Exploring Metarhabditis blumi as a Model for Anthelmintic Drug Discovery

Abstract

Helminth infections pose a significant global health challenge, as existing drugs often lack efficacy and may be contraindicated in some populations. Progress in the development of new drugs is hindered by the lack of innovative models for use in drug research. Metarhabditis blumi nematodes, which are associated with parasitic otitis in cattle, can severely affect the nervous system, leading to death. The treatment and control of this pathology face similar limitations to those for other parasitic diseases. Our study aimed to standardize M. blumi as a model for evaluating the anthelmintic activity of new drugs. Larvae (L3) and adult worms were treated with the reference drugs albendazole (16 µM) and ivermectin (2.5 µM) diluted in an NGM medium for 24 h, and various parameters were evaluated. Motility and mobility were analyzed using a video tracking and analysis program. Morphological and ultrastructural characterizations were performed after chemical fixation using light and scanning electron microscopy (SEM). The results showed that ivermectin was more effective than albendazole in treating M. blumi adults and L3. The SEM images revealed drug-induced ultrastructural changes. Compared to previous studies using the established Caenorhabditis elegans model, M. blumi demonstrated greater resistance to both albendazole and ivermectin. We conclude that M. blumi is a viable model for drug discovery assays and a valuable new experimental model for various biological studies, highlighting that, unlike C. elegans, M. blumi is associated with parasitism.

1. Introduction

Rhabditis is a genus of nematodes that is commonly associated with decomposing organic matter, as well as with fresh or marine environments [1,2]. These helminths are small in size, with females and males measuring approximately 1.5 mm and 1.2 mm, respectively [3]. In 2004, the genus Metarhabditis was proposed by Tahseen et al. [4], along with a description of the species Metarhabditis andrassyana. Some species are considered to be free living, such as Metarhabditis amsactae [5], while others are vertebrate parasites [2,6]. In cattle, these parasites, represented by rhabditiform nematodes, can cause parasitic otitis, as can ticks, flies, and mites of the genus Raillietia [7]. This disease was first reported in the coastal and inland regions of Tanzania and later in Botswana, Zimbabwe, Kenya, and Uganda [8]. Currently, it is prevalent in tropical regions—including Brazil—mostly in dairy herd breeds, particularly Gir (Bos primigenius indicus) and Indubrasil (Bos taurus indicus) [3,9]. In mild cases, these animals may experience local pain and ear secretion [10], while in severe cases, the parasite may rupture the tympanic membrane, resulting in hearing and facial nerve impairments, as well as the formation of a brain abscess, compromising the animal’s nervous system and leading to its death [9,11,12,13]. In addition, it has been suggested that parasitic otitis may be associated with secondary bacterial and/or fungal infections, intensifying these symptoms [7,10]. This affects milk production and, therefore, results in considerable economic losses [8,9]. This parasitosis was also reported in the outer ear canal of an agricultural worker in Germany in 2014 [14]. Regarding treatment, therapies are available, but they are not particularly effective [15].

Anthelmintics are used for infections caused by worms (flatworms and roundworms) [16]. A variety of drugs administered to treat these infections act on proteins that control the electrophysiological activity of neurons and muscles, specifically ion channels and neurotransmitter receptors. When these proteins are affected, the nematode’s performance is affected, resulting in paralysis, malnutrition, activation of the immune system, and expulsion of the parasite from the host [17]. However, the indiscriminate use of these drugs to treat these conditions can lead to anthelmintic resistance, which is a global problem [18]. Resistance to treatment strategies affects the efficacy not only of a specific drug but also of other drugs with similar mechanisms of action, rendering them equally ineffective and reducing the range of available treatments for these parasitic infections [19,20,21].

