In vitro culture of the parasitic stage larvae of hematophagous parasitic nematode Haemonchus contortus

Abstract

Current research on common parasitic nematodes is limited because their infective stages cannot be propagated in vitro. Here, we report a culture system for developing L4s of Haemonchus contortus, a blood-feeding nematode of ruminants. Our results demonstrated that a proportionate mixture of NCTC-109 to Luria-Bertini (1:2) media promoted the formation of early L4s and then into late L4s upon inclusion of 12.5% (v/v) defibrinated blood, albeit with a decline in survival. Adding antioxidants (0.3 mg/mL of L-glutathione or 200 nmol of vitamin C) improved survival of L4s, with approximately 90% developing to late L4s by 22 days. These L4s showed parallel morphological features (such as digestive and reproduction systems) compared with in vivo L4s at day 7 (following challenge infection), although with delayed development. Our work optimized the in vitro culture system for L4s while providing an important platform for in-depth molecular research on Haemonchus and other related parasitic nematodes.

1. Introduction

Parasitic nematodes, a heterogeneous group of roundworms, exhibit a broad spectrum of hosts, spanning humans and animals (Blaxter, 2011), and resulting in devastating diseases and socioeconomic losses. Approximately 20% of the human population is infected with parasitic nematodes, primarily in areas with scarce resources (Moser and Ilem, 2024). Further, all grazing ruminants are exposed to gastrointestinal nematodes, at a cost of billions of US dollars annually in production losses (Charlier et al., 2014, 2020; McRae et al., 2014; Rashid et al., 2019). Infections by these parasites continue to challenge humans and livestock due to the lack of commercial vaccines (Hotez et al., 2009; Nisbet et al., 2013; González-Hernández et al., 2016; Loukas et al., 2016; Leung et al., 2020; Mustafa et al., 2024) and widespread drug resistance, especially for animals (Kaplan and Vidyashankar, 2012; Stromberg et al., 2012; King, 2019; Lanusse et al., 2018; Nielsen 2022; Alaro et al., 2023). Indeed, these parasites infecting livestock have become resistant to almost all classes of anthelminthic drugs (Kelleher et al., 2020; Ahuir-Baraja et al., 2021; Belecke et al., 2021). Thus, it is imperative to develop new intervention strategies.

An in vitro culture system promotes a deep understanding of parasite biology. Research on most parasitic helminths including parasitic nematodes remains limited because certain parasite stages cannot be grown in vitro (Njouendou et al., 2017; Wang et al., 2019; Moratal et al., 2023). The greatest challenge is that the host environment is complex with regard to nutrients, temperature, pH, CO₂, and host signaling (e.g. host progesterone), which are required to maintain parasite growth and stage differentiation (Mapes, 1970; Dick and Leland, 1973; Douvres, 1980, 1983; Douvres and Urban, 1983; Urban et al., 1984; Feather et al., 2017; Gutiérrez-Amézquita et al., 2017). Understanding and replicating the host environment is necessary to culture parasitic worms outside the host. For hematophagous helminth species, blood is an indispensable culture component that provides essential nutrients such as amino acids (Zečić et al., 2019), fatty acids (Kurzchalia and Ward, 2003), and heme (Bouchery et al., 2018). Heme is a byproduct of hemoglobin degradation, vital for sexual maturation and egg production in Schistosoma spp. (Toh et al., 2015). Free heme, however, also triggers generation of reactive oxygen species (ROS) (Ponka, 1999; Kehrer, 2000), resulting in cell damage and even organism death (Schmitt et al., 1993; Aft and Mueller, 1984; Kumar and Bandyopadhyay, 2005). Thus, optimization of blood-supplemented culture systems is critical for blood-sucking parasitic worms.

