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International Journal for Parasitology: Drugs and Drug Resistance logoLink to International Journal for Parasitology: Drugs and Drug Resistance
. 2023 Oct 9;23:63–70. doi: 10.1016/j.ijpddr.2023.10.001

Trichuris muris egg-hatching assay for anthelminthic drug discovery and characterization

Anastasia Schärer a,b,1, Stefan Biendl a,b,1, Jennifer Keiser a,b,∗
PMCID: PMC10590722  PMID: 37856948

Abstract

Trichuriasis is a neglected tropical disease widely distributed among tropical and sub-tropical areas and associated with poverty and lack of access to safe drinking water, sanitation and hygiene. Existing drugs have limited efficacy and face a constant risk of developing resistance, necessitating the search for alternative treatments. However, drug discovery efforts are sparse and little research has been performed on anthelminthic effects on embryonated eggs, the infectious life stage of Trichuris spp.

We examined bacterial species dependent egg hatching of the murine model parasite Trichuris muris and identified Escherichia coli, Pseudomonas aeruginosa and Enterobacter hormaechei effective as hatching inducers, resulting in hatching yields of 50–70%. Streptococcus salivarius, reported to be associated with reduced drug efficacy of ivermectin-albendazole coadministration in Trichuris trichiura infected patients, did not promote egg hatching in vitro. We optimized hatching conditions using E. coli grown in luria broth or brain-heart infusion media to reach consistently high hatching yields to provide a sensitive, robust and simple egg-hatching assay. Oxantel pamoate demonstrated the strongest potency in preventing hatching, with an EC50 value of 2–4 μM after 24 h, while pyrantel pamoate, levamisole and tribendimidine exhibited only moderate to weak inhibitory effects. Conversely, all tested benzimidazoles and macrolide anthelminthics as well as emodepside failed to prevent hatching (EC50 > 100 μM).

Our study demonstrates that egg-hatching assays complement larval and adult stage drug sensitivity assays, to expand knowledge about effects of current anthelminthics on Trichuris spp. Further, the developed T. muris egg-hatching assay provides a simple and cheap screening tool that could potentially lead to the discovery of novel anthelminthic compounds.

Keywords: Anthelminthics, Drug discovery, Egg hatching, Trichuris muris, Trichuris spp.

Graphical abstract

Image 1

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Highlights

  • •

    E. coli, P. aeruginosa and E. hormaechei are effective hatching inducers of T. muris.

  • •

    Distinct streptococci species show varying T. muris egg hatching inducing potential.

  • •

    Oxantel pamoate inhibits T. muris hatching with an EC50 value of 2–4 μM.

  • •

    Benzimidazoles and macrolide anthelminthics failed to prevent hatching of Trichuris.

  • •

    Egg-hatching assays complement larval and adult stage drug sensitivity assays.

1. Introduction

Trichuriasis is a neglected tropical disease widely distributed among tropical and sub-tropical areas and associated with poverty and lack of access to safe drinking water, sanitation and hygiene (Else et al., 2020). The disease constitutes a global public health problem affecting around 500 million people worldwide, with children bearing the highest burden (GBD DALYs and HALE Collaborators, 2017; Fauziah et al., 2022). Trichuris trichiura, often referred to as whipworm, is a gut-dwelling parasitic nematode that infects humans by contact or ingestion of embryonated eggs. To control infections and reduce morbidity, preventive chemotherapy programs regularly distribute vast amounts of the benzimidazoles albendazole and mebendazole to at-risk populations in endemic areas (World Health Organization, 2019; Montresor et al., 2020). Treatment efficacy of these anthelminthics is insufficient and coadministration with the macrocyclic lactone ivermectin shows setting-dependent variability in efficacy (Moser et al., 2017; Hürlimann et al., 2022). While recent investigations have identified emodepside as promising tool in the treatment of trichuriasis (Mrimi et al., 2023), the risk of parasite resistance to the few available drugs, warrants the discovery and development of novel anthelminthic drugs active against T. trichiura infections.

Current drug discovery for trichuriasis relies commonly on in vitro motility-based activity assays on larval and adult stages of the mouse model organism T. muris (Silbereisen et al., 2011; Tritten et al., 2011; Wimmersberger et al., 2013). In contrast, nematode egg-hatching assays were initially developed to detect drug resistance in the veterinary field (Calvete et al., 2014) and little research was performed to utilize such set-ups for drug discovery. We have recently reported on the development of an egg-hatching assay for the hookworms Heligmosomoides polygyrus, Ancylostoma duodenale and Necator americanus (Easland et al., 2023), showcasing a simple, fast and cost-effective tool to reliably determine ovicidal properties of drugs. While hookworm larvae hatch spontaneously, embryonated Trichuris eggs (Forman et al., 2021) exploit and require the presence of host microbiota to hatch and invade the host (Hayes et al., 2010; Koyama, 2013; Lawson et al., 2021). Although several bacterial species have been found to induce hatching (Hayes et al., 2010; Koyama, 2013; Wimmersberger et al., 2013; Lawson et al., 2021; Sargsian et al., 2022; Robertson et al., 2023) or modify treatment (Schneeberger et al., 2022), the ideal conditions for hatching remain uncertain, resulting in highly variable hatching yields and difficulties to compare results between laboratories.