In vivo and in vitro models are used to discover and monitor anthelmintic resistance in animals, with Caenorhabditis elegans being a widely used in vitro model [22]. This model is a free-living nematode and one of the most well-characterized organisms in the world, which has contributed to the understanding of the modes of action of all the main classes of anthelmintics [23]. Nematodes of the genus Metarhabditis share morphological, morphometric, and biological similarities with C. elegans, as both belong to the Rhabditidae family. These similarities present a valuable opportunity to utilize these parasitic nematodes as an experimental model. Nematodes of the genus Metarhabditis, due to their parasitic lifestyle and shorter life cycle, offer a valuable complementary model to C. elegans for evaluating the effects of anthelmintic drugs and conducting other biological assays. The extensive research conducted over the years using only C. elegans provides a solid foundation for exploring these two models together in the search for new anthelminthic drugs.

This study aimed to demonstrate the potential of Metarhabditis blumi as an in vitro parasitic nematode model for studies of new drug candidates and mechanisms of action. This approach also opens up new opportunities to explore and amplify experiments traditionally focused on free-living nematodes or invertebrate parasites to a vertebrate parasite model.

2. Results

2.1. Impact of Treatments on Motility and Mobility of M. blumi Adults and Third-Stage Larvae (L3)

M. blumi adults treated with ivermectin (2.5 µM) for 24 h showed significant paralysis and reduced motility within the first 2 h, with an average of 45.5% of nematodes becoming immobile by the end of the experiment. In contrast, M. blumi treated with albendazole (16 µM) did not exhibit paralysis, resulting in higher motility without significant immobilization. These results suggest that albendazole was less effective than ivermectin in reducing motility in adult M. blumi nematodes (

Table 1. Motility and mobility of M. blumi adults treated with albendazole (16 μM) or ivermectin (2.5 μM) over a 24 h period.

	Control	Albendazole	Ivermectin
Motility	83.6 ± 2.03	84.6 ± 2.43	54.5 ± 2.03 *
Velocity	782.05 ± 123.8	310.13 ± 106.9	100.5 ± 14.38 *
Displacement	6.77 ± 1.61	3.2 ± 0.91	1.6 ± 0.11 *

Data are expressed as follows: motility is represented as the percentage of motile parasites, mobility is expressed as the velocity (µm/s), and displacement is measured in millimeters (mm). * p < 0.05 (mean ± SD, n = 3).

; Figure 1).

For M. blumi L3, treatment with ivermectin for 120 min led to a significant reduction in motility, with an overall average of 31.77% immobile nematodes. Conversely, albendazole treatment resulted in 86.3% of L3 retaining motility, showing a minimal reduction in movement. These findings further demonstrate the greater efficacy of ivermectin compared to albendazole in affecting the motility of M. blumi larvae (

Table 2. Motility and mobility of M. blumi L3 treated with albendazole (16 μM) or ivermectin (2.5 μM) for 120 min.

	Control	Albendazole	Ivermectin
Motility	93.5 ± 3.78	86.3 ± 2.82	68.23 ± 2.17 *
Velocity	324.06 ± 53.99	130.3 ± 40.73	126.8 ± 60.8
Displacement	3.30 ± 0.54	1.34 ± 0.39	1.2 ± 0.61

Data are expressed as follows: motility is represented as the percentage of motile parasites, mobility is expressed as the velocity (µm/s), and displacement is measured in millimeters (mm). * p < 0.05 (mean ± SD, n = 3).

; Figure 2).

Using video microscopy, it was possible to evaluate the impact of the treatment on nematodes that exhibited continuous, albeit slower, movement before becoming completely paralyzed. The variability in the range of movement made it difficult to establish a consistent scoring. To address this limitation, we applied software that tracked the recorded movement of M. blumi, generating the two key parameters of velocity (µm/s) and displacement (mm) for the larvae and adult worms during the experiments (Supplementary Materials, Videos S1–S9).

The velocity and displacement of M. blumi adult worms were significantly reduced in the treated groups compared to the untreated control. A reduction in velocity was most prominent at 8, 12, and 16 h and coincided with the rising curve in the control group. In the groups treated with ivermectin, significant reductions were noted at 8 and 16 h (p = 0.0107 and p = 0.0036, respectively). The velocity in the control group began to decrease after 16 h, with a minimum velocity recorded at 20 h, while still maintaining a distance from the treated groups (Table 1; Figure 3).