Haemonchus contortus (barber’s pole worm), a model gastrointestinal nematode, is a prevalent blood-feeding parasite of livestock (Zheng et al., 2023). This parasite is transmitted orally from contaminated pasture to the host through a complex 3 week life cycle (Fig. 1). The eggs are excreted in the host feces, L1s develop inside the egg, then hatch and molt through to L2 and L3 stages within a week. The L3s are infective and, upon ingestion by the host, exsheath, and after a histotrophic phase, develop to L4s and then to dioecious adults (Wang et al., 2023). The two last stages feed on the host’s blood. Only the free-living and early L4 stages can be cultured using a Luria-Bertani (LB)-based medium (Preston et al., 2015). Despite nearly a century of effort to achieve an optimized culture of the blood-feeding stages (Lapage, 1993; Sommerville, 1976, 1977; Stringfellow, 1984, 1986; Niciura et al., 2023), there have been few breakthroughs beyond the early L4 stage (Silverman, 1959; Schulz, 1967; Stringfellow, 1986), and particularly in the past 30 years, there has been no replication of this parasitic stage development, which inspired us to undertake the present study.

Fig. 1. Life cycle of the parasitic nematode Haemonchus contortus. Haemonchus contortus exists in five developmental stages including three free-living and two parasitic stages.

The free-living stages comprise the development from eggs to L3s via L1s and L2s, while the parasitic stages consist of L4s and adults. During parasitic stages, the nematodes feed on the blood of their host.

2. Materials and methods

2.1. Ethics statement

Animal experiments were approved by the Experimental Animals Ethics Committee of Huazhong Agricultural University, China (HZAUGO-2019-008). The animals were maintained according to the guidelines established by the Committee.

2.2. Parasite and animal materials

Haemonchus contortus (Haecon-5 strain) was propagated in experimental goats, as described previously (Liang et al., 2022). The helminth-free Boer goats (3–4 months of age) were maintained in a ventilated cage to prevent the emergence of accidental nematode infections. They were provided with alfalfa pellets twice each day and always had access to free water. Then the goat was gavaged with 7000 infective L3s (iL3s) of H. contortus. Fecal samples were collected daily from day 28 p.i., followed by incubation at 25 °C for 1 week under saturated humidity. iL3s were collected from feces utilizing the Baermann collection procedure (Palevich et al., 2022). Late L4s were collected from the infected goat’s abomasum on day 7p.i. according to the method reported (Liang et al., 2022).

2.3. Preparation of basal media

LB medium was prepared as described previously (Preston et al., 2015), with a few modifications. In brief, 5 g of yeast extract, 10 g of tryptone, 5 g of NaCl, 2.38 g of HEPES, and 3.7 g of NaHCO₃ (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in 800 mL of double distilled H₂O (ddH₂O). The pH was adjusted to 6.8 using HCl, then the volume was increased to 1 L, followed by heat sterilization. M-199 or NCTC-109 (Sigma-Aldrich, St. Louis, MO, USA) were supplemented with 2.38 g of HEPES and 3.7 g of NaHCO₃. The pH was adjusted to 6.8 using HCl, and the final media were filter-sterilized (0.22 μm).

2.4. In vitro exsheathment and the xL3 cultivation

iL3s were cultured based on a published procedure (Preston et al., 2015). Larvae were exsheathed and sterilized by incubation in 0.15% v/v sodium hypochlorite (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 10 min. Exsheatheed L3s (xL3s) were immediately washed five times in 0.85% NaCl solution containing 100 IU/mL of penicillin, 100 μg/mL of streptomycin and 0.25 μg/mL of amphotericin B (Sigma-Aldrich, St. Louis, MO, USA; centrifuged at 1300g for 3 min at room temperature). These xL3s were then suspended in different media (M-199, NCTC-109 and LB) supplemented with 20% FBS (Invitrogen, CA, USA), 100 IU/mL of penicillin, 100 μg/mL of streptomycin, and 0.25 μg/mL of amphotericin, 1% L-glutamine (100×), 1% sodium pyruvate (100×), and 1% MEM non-essential amino acids solution (100×) (Sigma-Aldrich, St. Louis, MO, USA). Later, to select the best medium for xL3 differentiation to L4 stage, xL3s were cultured in 24-well plates (100 xL3s/well) in 500 μ L of M-199, NCTC-109 or LB medium with supplements as described above, and those worms were incubated at 39 °C with 20% CO₂ for 7 days.

