30th Annual Meeting of the German Society for Parasitology

Prof.

Prof. Dr.

Objective in vitro drug testing for Echinococcus granulosus

Abstract text Introduction Cestodes of the genus Echinococcus cause the zoonotic diseases alveolar echinococcosis (AE) and cystic echinococcosis (CE). An in vitro drug screening pipeline is established for AE, but drug efficacy assessments against E. granulosus are mainly performed via subjective eosin exclusion test on protoscoleces (PSCs), which are not the disease-causing stage. Thus, there is the urgent need for unbiased drug tests against E. granulosus. Objectives Our objectives were to validate whether the current in vitro drug screening assays for E. multilocularis metacestodes, protoscoleces and primary cells can be applied to E. granulosus. Furthermore, we aimed to establish E. granulosus metacestodes cultures that can be maintained in vitro for prolonged periods of time and to investigate whether proliferative primary cell cultures of E. granulosus could form new metacestode vesicles. Material and methods We applied an in vitro screening cascade established for E. multilocularis to E. granulosus to compare the efficacy of several standard drugs (niclosamide, nitazoxanide, albendazole, monepantel, mefloquine, buparvaquone and MMV665807). Efficacy against metacestodes was measured by damage marker release and vesicle viability assays. Efficacy against PSCs was assessed by motility assay, and viability assay was employed to investigate the impact on stem cells. Proliferation of primary cells was assessed by EdU incorporation and metacestode vesicle formation assays. Results In vitro cultured E. granulosus metacestodes exhibited an intact laminated layer. Comparing drug efficacy against E. multilocularis and E. granulosus in vitro, the damage marker release assay and the metacestode viability assay showed similar drug responses for most tested drugs, while buparvaquone and MMV665807 showed significantly higher activity to E. multilocularis metacestodes. Albendazole had a higher impact on E. granulosus PSCs. Stem cell assays showed similar activities for most tested drugs, but nitazoxanide, albendazole and monepantel showed higher activity to E. multilocularis stem cells. E. granulosus primary cells were successfully cultured for four weeks, underwent proliferation and formed novel metacestodes. Conclusion Our results show that established drug screening assays can be applied to E. granulosus and that its stem cells can be cultured to form novel metacestodes in vitro. This allows in vitro screening of drugs against E. granulosus in an unbiased and objective manner.

Drug Development/Target Identification

One Health/NTD/Zoonoses

Talk

Priv. Doz. Dr.

Efficient method of extracting off-host stages of Tunga penetrans (sand flea) for the identification of transmission hotspots

Abstract text Introduction Tunga penetrans, (sand flea) is the cause of a severely neglected parasitic skin disease in the tropics and received little attention from entomologists to understand the transmission ecology. Objective Like all fleas, T. penetrans has environmental off-host stages and we used the Berlese-Tullgren funnel method to extract these off-host stages from soil samples to identify the disease transmission hotspots within rural households in Uganda and Kenya. Material and Methods The Berlese-Tullgren Funnel extractor was developed from locally available material in a small size with several units to fit the purpose of our work. We calibrated the apparatus by investigating the influence of different bulb powers as heat source and two extraction times. Field surveys were conducted in 50 tungiasis affected households. A total of 65 outdoor samples and 160 indoor samples were collected and extracted. The odds ratios or rate ratios and their 95% confidence intervals were calculated using generalized models in R software. Results The impact of heat (bulb wattage) and time (hours) on the efficiency of extraction was demonstrated and through a stepwise approach standard operating conditions defined for parallel extraction of 80% (95% CI 71-87%) of all present off-host stages from any given soil sample alive. The odds of finding off-host stages from indoor samples associated with infected individuals sleeping places was 7-fold higher (95% CI 2.4-21.5) than finding them from outdoor samples and for every larva found in the outdoor environment, 29 larvae were found indoors (95% CI 7.7-110.2). Conclusions Our findings highlight that the indoor sleeping areas are the transmission hotspots for tungiasis and can be targeted for disease control and prevention measures.

Parasite-Host Interaction

Efficacy of natural and synthetic insect growth regulators for the control of sand fleas (Tunga penetrans) in laboratory bioassays

Abstract text Introduction:Tunga penetrans, (sand flea) is the cause of a tungiasis, a severely neglected tropical parasitic skin disease. Even if the disease is treated, re-infection occurs almost immediately due to off-host stages. It is, therefore, vital to identify targets for parasite control as treatment and prevention tools. Insect growth regulators such as pryriproxyfen have the ability to mimic a natural hormone in insects and disrupt their growth and is used widely for parasite control in pets and livestock, and is available in low-income countries for plant pest control. Neem oil, a natural product available in many tropical regions, has similar properties on a range of insects.Objective:To implement dose-response bioassays under controlled laboratory conditions to assess the efficacy of both products against Tunga off-host stages and to establish the optimum concentration for field evaluations.Material and Methods:Dose-response tests with four increasing exposure amounts of neem and pyriproxyfen were implemented for their impact on off-host stage development and adult emergence. These experiments were replicated 10 times on different dates containing 10 larvae each from different collection batches.Results:Pyriproxyfen was confirmed as a powerful insect growth regulator. Whilst in the unexposed controls 100% of all larvae successfully developed into adults, having pupated at day 10 after collection and taking a median of 9.5 (IQR 4) days in the cocoon until adult emergence on day 30 (IQR 4) after collection. Pyriproxyfen prevented 100% of the larvae to pupate in as low concentrations as 0.0075 parts per million (ppm) active ingredients. Neem oil was only effective at relatively high dosages. The mode of action for the neem oil, was consistently different from pyriproxyfen since larvae died in the process of trying to pupate around 10-12 days after collection and out of the larvae that managed to pupate, pupation took place by day 10 as in the controls but only 30% managed to emerge as adults. However, the effective dose of 15 ppm for LD50 would translate in an application of 60 ml of neem oil per square metre of floor area in the field.ConclusionsPyriproxyfen-based products for spray application on infested household floors should be tested for potential disease control. The impact of neem oil was only achieved at extremely high application rates and hence would not be a feasible product to take forward.