The aim of this study was to develop a sensitive, robust and simple egg-hatching assay of the murine model parasite T. muris, to enable the complete characterization of drug effects on all life stages of Trichuris. We examined for the first time a wide range of bacterial species to trigger egg-hatching and optimized hatching conditions using Escherichia coli to reach appropriate, reproducible and standardized hatching yields. We further utilized the developed egg-hatching assay to test commercially available anthelminthic drugs from different compound classes on their ability to prevent hatching of Trichuris larvae from embryonated eggs. The anthelminthics assessed in this study had previously been evaluated in vitro against larval and adult stages of T. muris (Keiser and Häberli, 2021) as well as against all life stages of hookworm parasites (Keiser and Häberli, 2021; Easland et al., 2023) and included key benzimidazoles and macrolides as well as further commercially available anthelminthics, namely monepantel, levamisole, tribendimidine, emodepside, oxantel pamoate, and pyrantel pamoate.

2. Materials and methods

2.1. Animals

Female mice (C57BL/6NRj strain; age 3 weeks; weight ca. 15−17 g) were purchased from Janvier Laboratories (Le Genest-Saint-Isle, France). Rodents were kept in individually ventilated cages (IVC) cages under environmentally controlled conditions (temperature ∼21–22 °C; humidity ∼55% (±15%); 12 h light and 12 h dark cycle) with free access to water and food. The rodents were acclimatized for one week before infection.

2.2. Compounds and culture media

Abamectin, albendazole, doramectin, eprinomectin, fenbendazole, flubendazole, ivermectin, levamisole hydrochloride, mebendazole, oxfendazole, oxibendazole, pyrantel pamoate, ricobendazole, and thiabendazole were the products of Sigma-Aldrich (Buchs, Switzerland). Milbemycin oxime and moxidectin were purchased from Hangzhou Dingyan Chem. Co, Ltd. (Hangzhou, China). Tribendimidine was obtained from Shandong Xinhua Pharmaceutical Company Limited (Zibo, China). Emodepside was obtained from Bayer AG (Leverkusen, Germany).

The compounds were dissolved in pure dimethyl sulfoxide (DMSO, Sigma–Aldrich, Switzerland) at a concentration of 10 mM, aliquoted and stored at −20 °C until further use.

Luria Broth media (LB) was prepared by dissolving 2 g/100 mL dehydrated media concentrate (Thermo Fisher Scientific) in purified water. Brain Heart Infusion media (BHI) was prepared by dissolving 3.7 g/100 mL dehydrated media concentrate (Thermo Fisher Scientific) in purified water and subsequent autoclaving at 121 °C.

Roswell Park Memorial Institute 1640 culture medium (RPMI, Gibco, Waltham MA, USA) used as hatching media was prepared by supplementing with 5% tetracyclin (5 μM) solution (Lubio, Zürich, Switzerland) and 20% fetal calf serum (FCS, BioConcept, Allschwil, Switzerland).

All media were sterilized by filtration using a 0.22 μm filter bottle (Vitaris AG, Baar, Switzerland) under laminar flow.

2.3. Parasites

The life cycle of Trichuris muris is established and maintained at Swiss TPH (Keiser and Häberli, 2021). Female C57BL/6NRj mice were infected per os with 200–250 T. muris eggs showing around 90−95% embryonation (checked microscopically). After 41 days post-infection, unembryonated eggs were collected by filtering and centrifuging the feces of infected mice. After isolation, eggs were stored in purified water (Mili-Q) at room temperature (RT) in the dark for at least 3 months to allow for complete embryonation. For in vitro assays embryonated T. muris eggs were utilized and washed three times with freshly prepared hatching media.