2.5. Optimization of culture medium for L4 development

L4s were cultured using a published method with some modifications (Preston et al., 2015). Briefly, M-199, NCTC-109 and LB media containing indicated (see Section 2.4) additives were used individually or in combination (NCTC-109:LB as 1:1, 1:2, 2:1). Of the 500 μL culture, 200 μL of supernatant was replaced by fresh medium every 2 days. Varying amounts (0–12.5% v/v) of defibrinated blood (DFB) from helminth-free sheep (3–4 months age) (Dening Bio, Henan, China) were added to NCTC-109:LB (1:2) medium on day 7 to assess its effect on L4 development. The antioxidants, L-glutathione (L-GSH) (0.15–0.45 mg/mL) or vitamin C (Vc) (100–300 nmol) (Sigma-Aldrich, St. Louis, MO, USA), which was dissolved in ddH₂O, were supplemented to the medium with 12.5% DFB (Table 1) from day 7 to day 22. Developing worms were scored by inverted microscopy for (i) survival rates (n ≥ 100) on days 12, 17, and 22; (ii) body length (from mouth to tail tip) and body width (represented by the pharynx, n ≥ 10) on days 12, 17, and 22; and (iii) L4 developmental rate (n ≥ 100) on day 22. Three independent experiments were carried out.

Table 1. Culture conditions and medium components for the in vitro development of Haemonchus contortus L4s.

Culture component	Culture concentration
Medium	NCTC-109:LB (1:2)
Antibiotics	1% Penicillin streptomycin-amphotericin (100X)
L-glutamine	1% (100×)
Pyruvate sodium	1% (100×)
Nonessential amino acids	1% (100×)
FBS	20%
DFB	12.5%
AntioxidantspH	0.3 mg/mL L-GSH or 200 nmol Vc6.8

FBS, foetal bovine serum; DFB, defibrinised blood.

2.6. Measurement of ROS

In order to verify whether the DFB triggered ROS production in L4s (Jasmer et al., 2021), worms were treated with variable concentrations of DFB (0–12.5% v/v) or 2.56 mg/mL of hemin (Sigma-Aldrich, St. Louis, MO, USA), and cultured for 2 days. Larvae were washed using NCTC-109:LB (1:2) three times to remove hemin and treated with 100 nmol of DCFH-DA (2′, 7′ -dichlorofluorescein diacetate) (Beyotime, Shanghai, China) (39 °C, 20% CO₂, 30 min, in the dark). Samples were washed thrice with LB medium (centrifuged at 1300g for 3 min), and larvae were anesthetized with 1% ivermectin on slides for fluorescent imaging (green) using a Cytation5 cell imager. Fluorescence was quantified by a spectrophotometer (excitation: 488 nm, emission: 525 nm) in 96-well plates (20 L4s/well). Two technical replicates and three independent experiments were carried out.

2.7. Assessment of intestinal cell death

L4s were cultured in medium with 0–12.5% DFB for 2 days. Propidium iodide (PI, Beyotime, Shanghai, China) was added to a concentration of 100 μmol, and samples were incubated for 4 h (39 °C, 20% CO₂). The floating color was rinsed off by washing larvae with 0.85% NaCl (centrifuged at 1300g for 3 min). In the final step, worms were anesthetized on slides with 1% ivermectin and PI labeling was quantified by fluorescent imaging (400× magnification, red, 594 nm). A stringent PI scoring system was employed where only larvae with more than four PI-labelled nuclei were considered positive. The average count of PI-stained larvae (n ≥ 10) was estimated for all treatments. Three independent experiments were carried out in this test.

2.8. Haem aggregation assay

As reported previously (Bouchery et al., 2018), L4s were cultured in DFB for 72 h. We adjusted the DFB concentration to 12.5% in all groups after parasite collection to eliminate errors caused by traces of heme in the cell membrane at different DFB concentrations. Samples were washed thrice with ddH₂O (centrifuged at 1300g for 3 min) to remove hemoglobin, and then parasites were solubilized in 0.1 mol of NaOH solution (37 °C, 24 h). The hemozoin was quantified at 400 nm using a multi-mode plate reader (BioTek Cytation 5, Winooski, Vermont, USA). Two technical replicates and three independent experiments were carried out in this test.