Population biology of Strongyloides stercoralis (nematoda) – How many different species and hosts are there?

Abstract text The estimated number of people infected with Strongyloides stercoralis, the species which causes the vast majority of human strongyloidiasis cases, has been corrected upwards over the last few years and has now surpassed the 600 millions. Recent studies by us and others revealed considerable genetic and genomic diversity and variability in the biology, in particular life cycle and host preferences, of Strongyloides stercoralis (s.l). Therefore, it is questionable that what is currently considered to be Strongyloides stercoralis is really only one species. In order to complement the previous studies, we are currently collecting S. stercoralis from human and animal (i.e. canine and non-human primate) hosts is Ghana and in Bangladesh. Results from these collection efforts and an update on the establishment of a collection of live Strongyloides stercoralis isolates will be presented.

Soil-transmitted Parasitoses

Univ.-Prof. Dr. rer. nat.

In Vitro Effect of Artemisinin and Its Derivatives on the Metabolic Processes of Ascaris suum Third Stage Larvae

Abstract text Soil transmitted helminths (STH) infect a total of 807 million – 1.2 billion people in the world, Ascaris lumbricoidesbeing the most prevalent gut nematode in sub-Saharan Africa. Important to note is that co-infection with malaria, caused by Plasmodium falciparum, and STH is common in areas with overlapping endemicity. WHO recommends the use of artemisinin-based combination therapy (ACTs) as the first-line treatment of symptomatic malaria. Artemisinin and its derivatives; artemether, artesunate and dihydroartemisinin (ARTs), have pharmacological activity not only against Plasmodium falciparum but also helminths i.e., schistosome spp., viruses i.e., SARs Cov-2, cancers like lung carcinoma and autoimmune diseases i.e., inflammatory bowel syndrome. Our study aims to investigate the in vitro antinematode activity of artemisinin, artesunate and artemether on the energy metabolism of A. suum L3. A. suum L3 were exposed to artemisinin, artesunate or artemether for 24 hours and evaluated for general metabolic activity or ATP production using the resazurin assay or bioluminescence ATP assay respectively. We detect a dose dependent inhibition by artesunate and artemether on the metabolic activity of A. suum L3. Thus, the derivatives, artemether and artesunate, were more effective when compared to artemisinin. Resazurin has been reported to detect mitochondrial function, therefore, a potential mechanism of action of artesunate and artemether could be an interference with the electron transport chain of the mitochondria. Consequently, fluorescence lifetime imaging microscopy is used in current experiments to further investigate metabolic enzymes affected in artesunate and artemether exposed A. suum L3. Thus far, our data indicate that ARTs may have a direct inhibitory effect on the nematode metabolism.

Dr.rer.nat

Identification of a novel anti-schistosomal multi-stage Component X derived from mouse serum

Abstract text Praziquantel (PZQ) is the drug of choice for the treatment and transmission control of schistosomiasis, the most important so-called neglected tropical disease (NTD). PZQ solely targets the adult worms but is ineffective on early migrating larval stages and, thus, does not protect from reinfection. The widespread use of PZQ favors the risk of the development and spread of drug resistance. There is a need to develop novel drugs effective against immature, juvenile, and adult worm stages. We have recently demonstrated the presence of not yet identified soluble multi-stage schistosomicidal factors in the serum of mice, the most widely used experimental laboratory host. The present study aimed to build on these findings to identify the active compound(s) in mouse serum and characterize the anti-schistosome properties. We used large-scale fractionation of mouse serum to investigate the susceptibility of all schistosome stages in our novel in vitro culture platform that supports long-term larval survival and development. Subsequent comparative mass spectrometry analysis revealed a set of candidates we selected based on enzymatic properties and screened for their multi-stage schistosomicidal efficacy. The hit compound mode of action was defined via confocal microscopy. Through in vitro screening, we observed that a phospholipase which we named Component X shared a similar schistosome-killing phenotype with mouse serum. Component X lethally affected parasites at all stages, including ex vivo adult worms, in a concentration- and time-dependent manner and is upregulated in infected animals compared to controls. Moreover, the treatment with sublethal doses of Component X affected tegument integrity and disrupted lipid layers and the structure of the parasite"s reproductive system. Overall, our data reveal that Component X represents a multi-stage schistosomicidal host component, essentially produced in response to infection which could potentially be explored further for the development of novel therapeutic strategies.

Session XXI
One Health/ NTD 2

Appointment

Chair

Programme

Session XXI One Health/ NTD 2

Appointment

Chair

Programme

Session XXI
One Health/ NTD 2