2.4. Bacteria

Escherichia coli (DSM 30083), Staphylococcus aureus (DSM, 20231), Streptococcus salivarius (DSM, 20067), Streptococcus parasanguinis (DSM 6778), Enterobacter hormaechei (DSM 12409) and Pseudomonas aeruginosa (DMS 1128) were purchased as freeze-dried pellets from the German Collection of Microorganisms and Cell Cultures (Leibniz Institute DSMZ, Braunschweig, Germany). Streptococcus equi (6812.72), Actinomyces odontolyticus (7311.53), Streptococcus mitis (6709.54), Streptococcus dysgalactiae (7502.58), Streptococcus oralis (6712.50) and Streptococcus pneumonia (1149.80) were received as freeze-dried pellets from the Institute for Infectious Diseases (IFIK, Bern, Switzerland). Paraclostridium spp. were cultivated in-house, stored as glycerol samples and were identified as P. benzoelyticum or P. bifermentans but not further specified.

2.5. Bacterial cultivation

All bacterial cultures were prepared under aseptic conditions and laminar flow.

2.5.1. From freeze-dried samples

Delivered glass ampoules containing the freeze-dried pellets were opened according to the DSMZ recommendation under aerobic conditions. The pellet was suspended in 500 μL BHI supplemented with 5% yeast broth. Two 15 mL tubes containing 10 mL broth were prepared through inoculation with 150 μL of the suspended bacteria pellet, gentle mixing on a mechanical mixer and cultured overnight at 37 °C and 5% CO2. Additionally, in order to control for contamination, two Columbia agar plates (Thermo Fisher Scientific) were streaked with 50 μL bacterial suspension each. After 24 h of cultivation, the plates were evaluated for single cell growth and glycerol stocks were prepared by mixing 100 μL of 40% glycerol and 100 μL of the bacterial suspension.

2.5.2. From glycerol samples

An overnight culture was prepared by transferring 100 μL of a thawed bacterial glycerol stock to 5 mL BHI without yeast supplementation or LB media in a 15 mL tube. The bacteria suspension was mixed shortly on a mechanical mixer, placed in an incubator at 37 °C and 5% CO2, and continuously shaken at 160 rpm. To ensure the purity of the bacterial culture a 10 μL loop was immersed into the prepared bacterial culture and streaked on a Columbia agar plate. The plate was placed into the same incubator and evaluated after 24 h.

2.6. Bacterial growth monitoring

To identify the bacterial-species specific time-point of transition from the exponential (log)- growth phase to the stationary phase, bacterial daily cultures were prepared by transferring 100 μL of the overnight culture into 9.9 mL pure culture media. 200 μL of the daily culture was transferred into an optical clear 96-well plate in triplicates. For baseline determination, the same volume of pure broth media was measured. The plate was sealed with a clear seal and placed into the spectrophotometer. The absorbance (Abs) was measured every 30 min at a wavelength of 600 nm and the temperature was set to 37 °C.

For the in vitro assays, the bacterial daily culture and bacterial growth monitoring was performed as described above, with the exception that the absorbance measurement was continued for an appropriate amount of time depending on the bacterial species (measurement time was ca. 1–2 h longer than the approximate time to reach the stationary phase). The remaining bacterial culture was continuously shaken at 120 rpm in an incubator set to 37 °C and ambient CO2.

Antibacterial action of the assayed anthelminthics could influence the results of the egg-hatching assay without direct action of the drug on T. muris eggs. To rule-out such secondary effects, we tested effects of anthelminthic drugs on E. coli by monitoring bacterial growth as described above, with the exception that 50 μL instead of 200 μL of bacterial daily culture or the broth media (baseline) were transferred into an optic clear 96-well plate. Shortly before reaching the stationary phase (ca. 4 h after inoculation), the measurement was for a moment paused, to allow for addition of 140 μL of sterile RPMI media and 10 μL of the corresponding drug at the specified concentration. The growth measurement was resumed and continued overnight. The baseline consisted of 50 μL of pure broth media mixed with 130 μL sterile RPMI and 20 μL DMSO (10%). Negative control samples containing the bacterial suspension, RPMI and DMSO were measured simultaneously. E. coli bacteria were grown in LB and BHI without yeast supplementation.

2.7. Characterization and optimization of determinants of Trichuris muris egg hatching

All in vitro egg hatching assays were performed under aseptic conditions and laminar flow. 50–60 embryonated eggs (in 20 μL of the egg suspension) were suspended in hatching media to reach a final volume of 200 μL within sterile 96-well flat bottom plates. To control for uniform distribution and quantity, the eggs were counted under the microscope at 10× magnification (Carl Zeiss, Germany). Plates were incubated at 37 °C and 5% CO2. The hatching percentage was evaluated after 2, 4, 6 and 24 h by quantifying the number of empty eggshells under an inverted transmitted-light microscope at 10× magnification.

2.7.1. Bacterial quantity

Shortly before reaching the stationary phase (ca. 4 h after inoculation), an E. coli culture was adjusted to 3 million cells/μL (BHI) or 0.8 million cells/μL (LB). 50–60 eggs were suspended in hatching media within sterile 96-well flat bottom plates and 100, 75, 50, 25 and 10 μL of the adjusted E. coli culture was transferred to each well. Each experimental condition was performed in triplicates and repeated two times.