2.9. Comparison of in vitro and ex vivo L4s

Goats were infected with H. contortus iL3s, and L4s were collected from the abomasum on day 7 p.i. In parallel, L4s were developed from early L4s in vitro in the medium containing 12.5% DFB and 0.30 mg/mL of L-GSH, collected on day 22 and then cleaned with 0.85% NaCl. Larvae were fixed on slides with 1% ivermectin in cold 0.85% NaCl to inhibit body shrinkage. Morphological characteristics were analyzed by differential interference contrast (DIC) microscopy (Veglia, 1915).

2.10. Statistical analyses

Statistical analyses were performed using GraphPad Prism 8.0 (San Diego, CA, USA). The correlation between the ROS content and the intestinal cell death was analysed. Differences were compared by one-way ANOVA and Student’s t-test. Significance was determined as: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

3. Results

3.1. Morphological characteristics of H. contortus L4s

Based on a previous report (Preston et al., 2015), we defined the L4 development into three phases: early L4, middle L4, and late L4 (Supplementary Fig. S1). In the early L4 stage, the mouth and sexual differentiation were observed. The mouth cavity occurred once again (Supplementary Fig. S1A). The female tail was tapered slightly and longer than the male worms. The male worms could be identified by a thick posterior end. The tail was short, curled posteriorly, and bent dorsally (Supplementary Fig. S1B and C). The middle L4s had developed a reproductive system (Supplementary Fig. S1D), defined as a tube with a blind end, and the tails became more distinct (Supplementary Fig. S1E and F). The female vulva was visible in the late-phase L4s (Supplementary Fig. S1H), while the male spicule tip was shielded by a hook-shaped protrusion of the cloacal dorsal wall (Supplementary Fig. S1H and I). These characteristics enabled a systematic assessment of the L4s, as described below.

3.2. NCTC-109:LB (1:2) mixed medium supports the development of L4s

We first compared the development of the early L4s on days 12, 17, and 22 in basal media (M-199, NCTC-109, and LB) used previously (Meng and Kong, 1993; Preston et al., 2015). Worms cultured in NCTC-109 had the highest proportion of tail development on day 22 (27%, Fig. 2A), and those incubated in LB displayed greater body length and width (Fig. 2B and C). We then examined different proportions of NCTC-109 and LB media (1:1, 2:1, and 1:2), and found that worms achieved similar body lengths and widths on specified days. The NCTC-109:LB (1:2) formulation resulted in longer body lengths and widths on day 17 and better tail development (day 22) than other groups (Fig. 2D–F), therefore we selected NCTC-109:LB (1:2) as a basal medium for further assays.

Fig. 2. NCTC-109:Luria Bertani (LB) (1:2) mixed medium supports Haemonchus contortus L4 growth.

(A, D) The tail developmental rate of L4s (n ≥ 100) cultured in three media, was counted on day 22. (B, E) Body length (the length from buccal capsule to tail) and (C, F) body width (the width of the pharynx) of L4s (n ≥ 10) cultured in three different media, were measured at specific time points after the culture began. Data pooled with mean ± S.E.M. from three independent experiments, and the number inside each column represents each group’s mean (A, D), by one-way ANOVA, compared with each other. Each replicate is shown as a dot positioned above the bars. N, NCTC-109; L, LB. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

