Background

The human hookworms Necator americanus and Ancylostoma duodenale infect about 470 million people worldwide and are primarily endemic in poverty-stricken areas of tropical and sub-tropical climates [1, 2]. Infection initiated by skin penetration leads to invasion of the lungs and intestine, resulting in painful lung and intestinal manifestations and anemia [1,2,3]. Within the host, adult hookworms release thousands of eggs daily, which exit through faeces and continue the life cycle and cause subsequent infections [1]. Currently, mass drug administration (MDA) is fundamental to control infection and reduce morbidity [1, 4]; however, only few chemotherapeutic agents are currently available against these helminths [5, 6]. The standard therapy used for treatment includes drugs derived from anthelminthics for veterinary use, primarily albendazole and mebendazole of the benzimidazole family [5]. Monotherapies administered at single doses display only moderate cure rates and efficacy varies over studied regions [7]. Additionally, pharmacotherapy does not prevent reinfection [8] and no licensed vaccines are currently available [9, 10]. Since there is an urgent need for new treatment options, and anthelminthic drug discovery is underfunded and neglected [4, 6, 11], it is essential to optimise drug discovery.

Current methods in helminth drug discovery overview a variety of phenotypic [12,13,14] and motility [14,15,16,17,18] based assays that have been developed and advanced to detect nematocidal activity primarily using larval and adult stages [10]. However, only few studies published incorporate the use of egg hatching to determine ovicidal abilities in drug discovery, omitting a part of the life cycle [10, 19]. Nevertheless, similar methodologies have been of use for over 40 years in the detection of helminth strains resistant to treatment [20,21,22]. The establishment of an egg-hatching assay platform for human hookworms harbors the potential to detect cryptic ovicidal effects of extant compounds, complementing anthelminthic drug screening.

The aim of our study was to enable the complete characterisation of anthelminthics against all life stages of hookworms by targeting the understudied egg stage. We developed an in vitro assay for drug evaluation against the egg stage through optimisation of laboratory conditions of hookworm egg maturation and hatching of Heligosomoides polygyrus, Ancylostoma ceylanicum, and N. americanus. We evaluated both unembryonated and embryonated eggs to consider drug sensitivity differences based on egg maturity. The anthelminthics assessed in this study included key benzimidazoles and macrolides and further commercially available anthelminthics, namely monepantel, levamisole, tribendimidine, emodepside, oxantel pamoate, and pyrantel pamoate. Full characterisation of the potency of broadly employed anthelminthics will help answer why drug action differs over life stages and in translation between host species as well as test whether this assay could be integrated in hookworm drug discovery [23].

Methods

Animals

Female mice (NMRI strain; age 3 weeks; weight ca. 20 − 22 g) were purchased from Charles River (Sulzfeld, Germany) and male Syrian Golden hamsters were purchased from Janvier Laboratories (Le Genest-Saint-Isle, France). Rodents were kept in polycarbonate cages under environmentally controlled conditions (temperature ∼25 °C; humidity ∼70%; 12 h light:12 h dark cycle) with free access to water and food. The rodents were acclimatised for 1 week before infection.

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 Chemical Co., Ltd. (Hangzhou, China). Tribendimidine was obtained from Shandong ** larvae within, the egg stage is particularly sensitive to benzimidazole exposure.

Fig. 2
figure 2

Benzimidazole activity against unembryonated N. americanus, A. ceylanicum, and H. polygyrus eggs and embryonated H. polygyrus eggs after 72 h of drug exposure. Each point represents the mean EC50 value [µM] with error bars representing the standard error (s.e.) of the mean

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Fig. 3
figure 3

Effect of benzimidazoles, macrolides, and miscellaneous anthelminthics on unembryonated N. americanus, A. ceylanicum, and H. polygyrus and embryonated H. polygyrus eggs after 72 h of drug exposure at 100 µM. Eggs are depicted for active drugs preventing hatching, while larvae are depicted for inactive drugs

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All six macrolides tested displayed no activity against the hookworm eggs (all EC50 values > 100 µM). Complete hatching was observed among these compounds after 72 h (Fig. 3). Macrolides feature much larger structures than the benzimidazoles (Additional file 1: Fig S9b), revealing a possible steric cause for the lack of ovicidal activity. In contrast to the benzimidazoles the protective barrier of the shell may be preventing entry of the drug and exposure of the develo** larvae within. Additionally and in contrast to hookworm larvae susceptible to macrolides [23, 30], the develo** larvae within the egg likely do not depend on the feeding and motility facilitated by the macrolide target glutamate and GABA channels [30].

Among the miscellaneous anthelminthic compounds tested, monepantel, levamisole, and tribendimidine displayed varied potencies among the hookworm species. Monepantel has been previously described with activity only against A. ceylanicum L3 [23], correlating with the highest potency observed among A. ceylanicum eggs. High potency was also observed against embryonated H. polygyrus eggs, although no activity was displayed against the unembryonated eggs. Lower potency of monepantel was also observed against N. americanus eggs, not seen previously against its L3 or adult stages [23]. Levamisole and tribendimidine act on the same receptor [31] presenting overall high potencies among hookworm L3 and adult parasites [23]. This activity was also seen among the hookworm eggs with overall lower potency of tribendimidine compared to levamisole. These three active compounds did not display the level of embryonic deterioration observed among the active benzimidazoles as many remaining unhatched eggs exhibited clear embryonation (Fig. 3). Emodepside, oxantel pamoate, and pyrantel pamoate displayed no activity against the hookworm eggs (all EC50 values > 100 µM) with complete hatching observed by 72 h (Fig. 3). Further research is needed to describe the lack of activity seen with these compounds which may be of interest in explaining the difference in activity seen compared to compounds such as levamisole and tribendimidine found active with similar mechanisms of action [32].

Conclusion

In summary, we developed and utilised egg-hatching assays to broaden the understanding of anthelminthic drug activity on hookworm egg maturation and hatching. Hookworms hatched optimally at room temperature in PBS achieving > 75% hatching over 34 h. Storing eggs at 4 °C proved beneficial to study drug effect on embryonated eggs by delaying maturation and hatching while preserving egg viability with > 95% hatching observed within 2 weeks or when returned to room temperature.

The in vitro assessment of the hookworm eggs showed further differences of drug action compared to larval and adult stages, displaying a changed sensitivity to the tested compounds within the eggshell. The benzimidazoles displayed the strongest potency against the egg stage, in particular thiabendazole and albendazole with EC50 values < 1 µM. Conversely, the macrolides displayed no activity although previously activity was seen against adults and particular high potency against larvae (23). Emodepside, oxantel pamoate, and pyrantel pamoate were inactive while monepantel, levamisole, and tribendimidine displayed varied potencies among the hookworm species. The activity discrepancies of currently profiled anthelminthics observed between life stages highlight the importance of profiling drug activity against all life stages, including the egg-stage model, in drug discovery for human hookworms. Likewise, we would not recommend screening compounds against the egg stage solely, even though this might be the most cost-effective method. Also, in adapting the developed assay from H. polygyrus to N. americanus and A. ceylanicum, assay conditions and determinants did not need to be altered, displaying good accordance among the three parasite models. Additionally, the resulting data from the assays were predominantly similar among the three species, further verifying H. polygyrus and A. ceylanicum as excellent models in hookworm drug discovery.

Continuing application of this egg-hatching assay will aid in enabling further drug discovery by identifying ovicidal abilities of known anthelminthics and potential drug candidates.