2.7.2. Bacterial species ability to hatch Trichuris muris eggs

Different bacterial isolates were tested on their ability to promote hatching of T. muris eggs. 50–60 eggs were suspended in 130 μL hatching media. Subsequently, 50 μL of a prepared bacterial daily culture shortly before reaching the stationary phase were added. Each experimental condition was performed in quadruplicates and repeated two times.

2.8. In vitro drug sensitivity assays

A range of anthelminthic compounds and derivatives were screened against embryonated eggs of T. muris. To determine activity at a single high concentration, 50 to 60 eggs were exposed to 100 μM drug concentrations. To determine EC50 values, 50 to 60 eggs were incubated in drug concentrations obtained from two-fold serial dilutions for a total of at least ten concentrations starting at 100 μM. Drug serial dilutions were prepared in DMSO from the stock concentration (10 mM) to 100× of the final concentration. A subsequent dilution step to 10× of the final concentration was prepared in culture medium. For each prepared assay, 1% of DMSO in culture medium served as negative control, corresponding to the amount of DMSO present in each drug well. Additionally a media control containing no DMSO was prepared. Assay plates were incubated at 37 °C and 5% CO2, and evaluated at 24 and 48 h. The total number of empty eggs shells were quantified under an inverted transmitted-light microscope at 10× magnification (Carl Zeiss, Germany). Each experimental condition was tested in triplicates and experiments were repeated at least once.

2.9. Data analysis

Egg development and hatching percentage was determined by counting the number of hatched eggs compared to the total number of eggs. Drug activity was determined by normalizing hatching counts of the eggs exposed to drugs to hatching counts of the vehicle-control and averaging across replicates.

The EC50 values of the tested drugs were determined by applying a nonlinear least-squares analysis using a four-parameter sigmoid function. Drug activities and EC50 values were calculated in R (version 4.1.3) (R Core Team, 2020) as previously described (Biendl et al., 2022). Visualizations were created in R.

3. Results and discussion

3.1. Assessment and optimization of in vitro Trichuris muris egg hatching

3.1.1. Selection and culture of bacterial species

Hatching of T. muris eggs for harvesting of first-stage larvae (L1) (Additional file 1: Fig. S1) for drug assays has traditionally been done using E. coli cultured in LB medium as hatching inducers (Wimmersberger et al., 2013). We evaluated the hatching inducing potential of bacterial species found to indwell the T. muris hatching site within the small intestine (Escherichia coli and Enterobacter hormaechei) and with reported sufficient hatching results in comparable studies (Staphylococcus aureus, Paraclostridium spp. and Pseudomonas aureginosa) (Sargsian et al., 2022; Robertson et al., 2023). We further selected S. salivarius that was previously associated with reduced drug efficacy in Trichuris trichiura infected patients (Schneeberger et al., 2022), which may indicate a role of the bacterium in promoting the early life cycle of the parasite. We expanded the evaluation of streptococci to species not found to be associated with treatment outcome, namely to S. dysgalactiae, S. equi, S. mitis, S. oralis, S. parasanguinis and S. pneumonia, in order to differentiate between species and genus level effects. Finally, we included Actinomyces odontolyticus, a bacterium predominantly found in the oral cavity (Li et al., 2018), to investigate our hypothesis that non-intestinal commensal bacteria fail at inducing T. muris egg hatching.

As a starting point, based on our in-house protocol for T. muris first-stage larvae harvesting (Wimmersberger et al., 2013), we initially evaluated growth compatibility of all selected bacterial species in LB medium (Lessard, 2013) (Additional file 1: Fig. S2), but did not observe growth of A. odontolyticus, S. mitis, S. oralis, S. pneumoniae and S. salivarius. Therefore, we additionally cultured all selected bacteria in BHI medium. In BHI all selected bacteria proliferated and we observed generally superior growth compared to LB (Additional file 1: Fig. S2), e.g. 3·million E. coli cells/μL after 4 h in BHI, compared to 0.8 million cells/μL after 4 h in LB.

We further aimed at exposing embryonated T. muris eggs to a maximally high concentration of healthy and viable bacterial cells, since the importance of intact bacterial cell walls during the hatching process was highlighted by Hayes and colleagues who proposed outer membrane fimbriae type I of E. coli to be mediating hatching (Hayes et al., 2010). Thus, we followed species-specific bacterial growth over time and transferred the bacteria when the terminus of their respective exponential growth phase was reached (Additional file 1: Fig. S2), e.g. after ca. 4 h for E. coli in both BHI and LB medium.