3.3. DFB promotes L4s but also increases ROS

We optimized the basal culture medium by adding DFB on day 7 after xL3s developed into early L4s. The survival rates, body lengths and widths of L4s on days 12, 17, and 22 with 0–12.5% DFB (Fig. 3A–C and Supplementray Fig. S2) were compared. The survival of L4s decreased significantly in the presence of DFB (Fig. 3A and Supplementary Fig. S2), but the growth of the surviving larvae was progressively improved with increasing DFB (Fig. 3B and C). To determine the basis of poor survival, we considered that degradation of DFB-derived hemoglobin produces free heme, which could generate ROS. Therefore, we measured ROS production in the early L4s. Indeed, abundant ROS was detected in the intestine of DFB-exposed L4s (Fig. 4A and B), which gradually increased and then declined at higher concentrations (Fig. 4C). A strong correlation between DFB-dependent ROS and death of L4 intestinal cells (R² = 0.926, P = 0.0023) was evident (Fig. 4D and E). Hemozoin production in L4s was also elevated; hemozoin production rose with increasing DFB concentrations (Fig. 4F).

Fig. 3. Defibrinated blood (DFB) improves Haemonchus contortus L4 development.

(A) Survival rates of L4s (n ≥ 100) cultured in different concentrations of DFB. (B) The body length and (C) body width of L4s (n ≥ 10) were measured. Data were pooled with the mean ± S.E.M. from three independent experiments, and the number inside each column represents each group’s mean (A), by one-way ANOVA analysis. Each replicate is shown as a dot positioned above the bars. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Fig. 4. Defibrinated blood (DFB) increases reactive oxygen species (ROS) in Haemonchus contortus culture.

(A) Localization of ROS in L4s. Two days after adding DFB or hemin, L4s were harvested and stained with DCFH-DA (2′, 7′ – dichlorofluorescein diacetate), then the location of ROS was observed using fluorescence microscopy (green). (B) ROS were detected in larvae cultured in the medium containing 2.56 mg/mL of hemin or 2.5% DFB or (C) ROS levels of L4s (n = 20) cultured in the media containing different DFB concentrations. (D) Intestinal cell necrotic proportion of each L4 in the media containing different DFB concentrations. L4s (n ≥ 10) were cultured for 2 days and then stained by propidium iodide (PI). Negative: necrotic cells < 5, positive: necrotic cells ≥ 5. (E) The correlation between ROS content and intestinal cell death. (F) The content of L4 hemozoin cultured in media containing different concentrations of DFB. Data were from three independent replicates. Bars represent the mean ± S.E.M. of two technical replicates and three independent experiments (B, C, F) or three independent experiments (A, D), with each replicate shown as a dot positioned above the bars, one-way ANOVA analysis. DIC, differential interference contrast; DCFH-DA, 2′, 7′ – dichlorofluorescein diacetate. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.4. Antioxidants can reduce ROS and enable development of L4s

To control the ROS in L4s, we used common antioxidants, L-GSH and Vc, with endogenous and exogenous detoxifying properties, respectively. L-GSH reduced the ROS level in L4s with an optimal added concentration of 0.30 mg/mL (Fig. 5A). Similarly, the death of L4 intestinal cells decreased in the presence of L-GSH compared with the control group (Fig. 5B). Vc also reduced the ROS and the death of intestinal cells similarly to L-GSH (Supplementary Fig. S3A and B). The survival rate of the worms in the presence of 0.3 mg/mL L-GSH was just over double the number of surviving worms of the control on day 22 (Fig. 5C). Equally, the body lengths and widths of the surviving L4s also increased (Fig. 5D and E). Notably, >90% of surviving L4s developed to the middle and late stages (Fig. 5F). Similar to L-GSH (0.3 mg/mL) on day 22, Vc promoted L4 survival at tested time points with the highest survival rate of 60% (day 22) in the group containing 200 nmol of Vc (Supplementary Fig. S3C). Not least, it supported L4 growth (Supplementary Fig. S3D and E) and ∼55% of surviving L4s developed to the late L4 stage (200 nmol of Vc on day 22), approximately three times as many as the controls (18%) (Supplementary Fig. S3F).

Fig. 5. L-glutathione (L-GSH) reduces oxidative damage and improves the development of Haemonchus contortus L4s.

