In vitro culture of parasitic stages of Haemonchus contortus

Abstract Haemonchus contortus is a constraint to sheep production. Seeking to reduce the use of hosts and produce parasitic stages in large-scale, a 42-day in vitro culture protocol of H. contortus third-stage larvae was optimized using Dulbecco’s modified Eagle’s medium (DMEM). In cell-free culture, larvae were maintained at 39.6°C, in acidic media (pH 6.1) for 3 or 6 days with Δ4-dafachronic acid followed by DMEM pH 7.4 supplemented or not with Fildes’ reagent. In DMEM pH 7.4 at 37°C, supplementation with Caco-2 cells was compared to Fildes. On Day 14, fourth-stage larvae (L4) development rates in acidic media supplemented (86.8-88.4%) or not (74.4-77.8%) with Fildes and in Caco-2 cell co-culture (92.6%) were similar, and superior to DMEM pH 7.4 with Fildes (0.0%). On Day 21, Caco-2 cell co-culture resulted in higher larvae differentiation (25.0%) and lower degeneration (13.9%) compared to acidic media (1.5-8.1% and 48.6-69.9%, respectively). This is the first report of prolonged in vitro culture of H. contortus larvae using commercial media in co-culture with Caco-2 cells. Although no progression to the adult stage, Caco-2 cell co-culture resulted in morphological differentiation of H. contortus L4 and larval viability for up to 28 days.


Introduction
Sheep production provides meat, milk, and wool for consumers, takes advantage of areas unsuitable for other forms of agriculture (Sargison, 2012), and is predicted to increase in scenarios of global warming in South America (Seo et al., 2010).The challenges faced by small ruminant producers in tropical regions include adverse climate conditions, scarcity or competition for water and food resources, and a high prevalence of gastrointestinal nematodes (McManus et al., 2011).Gastrointestinal nematodes are responsible for annual losses estimated at AUD$ 369-436 million in Australia (Hosking et al., 2009;Emery et al., 2016) and US$ 107.5 million in Brazil (Chagas et al., 2022).Economic losses are usually secondary to health issues and productive deficits resulting from nutrient spoliation, anemia, reduced weight gain, and deaths triggered by parasites, mainly when ineffective anthelmintics are used in flocks (Miller et al., 2012).
Haemonchus contortus is the most pathogenic parasite in small ruminants in tropical and subtropical climates.H. contortus, a hematophagous nematode of the abomasum, can remove up to 30 µL of blood per day and has a short pre patency period of 18-21 days.In addition, each adult female can reproduce with four to eight males and shed 1,300 eggs per day (Redman et al., 2008;Emery et al., 2016;Naeem et al., 2021), resulting in high mutation rates and genetic diversity in this species (Gilleard, 2013;Doyle et al., 2018).Anthelmintic treatment is the main approach used to control nematodes in sheep to reduce production and economic losses.However, treatments select helminths with genetic polymorphisms that confer resistance, and this subsequently leads to treatment failure (Barnes et al., 1995).
Therefore, the development of alternative and sustainable measures to control gastrointestinal helminths in flocks is of great interest.Parasite cultivation in vitro could provide large-scale production of parasites across diverse life cycle stages, which may be useful for vaccine development (Preston et al., 2015;Naeem et al., 2021), parasitic crosses to map resistance-related polymorphisms (Redman et al., 2012;Niciura et al., 2019), in vitro assays of new anthelmintic products, and gene silencing (Kotze & Bagnall, 2006).In addition, parasite culture improves understanding of antigenic molecules in excretory and secretory products, which induce increased systemic humoral responses and protection against H. contortus (Arunkumar, 2012;Lu et al., 2021) and serve as potential targets for immune or chemical control (Gamble & Mansfield, 1996).Additionally, since current methods used to extract native proteins for immunization against H. contortus rely on host infection and slaughter (González-Sánchez et al., 2018), the development of in vitro methods is desirable.
The free-living H. contortus stages (eggs, L 1 , L 2 , and L 3 ) can be easily collected from sheep host feces or fecal cultures, whereas parasitic stages (L 3 in hosts, L 4 , and adult L 5 ) are only recovered after the host has been slaughtered.An alternative host model for H. contortus growth, using immunosuppressed jirds, resulted in slower and incomplete development (Conder et al., 1992).Thus, the lack of a protocol for obtaining all life stages of H. contortus by in vitro culture (Rufener et al., 2009) or by using laboratory animals as hosts (Gilleard, 2013) leads to dependency on small ruminants and limits the development of new control strategies.Nevertheless, there is a global demand for research methods that replace, reduce, or refine the use of animals in experiments (Liebsch et al., 2011), and models need to be developed and adapted to ensure the replacement of in vivo methods by in vitro methods (Chagas, 2015;Shivam et al., 2021).
Studies investigating H. contortus in vitro development resulting in male and female adults were conducted in the 1980s, and rely on complex and homemade media (Stringfellow, 1984(Stringfellow, , 1986)), but these proved difficult to replicate.Considering recent advances in culture techniques, attempts to improve culture conditions and achieve development of H. contortus in vitro without the use of sheep hosts are sought (Geary, 2016).Thus, to improve culture conditions and large-scale in vitro cultures of parasitic stages of H. contortus, the objective of this study was to optimize an in vitro protocol using commercial media to obtain larvae of a more advanced developmental stage than other studies have achieved, which could be maintained in vitro for longer periods than currently possible.We sought to evaluate H. contortus in vitro development, differentiation, and viability by comparing cell-free cultures to co-cultures with Caco-2 cells using different supplements, pH, temperatures, and atmospheres.