3.1.2. Bacteria induced hatching of embryonated Trichuris muris eggs

The bacteria induced hatching of T. muris from embryonated eggs demonstrated similarities across the different bacterial species in regards to time course, but varied considerably in regards to hatching percentage (Fig. 1). Within the initial 6 h we observed a rapid increase in hatching percentage. Subsequently, there was only a slight increase in hatching beyond 6 h. Finally, we did not observe any additional hatching beyond 24 h, indicating that all viable larvae capable of hatching do so within a 24 h period in the presence of hatching inducing bacteria.

Fig. 1.

Fig. 1

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Bacteria induced in vitro hatching of T. muris. Hatching performance of embryonated T. muris over time (left) and at 24 h (right) in dependence of the bacterial species transferred from brain-heart infusion (BHI, top) or luria broth (LB, bottom) medium. Experiments were performed in at least three independent repeats; each black diamond represents one well of eggs and bars together with coloured round points represent the mean percentage of eggs hatched with error bars representing the standard error (s.e.) of the mean.

We identified E. coli, P. aeruginosa and E. hormaechei as effective hatching inducers, resulting in mean hatching yields of 40–70% after 24 h, when bacteria were transferred from BHI or LB culture (Fig. 1). When bacteria were transferred from BHI culture, E. coli induced hatching most reliably and in the highest quantities (>60% mean hatching), followed by E. hormaechei and P. aeruginosa which yielded moderate hatching percentages (>40%). When bacteria were transferred from LB culture, P. aeruginosa yielded the highest hatching percentage (>60%), followed by E. coli (>50%) and E. hormaechei (>40%). Unsurprisingly, E. coli and E. hormaechei trigger hatching, since T. muris can utilize locally increased concentrations of bacteria comprised prevalently in the mammalian intestinal microbiota as possible locational cues. S. aureus from BHI and LB culture resulted in insufficiently low hatching between 30 and 10%. Similarly low hatching yields (<30%) were obtained when eggs were exposed with S. dysgalactiae, S. equi grown in LB medium, which was further reduced when these bacteria were transferred from BHI culture (<10% for S. dysgalactiae, <5% for S. equi).

No or only isolated spontaneous hatching (<5% hatching) was observed using the bacterial species Streptocccus salivarius, S. parasanguinis, S. pneumonia, S. mitis, S. oralis, Actinomyces odontolyticus, Paraclostridium spp. or when no bacteria were added (Additional file 1: Fig. S3). The failure of S. salivarius to induce egg hatching hints that the promotion of the early life cycle of the parasite is likely not a factor involved in the linkage between this species and reduced drug efficacy in infected patients. Importantly, the differences in the potential to act as hatching inducers of the various streptococci tested indicates the relevance of species specific factors that are not necessarily shared among all members of a bacterial genus. The discrepancy of previously reported hatching potential of paraclostridia (Sargsian et al., 2022) to our observations further accentuates this observation. Ultimately, the failure of A. odontolyticus to promote hatching was anticipated, given its natural inhabitancy within the oral cavity and not the intestine.

We initially also tested hatching with bacteria transferred from culture in BHI media supplemented with 5% yeast extract. However, we did not observe any hatching (<5%) under these circumstances, even for bacterial species that induced hatching when transferred from BHI culture without yeast supplementation as e.g. E. coli. Therefore we concluded that yeast supplementation negatively affects T. muris hatching and we did not pursue this further.

Finally, we continued utilisation of E. coli as bacterial hatching inducer for the egg-hatching assay, due to the high hatching percentages (>50%) achieved when transferred from both culture media and its convenient and fast proliferation within 4 h after inoculation. While P. aeruginosa and E. hormaechei and to a lesser extent S. aureus and S. dysgalactiae were also able to promote hatching, their capabilities were less consistent and robust compared to E. coli.

In our study we focused on individual bacterial species acting as hatching inductors for T. muris eggs. As future prospect, establishing stable cultures of synthetic polymicrobial bacterial communities - potentially enriched with species described to influence treatment outcome - to more accurately reflect the gut microbiome, might prove to further enhance in vitro hatching of eggs and more accurately reflect the complex relationship between parasite, bacteria and drugs (Javdan et al., 2020; Schneeberger et al., 2022). Similarly, conducting hatching experiments under microaerophilic or anaerobic conditions represents physiological conditions more closely, but will add considerable experimental complexity.