(A) Reactive oxygen species (ROS) quantification and (B) intestinal cell death assay of L4s (n = 10) cultured in media containing 12.5% defibrinated blood (DFB) and different concentrations of L-GSH or without L-GSH (control). (C) The survival rate of L4s (n ≥ 100) was assessed after in vitro culture in media containing 12.5% DFB and three concentrations of L-GSH, and a control. (D) Body length and (E) body width of L4s (n ≥ 10) were measured. (F) The developmental progress of L4s (n ≥ 100) was recorded on day 22 based on the morphological characteristics representing early L4s, middle L4s, late L4s and middle plus late L4s, respectively. Data represented the mean ± S.E.M. of two technical replicates and three independent experiments (A), three independent experiments (B-F), and the number in each column represents each group’s mean (C, F), by one-way ANOVA, compared with the control. Each replicate is shown as a dot positioned above the bars.*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

3.5. Late L4s propagated in vitro mimic their in vivo counterpart

In the final step, we compared the morphological features of worms developed in vitro with those collected from the abomasum of a goat infected (day 7) with L3s (Fig. 6). The following morphological features reported earlier (Veglia, 1915) were comparable in both groups. (i) The mouths of worms were triangular (Fig. 6A and B); (ii) the anterior end of the ovaries was blind, and the uterus had a transverse endometrium (Fig. 6C and D); (iii) the ovijector consisted of an independent group of large cells (Fig. 6E and F); (iv) the vulval structure was evident in the female worm (Fig. 6G and H); (v) the rectum and the anus were observed (Fig. 6I and J); and (vi) the male spicule was well developed (Fig. 6K and L). Despite these shared features, the body lengths and widths of the L4s cultured in vitro (22 day) were smaller (Fig. 6M and N), indicating their precocious development.

Fig. 6. Late Haemonchus contortus L4s developed in vitro resemble the in vivo stage.

Representative images were taken using differential interference contrast (DIC) microscopy, showing female and male late L4s (n ≥ 10). Late L4s were collected from goat abomasum 7 days p.i. or at 22 days of in vitro culture. The worm organs of mouth (A, B), ovary and uterus (C, D), ovijector (E, F), vulva (G, H), rectum and anus (I, J), and spicule (K, L) are indicated. (M) The body length and (N) body width of late L4s (n ≥ 30) grown in vivo (7 days) and cultured in vitro (22 days) were compared. Data was analyzed using a student’s t-test. Each replicate is shown as a dot positioned above the bars. ***P ≤ 0.001. Scale bars = 20 μm.

4. Discussion

Haemonchus contortus is the most widely employed parasite model in drug discovery, vaccine development and anthelmintic resistance research (Lanusse et al., 2016). Establishing in vitro culture of parasitic stages will significantly facilitate our understanding of its biology. The early L4s were first cultured in 1933 (Lapage, 1993), but there has been very little breakthrough to promote parasite growth beyond this stage (Sommerville, 1966, 1976, 1977; Preston et al., 2015; Niciura et al., 2023). The current work has established a culture system to develop xL3s into late L4s (∼60%). A standardized medium consisting of NCTC-109 with LB and supplemented with DFB and antioxidants (L-GSH or Vc) promoted survival and development of L4s. Poor survival of L4s in DFB-supplemented medium is likely due to high levels of ROS, which L-GSH or Vc could reduce. In addition, the morphological features of cultured late L4s were identified based on those of late L4s developed in vivo.

There have been a few culture media used to H. contortus culture (Stringfellow, 1986; Meng and Kong, 1993; Niciura et al. 2023). Among those, NCTC-109 has shown promise with other parasitic helminths. e.g., Stephanurus dentatus (Douvres et al., 1966) and Angiostrongylus cantonensis (Moreau and Lagraulet, 1972). NCTC-109 contains more vitamins and cofactors than M-199, which may underlie the improved development of early L4s. Similarly, LB medium, composed of natural ingredients, is rich in amino acids and vitamin B (Jacob et al., 2019). Transcriptomic analysis showed that amino acid metabolism and transcript levels of vitamin B family binding protein-coding genes significantly increased in L4s isolated from sheep (Laing et al., 2013), which correlates with the growth-promoting activity of NCTC-109:LB in our assays.