A reduction in displacement was observed starting at 4 h, with the maximum impact at 12 h, when compared to the controls. It is important to highlight that the peak displacement of the control was observed at 12 h, a crucial moment where both treatments impacted displacement. Specifically, ivermectin treatment showed significant reductions at 4 and 8 h. After 12, 16, and 20 h, no significant differences in displacement were identified when comparing the treated groups with the control nematodes (p = 0.0036 and p = 0.0107) (Table 1; Figure 4).

Both the velocity and the displacement of M. blumi L3 nematodes were reduced in the treated groups compared to the larval control. A reduction in velocity was observed in the ivermectin-treated group at 90 and 120 min. Compared to those worms subjected to albendazole treatment, a lower nematode velocity was observed at 30 min, with a slight oscillation during the treatment period. The velocity in the control group began to decrease after 90 min and continued to decrease until the end of the treatment period (Table 2; Figure 5).

A reduced displacement in M. blumi L3 was observed from 60 min in the albendazole-treated group, followed by brief and noticeable variations that nevertheless remained lower than in the control group. In the ivermectin-treated group, a reduction in displacement was observed at 90 min, which persisted during the treatment period (Table 2; Figure 6).

2.2. Morphological Changes in Adult Worms after Treatment

The impacts of albendazole and ivermectin on the morphology of M. blumi adult worms were observed using light microscopy. Distinct morphological alterations in males were observed within a 24 h period in the testicles following albendazole treatment and in the isthmus after exposure to ivermectin. Shedding of the cuticle was observed in female worms during the 24 h treatment (Figure 7). No apparent morphological changes were detected for L3 under the same treatment conditions.

2.3. Evaluation of Ultrastructure

Scanning electron microscopy (SEM) provided detailed insights into the effects of treatment on the oral cavity of M. blumi. Specifically, alterations in the cephalic papillae and cuticle were observed following albendazole treatment. SEM revealed significant changes in the external topography of the anterior end and the cuticle surface, likely resulting from treatment-induced shedding, which was also observed in the light microscopy results. In contrast, the ivermectin treatment predominantly caused damage to the cuticle, with less pronounced effects on the cephalic papillae within a 24 h period (Figure 8).

3. Discussion

The free-living nematode C. elegans has proven invaluable in biological research, including in the search for new anthelmintics and studies of drug resistance [24,25]. The nematode model is used in toxicity testing and is particularly notable due to its complex biological systems, including a complete digestive system, functional immune system, endocrine processes, sensory system, and neuromuscular system [26]. Given the time-consuming and costly nature of drug development, C. elegans provides a cost-effective alternative in the early phases of drug discovery. Its application in high-throughput screenings has accelerated the identification of new drugs and targets for conditions, such as neurodegeneration and metabolic disorders, demonstrating how an in vitro metazoan model can contribute to both basic research and drug development. In this study, we suggested an in vitro protocol to establish the parasite nematode M. blumi as a novel and complementary experimental model using methodologies similar to those employed with C. elegans to test anthelmintic drugs, with a focus on assessing its morphology and motility.

Albendazole (16 µM) and ivermectin (2.5 µM) concentrations were selected based on previous studies that established IC₅₀ values using C. elegans as a model [27,28]. Analyses of motility and mobility in M. blumi treated with albendazole and ivermectin revealed that ivermectin caused a significant reduction in or complete cessation of movement in both adults and L3, whereas albendazole did not produce similar effects.

Based on velocity and displacement evaluations, it is possible to determine an optimal time interval in which to further explore this experimental model in other studies. The adult worms showed increasing adaptation to cultivation conditions, reaching a peak at 12 h in the in vitro assays for velocity and displacement. In both experiments, the control group exhibited relative stabilization of these parameters, while the treated groups showed significant variation. This suggests that under these conditions, using L3 for treatment tests may not be a reliable parameter for evaluating drug efficacy.

These results suggest that M. blumi shows significant susceptibility to ivermectin, providing a foundation for developing novel treatment strategies for bovine parasitic otitis. Additionally, ivermectin serves as a reference drug, positioning M. blumi as a valuable new model for testing the efficacy of other candidate anthelmintic molecules. Studies using the C. elegans model in anthelmintic drug testing have shown similar results, with ivermectin causing paralysis and albendazole demonstrating limited efficacy [29,30].