3.1.3. Quantitative effects of Escherichia coli on Trichuris muris egg hatching

We further evaluated the hatching performance of T. muris over time and at 24 h in dependence of the volume of E. coli suspension transferred from BHI infusion or LB medium. To ensure accurate comparison across experimental repetitions, we adjusted the bacterial culture for each trial to contain 3 million E. coli cells/μL in BHI and 0.8 million E. coli cells/μL in LB directly before transferring bacteria to the wells containing the eggs. The standardized concentrations corresponded to the maximal consistently achievable concentrations of E. coli cells using the respective growth media within 4 h of culture. The percentage of eggs that hatched was relatively equal within a range of 100 to 10 μL E. coli suspension (Additional file 1: Fig. S4), corresponding to an E. coli cell count of 300 to 30 million (BHI) and 80 to 8 million (LB) (Fig. 2). However, we observed a slight delay (approximately 2 h) and overall greater variability in hatching at lower volumes (25 and 10 μL) and consequentially lower bacterial cell counts. When volumes of 100, 75 and 50 μL were transferred, hatching was higher after 24 h when using the BHI culture (68 ± 4%, 65 ± 4% and 63 ± 3% hatching) compared to the LB culture (60 ± 4%, 55 ± 3% and 58 ± 3% hatching), corresponding with the higher concentration of bacterial cells present in the BHI culture. As transferring quantities exceeding 50 μL did not distinctively enhance hatching, and lower quantities featured decreased consistency, we selected a volume of 50 μL E. coli culture containing 150 million cells (BHI) and 40 million cells (LB) to induce hatching within the developed T. muris egg-hatch drug activity assay.

Fig. 2.

Fig. 2

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Hatching performance of T. muris over time (left) and at 24 h (right) in dependence of the number of E. coli cells (in million cells) transferred from brain-heart infusion (BHI, top) or luria broth (LB, bottom) medium. Experiments were performed in at least three independent repeats; each black diamond represents one well of eggs and round points represent the mean percentage of eggs hatched with error bars representing the standard error (s.e.) of the mean.

3.2. Anthelminthic compound profiling on Trichuris muris eggs

An ideal anthelminthic should feature activity against all life-stages of the parasite and especially against the infective and disease-causing stage. We utilized the developed egg-hatch assay to expand our knowledge of important anthelminthics (Additional file 1: Fig. S5), to drug activity against the infective but understudied embryonated egg stage of Trichuris. None of the tested compounds influenced growth of E. coli in BHI or LB medium (Additional file 1: Figs. S6–8), ensuring that potential drug action on eggs is direct and not mediated through inhibition of the bacteria inducing egg-hatching.

We observed inhibitory effects of oxantel pamoate, pyrantel pamoate, levamisole and tribendimidine on T. muris egg-hatching and the corresponding EC50 values are summarized in Table 1. Differences in EC50 values between 24 and 48 h of drug exposure were low, indicating that compounds elicited ovicidal activity rather than solely delaying hatching. Oxantel pamoate displayed the strongest potency with single digit micromolar EC50 values and it was the only drug showing activity in assay media containing BHI (EC50 = 4 μM at 24 h of drug exposure) or LB (EC50 = 2 μM) (Fig. 3 and Additional file 1: Fig. S9).

Table 1.

In vitro activity (EC50 values [μM]) of anthelminthics against embryonated eggs of T. muris determined after 24 and 48 h of drug exposure and addition of Escherichia coli suspension transferred from BHI or LB media respectively.

Compound BHI
 LB
24 h
 48 h
 24 h
 48 h
EC50 [μM] (s.e.) EC50 [μM] (s.e.) EC50 [μM] (s.e.) EC50 [μM] (s.e.)
Oxantel pamoate 4.3 (0.5) 4.2 (0.4) 2.1 (0.2) 2.0 (0.2)
Pyrantel pamoate >100 >100 24 (7) 32 (9)
Levamisole >100 >100 24 (5) 21 (4)
Tribendimidine >100 >100 94 (7) 92 (7)
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(standard error of the mean in brackets).

Fig. 3.

Fig. 3

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In vitro concentration–response curve and EC50 value determination among embryonated T. muris egg-hatch assays conducted with oxantel pamoate after 24 h of drug exposure using E. coli transferred from brain-heart infusion (BHI, left) and luria broth (LB, right) medium. Each point represents the mean value of the activity [%] and error bars represent the standard error (s.e.) of the mean. The line represents the four-parameter sigmoid function used to determine the EC50 value and the dashed lines show the 95% confidence interval of the fit.

Pyrantel pamoate (EC50 = 24 μM), a compound closely related to oxantel pamoate, and levamisole (EC50 = 24 μM) showed moderate potency and tribendimidine (EC50 = 94 μM) was only weakly active (Additional file 1: Fig. S10). When bacteria where transferred from BHI growth media, all three compounds lost activity (Additional file 1: Fig. S11 A). BHI components, such as digested proteins derived from animal infusion (calf brain and beef heart), could scavenge drug molecules, thus lowering the amount of available free drug to act on the parasite. This unspecific mechanism is similar in nature to the loss of activity for drugs featuring strong plasma-protein binding (Pasche et al., 2018; Biendl et al., 2023). Our observation emphasize that the choice of the assay medium is of relevance to outcomes of drug-sensitivity assays and should be considered when evaluating drug activity.

