yet sensitive to other classes of anthelmintic drugs.

Drug Resistance

Faecal egg count reduction tests and nemabiome deep sequencing data reveal widespread resistance against benzimidazoles and macrocyclic lactones in sheep but not cattle parasitic nematodes in north-east Germany

Abstract text Introduction Anthelmintic resistance (AR) of strongyle nematodes against all available drug classes is a worldwide problem in small ruminants and horses and is rapidly spreading in cattle. Particularly, multi-drug resistant parasites pose a high risk for animal health and production. However, since most strongyle eggs are not distinguishable, it is difficult to identify the particular species that has evolved drug resistance. Objectives Since no recent data on the occurrence of AR in German ruminants are available, the faecal egg count reduction test (FECRT) and deep amplicon sequencing (nemabiome) were used to identify resistant strongyle populations and species. Materials and methods The FECRT was conducted using Mini-FLOTAC with fenbendazole (FEB), ivermectin (IVM) and moxidectin (MOX) on eight, eight and twelve sheep farms, respectively. Susceptibility to FEB and eprinomectin (EPR) was investigated with FLOTAC on eight and seven cattle farms, respectively. The FECR with 95% confidence intervals was calculated with the R package eggCounts. Internal transcribed spacer 2 PCR products with 5'-Illumina adapters were barcoded and sequenced on a MiSeq (V3 2x300). Reads were assigned to strongyle species using the nemabiome database and species composition was calculated. Results In sheep, resistance was found on 6/8 farms for FEB, 6/8 farms for IVM, 3/12 farms for MOX and 3/8 farms for all three drugs. Nemabiome data were obtained for eleven farms before treatment and Teladorsagia circumcincta (5.8-91.1% of the larvae) was found on all of them. Trichostrongylus colubriformis (≤41.6%) and Haemonchus contortus (≤42.1%) occurred on 10/11 farms. Chabertia ovina (51.5%) and Oesophagostomum venulosum (27.3%) represented the most abundant species on one farm each. On three farms, triple resistant H. contortus and T. circumcincta were detected. Resistance to FEB and IVM was detected in T. colubriformis, Trichostrongylus vitrinus, Trichostrongylus axei, Cooperia curticei and Cooperia fuelleborni. In cattle, FEB was fully active on all eleven farms while EPR resistance was detected on 1/7 farms. Ostertagia ostertagi and Cooperia oncophora were the most abundant species but Bunostomum phlebotomum and T. axei were the most abundant species on one farm each. No post treatment nemabiome data were obtained. Conclusions In sheep, highly pathogenic parasite species with triple resistance occurred on multiple farms while anthelmintics in cattle were still fully active on most farms.

Session III
Drug Development/ Resistance

Appointment

Chairs

Programme

Session IIIDrug Development/ Resistance

Appointment

Chairs

Programme

Session III
Drug Development/ Resistance