Adult worms of M. blumi treated with ivermectin for 24 h exhibited morphological changes in the esophagus and isthmus, similar to those observed in C. elegans by Ferreira et al. [24]. They reported that the inhibitory effect of ivermectin on C. elegans was evident only after 48 to 72 h of treatment, with approximately 100% of nematodes eventually becoming paralyzed. This suggests that the observed changes in the esophagus and isthmus may be related to the drug’s mechanism of action, which initially paralyzes pharyngeal pumping and subsequently affects the body wall muscles of the nematode, leading to paralysis and death. The initial impact on the esophagus may indicate where the drug action begins in both C. elegans and M. blumi. After 4 h of treatment with 2.5 µM ivermectin, adult M. blumi worms exhibited a partial reduction in motility (45.5%) and mobility (50%), which differed from previous findings using the C. elegans model, in which 1.6 µM of ivermectin paralyzed 100% of the worms in 30 min [31]. This comparison suggests that the parasite M. blumi is relatively more resistant to ivermectin than the free-living C. elegans. This difference in sensitivity may be due to several factors related to the metabolic adaptations of M. blumi to the parasitic life, which may reflect a more realistic result regarding the susceptibility of helminth parasites to drugs.

In an effort to control bovine parasitic otitis caused by nematodes of the Rhabditis spp., a previous study conducted in vivo treatments using a combination of chemical compounds and the nematophagous fungus Duddingtonia flagrans [32]. The authors concluded that the combination of the fungus, 10% DMSO, and 1.9% ivermectin were effective in controlling parasitic otitis in cattle, demonstrating that ivermectin shows promise as a treatment option in clinical trials for this condition.

Using both light and scanning electron microscopy, we revealed morphological changes in M. blumi after treatment with albendazole and ivermectin. These changes include roughness on the cuticle surface in M. blumi treated with ivermectin, cuticle detachment with both drugs, and slight swelling in the male reproductive system (testicles) within 24 h in the albendazole-treated group. Additionally, alterations in the digestive system, specifically in the isthmus and esophagus, were observed in adults treated with ivermectin within the same period. These findings are consistent with those of Souza [33], who reported similar changes in C. elegans exposed to the anthelmintic plant extract of Tocoyena bullata.

In this study, when comparing the effects of both treatments on M. blumi, ivermectin had a greater impact on motility than albendazole. These findings align with previous studies on C. elegans, which also demonstrated higher sensitivity to ivermectin compared to M. blumi. However, despite this difference in sensitivity, both nematodes showed similar responses when treated with the two drugs, with ivermectin consistently showing a stronger effect on motility compared to albendazole [24,29,30,31]. The present results highlight the potential of M. blumi, a nematode associated with parasitic infections in veterinary contexts, as a promising experimental model for in vitro drug discovery. It can be further developed and validated as a reliable tool for testing new anthelmintic drugs.

4. Materials and Methods

4.1. Isolation and Culture of M. blumi for Anthelmintic Evaluation Tests

Specimens of M. blumi were acquired from the Caenorhabditis Genetic Center (CGC, Minneapolis, MN, USA), which is funded by the NIH Office of Research Infrastructure Programs (P400D010440). The parasite was cultured according to the Stiernagle protocol [34] until adult worms were obtained, including gravid females. As third-stage larvae and adults were necessary for conducting the drug tests, nematode cultures were synchronized to last 24 and 48 h, respectively. All cultures were kept at 37 °C in a bacteriological incubator.

4.2. Anthelmintic Drug Tests

After L3 and adult worms were obtained as detailed in Section 4.1, samples were washed with 0.1 M PBS and then decontaminated for 60 min in 1 mL of liquid nematode growth medium (NGM) containing amphotericin B (Sigma, Saint Louis, MO, USA) (2.5 µg/mL) and gentamicin (Sigma, Saint Louis, MO, USA) (1 µg/mL).