The quantitative effect of reduction in hatching after oxantel pamoate and pyrantel pamoate exposure was also visualized through morphological deterioration of the embryonated T. muris egg (Fig. 4). Despite the eggshell retaining its morphological appearance, we observed a drug concentration dependent effect of stronger disintegration of the larva inside in the egg with increasing concentrations. The absence of one of the two bipolar plugs suggests that the hatching process was initiated normally. This suggests that the activity of oxantel pamoate and pyrantel pamoate is not based on permeating the eggshell but rather on its ability to rapidly arrest the larva inside the egg through muscular paralysis by binding to the nicotinic acetylcholine receptors.

Fig. 4.

Fig. 4

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Effect of oxantel pamoate, pyrantel pamoate, levamisole and tribendimidine on embryonated T. muris eggs after 48 h of drug exposure at 100 μM using E. coli transferred from brain-heart infusion (BHI) or luria broth (LB) medium.

For levamisole and tribendimidine we observed less pronounced morphological changes to the egg or larva within the egg. However, the larva remained within the egg and the opening of the polar plugs was apparently inhibited.

While both oxantel pamoate and pyrantel pamoate are highly potent against larval and adult T. muris stages in vitro, in vivo oxantel shows higher efficacy compared to pyrantel (Keiser et al., 2013; Keiser and Häberli, 2021). Likewise we observed the egg stage to be more sensitive to oxantel pamoate compared to pyrantel pamoate. In a previous study, both compounds did not show any activity against hookworm eggs (Easland et al., 2023), congruent to oxantel's lack of activity against hookworm larvae and adults in vitro (Keiser et al., 2013). Our study further supports the development of oxantel pamoate as drug combination partner for treatment and control of Trichuris infections (Palmeirim et al., 2021).

Like oxantel pamoate and pyrantel pamoate, both levamisole and tribendimidine act on nicotinic acetylcholine receptors (nAChR) (Bennett et al., 2019), however on differentially expressed subtypes (Robertson et al., 2015). Against T. muris eggs we observed an activity pattern similar to previous observations against hookworm eggs (Easland et al., 2023), with levamisole being more potent than tribendimidine and both drugs not eliciting strong morphological deterioration of the embryo.

Two of the miscellaneous compounds, namely emodepside and monepantel (Additional file 1: Fig. S5 A), as well as all tested benzimidazoles (albendazole, fenbendazole, flubendazole, mebendazole, oxfendazole, oxibendazole, ricobendazole, thiabendazole; Additional file 1: Fig. S5 B) and macrolide anthelminthics (abamectin, ivermectin, doramectin, eprinomectin, milbemycin oxime, moxidectin; Additional file 1: Fig. S5 C) failed to prevent hatching (EC50 > 100 μM) (Additional file 1: Fig. S11). Contrary to our findings, benzimidazoles were highly potent against hookworm eggs (Easland et al., 2023). Importantly, Easland and colleagues found active benzimidazoles to be more potent against unembryonated than against embryonated eggs of Heligmosomoides polygyrus (Easland et al., 2023), demonstrating drug sensitivity to be dependent on the developmental stage of the developing larva within the egg. Since in our set-up the T. muris larvae have already developed within the eggs, inhibiting the microtubule synthesis using benzimidazoles appears to be ineffective. Further, benzimidazoles perform generally poor in in vitro conditions and especially so against Trichuris species (Keiser and Häberli, 2021).

The observed lack of activity of the macrolide anthelminthics and emodepside, a promising tool in the treatment of soil-transmitted helminthiasis (Mrimi et al., 2023), was congruent with results of exposed hookworm eggs and is likely explained through similar underlying factors (Easland et al., 2023), such as steric hindrance of the large molecules preventing penetration of the protective eggshell and thus precluding drug exposure of the embryo. The reduced reliance of the embryo on feeding functionality mediated by glutamate gated chloride and gamma-aminobutyric acid (GABA) channels targeted by macrolides (Abongwa et al., 2017) further explains the discrepancy of activity between egg compared to larval and adult parasite life stages. Similarly, emodepside targets the presynaptic latrophilin-like receptors and subsequent interaction with slowpoke-1 channels (SLO-1), which are seemingly not significantly important for the hatching process.