Haemonchus contortus is a hematophagous parasite; hence, we considered blood an indispensable constituent for its in vitro culture. Indeed, in vivo, the growth and reproductive system development of its L4s are triggered by feeding on the host’s blood (Stringfellow, 1984). It has been shown in other hematophagous parasites such as schistosomes and hookworms, that erythrocytes or heme could promote L4 development and adult egg laying (Bouchery et al., 2018). In this work, supplementation of DFB promoted the development of early L4s to late L4s. Other blood-sucking helminths such as Schistosoma mansoni similarly necessitate the supplementation of red blood cells (RBCs) (despite adding only 0.2% v/v) for the sustenance of their normal biological function (Wang et al., 2019). At lower levels of DFB (0–2.5%), early L4s of H. contortus displayed poor survival and development (Supplementary Fig. S2). Notably, ROS generation and cell damage in L4s cultured in 12.5% DFB were lower than in 7.5% and 10.0% DFB. Additionally, more hemozoin (72 h after DFB was added) was produced when the L4s were exposed to 12.5% DFB. As the hemozoin formation is a critical detoxification mechanism in many blood-sucking parasites (Sullivan et al., 1996; Oliveira et al., 2000, 2005; Pisciotta et al., 2005), it may also be a vital factor in promoting L4 development under culture conditions with a high concentration of blood.

Studies on malaria and schistosomes have shown heme (a byproduct of hemoglobin degradation) could generate hydroxyl radicals and ROS, damaging cells (Oliveira et al., 2000; de Villiers and Egan, 2021). Equally, ROS production in DFB-exposed Haemonchus L4s correlates with intestinal cell death. The addition of L-GSH and Vc (after DFB was added for 2 days) reduced ROS production and enhanced the survival and development of L4s (on day 22). We also noted that additional supplementation of Vc (200 mmol) led to the death of all L4s after they had been cultured in its presence for only 2 days (data not shown). An early study demonstrated that highly reduced glutathione (80 mM) could inhibit L3 development to the L4 stage (Mapes, 1970). It may be because excessive antioxidants accelerate the redox cycle of Fe³⁺ and Fe²⁺ in the Fenton reaction, which leads to a significant rise in toxic hydroxyl radicals (He et al., 2020; Timoshnikov et al., 2020). This was consistent with the hypothesis that the reduction of disulphide bonding in a protein by L-glutathione could generate further reactive thiol groups in the reversible reaction. The data highlight the importance of oxidative homeostasis during in vitro culture of H. contortus.

In our experiments, the surviving L4s developed to middle or late L4s, reaching up to ∼90% on day 22 (∼40% middle, ∼50% late-phase), which is the first known time that H. contortus xL3s matured to this late stage. Thus far, hardly any culture system could consistently ensure xL3 development into late L4s. Previous studies have shown that few xL3s matured into adult stages under an extremely complex and unstable culturing system (Silverman, 1959; Schulz, 1967; Stringfellow, 1986). Comparison of morphology between the cultured L4s (day 22) and those isolated from animals (day 7 p.i.) revealed a high resemblance. Accumulation of blood clot-like material was observed within the pharynx of the worm, which was similar to that in the worms developed in vivo (Veglia, 1915). Thus far, although a breakthrough has been made in developing mature L4s, several concerns are yet to be addressed. For instance, (i) the in vitro system requires 17–22 days for xL3s to differentiate into late L4s as opposed to in vivo development of 7 days; (ii) the survival rate (∼60%) and the developmental rate of late L4s (∼50%) can be improved further; (iii) which of the blood ingredients damage the worms and how best to maintain oxidative homeostasis during the culture of parasitic stages?

In conclusion, the present study established a culture system for L4s of H. contortus. Despite nearly three decades of research, our streamlined protocol has facilitated the maturation of parasitic nematode larvae into the late L4 stage. Our work provides a valuable platform for in-depth studies on the molecular biology of Haemonchus and insight into developing culture systems for other parasitic helminths.