Parasites were then placed in 24-well plates containing liquid NGM and albendazole (Merck KGaA, Darmstadt, Germany) (16 µM) or ivermectin (Merck KGaA, Darmstadt, Germany) (2.5 µM) in 0.03% or 0.010% dimethyl sulfoxide (DMSO), respectively, and kept in a B.O.D. incubator (Marconi, SP, Brazil) at 22 °C for 24 h. Untreated controls were defined as parasites cultured using only liquid NGM with 0.03% DMSO (Merck KGaA, Darmstadt, Germany). The experiments with adult worms lasting 24 h were conducted in triplicates, while those with L3 were conducted in duplicates.

4.3. Motility Test

Adults and L3 of M. blumi were incubated in 24-well plates, treated with albendazole or ivermectin, and maintained at 22 °C in a B.O.D. incubator (Marconi, SP, Brazil). Adults were exposed to treatments for 24 h, while L3 were treated for 120 min. The motility of adults and L3 was assessed every 2 h and every 30 min, respectively, using an Olympus CK40 inverted light microscope (Olympus Corporation, Tokyo, Japan). Two motility parameters were defined, mobile and immobile, with immobile individuals defined as those exhibiting low motility or complete paralysis.

4.4. In Vitro Assessment of Mobility

The displacement and velocity of the M. blumi specimens were assessed by means of video microscopy using a Nikon Eclipse Ti-S inverted microscope (Nikon, Tokyo, Japan) every 4 h for a period of 20 h for adults and every 30 min for a period of 120 min for L3. To assess mobility and obtain reference parameters, ImageJ software (Fiji version 2.11.0) with the Manual-Tracking plug-in was used. This tool allows for the calculation of the velocity and displacement of moving objects by analyzing video frames and scaling the images. A tracking system was applied to the anterior end of the nematodes (larvae and adult worms) to monitor their movement. Nematodes were considered paralyzed when there was a total absence of any mobility, and the nematode immobility rate was determined. The units of velocity and displacement were µm/s and mm, respectively.

4.5. Light Microscopy

A total of 90 nematode adults and 15 L3 were fixed in Karnovsky solution and mounted between a slide and coverslip for subsequent image capture using an Olympus BX-53 light microscope (Olympus Corporation, Tokyo, Japan) equipped with an SC100 digital camera (Olympus Corporation, Tokyo, Japan) using the Olympus cellSens image digitization system software. The morphological changes in the nematodes subjected to different treatments were evaluated and compared to the results obtained in the controls.

4.6. Scanning Electron Microscopy (SEM)

The material, already chemically fixed, was post-fixed in 1% osmium tetroxide (OsO4), 0.8% potassium ferrocyanide, and 5 mM calcium chloride, dehydrated in an increasing series of ethanol (30%—absolute), dried using the critical point method in CO₂, mounted on metal supports with a carbo strip, and covered with a layer of gold. The samples were then analyzed using a Zeiss Auriga 600 Compact SEM electron microscope (Carl Zeiss Microscopy, Jena, Germany) (Urogenital Unit/IBRAG-UERJ).

4.7. Statistical Analysis

The Mann–Whitney test and analysis of variance (ANOVA), followed by Tukey’s post-test and multiple comparisons tests and the Kruskal–Wallis test followed by Dunn’s post-test, were performed using GraphPad Prism version 6.01 for Windows, GraphPad Software, Boston, MA, USA, www.graphpad.com. The results were considered statistically significant when p < 0.05 or p < 0.005.

5. Conclusions

This study introduced the nematode M. blumi as a promising in vitro experimental model for drug testing. The findings indicate that both M. blumi adults and L3 exhibit greater susceptibility to ivermectin than albendazole. Notably, considering the literature data for free-living C. elegans, the nematode parasite M. blumi demonstrated relatively lesser susceptibility to these drugs. Due to its parasitic nature and adaptation to a host environment (it was isolated from cattle), M. blumi is expected to be naturally more drug refractory than free-living nematodes. These characteristics underscore that M. blumi is a potentially valuable tool for exploring new anthelmintic therapies and other biological studies.

6. Patents

The in vitro cultivation of these nematode parasites was patented in accordance with Brazilian Industrial Property Law with registration number BR1020230011420.