Our findings demonstrate the potential of the egg-hatching assay, which will complement larval- and adult-stage drug sensitivity assays to expand our understanding of the anthelminthic effects of existing drugs and to facilitate the discovery of novel compounds for treatment. Further, together with methodological investigations of the processes involved in embryonation (Forman et al., 2021) and hatching of Trichuris eggs, consequently studying influences of compounds on these processes could promote the discovery of new drug targets. Targets associated with embryonation or hatching of eggs may likely be conserved within the Trichuris genus and distinct from mammal proteins providing opportunities for rational drug design.

4. Conclusion

In summary, we present the development of a simple, cost-effective and reliable phenotypic drug sensitivity assay for the infectious stage of Trichuris, the embryonated egg. Besides E. coli, we identified E. hormaechei and P. aeruginosa as bacterial inducers of T. muris hatching. Moreover, we show that some members of the bacterial genera Streptococcus and Paraclostridium do not promote hatching of T. muris in vitro. We further optimized the transferred volume and concentration of E. coli culture to render a robust egg-hatching assay.

Applying the developed egg-hatch assay to profile drug action of mainstay anthelminthics, we revealed oxantel pamoate to be uniquely potent against the infectious life stage of Trichuris. Oxantel pamoate inhibits hatching of T. muris eggs with an EC50 of 2 μM–4 μM after 24 h of drug exposure when bacteria were transferred from LB or BHI culture respectively. Pyrantel pamoate and levamisole were moderately potent, while tribendimidine exhibited only weak inhibitory effects. Benzimidazole and macrolide anthelminthics as well as emodepside were inactive against embryonated eggs. Contextualising our results with in vitro activity and in vivo efficacy data against larval and adult parasite life stages unveils considerable differences in drug sensitivity of different life stages. This highlights the importance of profiling drug activity on all life stages, and importantly also on the infectious life stage being embryonated eggs for Trichuris. With easy and ample access to this particular life stage, egg-hatching assays will prove valuable screening tools for anthelminthic drug discovery and characterization complementing existing larval and adult stage drug sensitivity assays.

Finally, with oxantel pamoate showing consistent activity against all Trichuris life stages, our study further supports the development of oxantel pamoate for treatment and control of Trichuris infections.

Supplementary information

The following supplemental material are available online:

Additional file 1: Fig. S1 Unembryonated and embryonated eggs and first-stage larva (L1) of T. muris observed under microscopic magnification. Fig. S2 Bacterial growth in brain-heart infusion and luria broth medium. Fig. S3 Bacteria induced in vitro hatching of T. muris. Fig. S4 Hatching performance of T. muris over time and at 24 h in dependence of the volume of E. coli suspension transferred from brain-heart infusion or luria broth medium. Fig. S5 Chemical structures of the evaluated anthelminthics. Fig. S6 E. coli growth in brain-heart infusion and luria broth medium in presence of oxantel pamoate and levamisole. Fig. S7 E. coli growth in brain-heart infusion and luria broth medium in presence of abamectin, ivermectin, albendazole and emodepside. Fig. S8 E. coli growth in brain-heart infusion and luria broth medium in presence of anthelminthics at 100 μM. Fig. S9 In vitro concentration–response curve and EC50 value determination among embryonated T. muris egg-hatch assays conducted with oxantel pamoate after 48 h of drug exposure. Fig. S10 In vitro concentration–response curve and EC50 value determination among embryonated T. muris egg-hatch assays conducted with pyrantel pamoate, levamisole and tribendimidine. Fig. S11 Effect of anthelminthics on embryonated T. muris eggs after 48 h of drug exposure at 100 μM.

Ethics approval

Animal studies were carried out in accordance with Swiss national and cantonal regulations on animal welfare at the Swiss Tropical and Public Health Institute (Allschwil, Switzerland; permission no. 520).

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analysed during this study are included in this published article and its supplementary information files.

Funding

We gratefully acknowledge financial support from the Swiss National Science Foundation (No. 175585).

Authors’ contributions

SB and JK conceived the experiments. SB and AS designed the experiments. AS performed the experiments. SB and AS analysed the data and drafted the first version of the manuscript. SB created the data visualisations. SB, AS and JK revised the manuscript. All authors read and approved the final manuscript.

Author details

1 Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Kreuzstrasse 2, 4123 Allschwil, Switzerland. 2 University of Basel, 4003 Basel, Switzerland.

Declaration of competing interest

The authors declare that they have no competing interests.

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Acknowledgements

Not applicable.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijpddr.2023.10.001.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

manuscript_tmuris_suppl.docx
mmc1.docx (15.8MB, docx)

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Associated Data

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Supplementary Materials

manuscript_tmuris_suppl.docx
mmc1.docx (15.8MB, docx)

Data Availability Statement

All data generated or analysed during this study are included in this published article and its supplementary information files.

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