Sheep hosts and parasitological tests
Two Santa Ines ewes were housed in barns with no access to pastures and fed corn silage ad libitum.Natural gastrointestinal nematode infections were eliminated using a combination of three anthelmintics (9.4 mg/kg levamisole, 20 mg/kg albendazole, and 2.5 mg/kg monepantel) for three consecutive days, according to a protocol Braz J Vet Parasitol 2023; 32(1): e010122

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Haemonchus contortus in vitro culture used to eliminate infection with multiresistant populations (Almeida et al., 2020).Nematode elimination was confirmed by fecal egg count (FEC) using the McMaster technique with a sensitivity of 50 eggs per gram (Ueno & Gonçalves, 1998) at 7 and 14 days after the last treatment.Ewes were experimentally infected by oral administration of 4,000 third-stage larvae (L 3 ) of the H. contortus Echevarria isolate, which is susceptible to albendazole, ivermectin, and levamisole (Echevarria et al., 1991).FEC was assessed weekly up to the detection of eggs in feces on Day 28.Feces were cultured in glasses (Roberts & O'Sullivan, 1950) at 27°C for 7 days, and H. contortus L 3 migrating into water were recovered, kept at 4°C and used for in vitro culture within 15 days.

H. contortus third-stage larvae (L 3 ) decontamination
To ensure decontamination prior to in vitro culture, H. contortus L 3 were washed five times by centrifugation (1,100 × g for 5 min) in saline solution (0.85% NaCl).Larvae were then exsheathed with sodium hypochlorite (0.15% for 5 min) followed by four washes in saline with 1% antibiotics-antimycotic (100 units penicillin, 0.1 mg streptomycin, and 0.25 μg amphotericin B; Sigma A5955).Approximately 1,000-5,000 larvae in 10 mL of media were transferred to 25 or 75 cm 2 flasks with non-treated (for cell-free suspension culture) or treated (for co-culture with cells) surfaces, and flasks were laid in an incubator.Sterile conditions were maintained by manipulation under laminar flow and media filtering through 0.22 μm PES membranes.
H. contortus lack an endogenous pathway for heme biosynthesis (Toh et al., 2010); therefore, this compound needs to be provided in the media through the addition of hemoglobin, hemin, tissue extracts, or blood cell lysates (Bolla, 1979).Thus, two supplements were tested: Fildes' reagent, which is a pepsin-digested defibrinated sheep blood medium, and Caco-2 cells (human colorectal adenocarcinoma-derived intestinal epithelial cells).Furthermore, acidic media (or media without bicarbonate) was used considering that the pH in the sheep abomasum varies from 1 to 6 (Harder, 2016) and that pH 4-4.5 resulted in maximum egg production by H. contortus (Honde & Bueno, 1982).Additionally, larvae were incubated at 39.6°C to simulate sheep average temperature, which is 39.9°C in the rumen, 39.8°C in the aorta, 39.6°C in the rectum, and 39.1°C in the abomasum (Bailey et al., 1962;Sommerville, 1966).In addition, the CO 2 concentration in the incubator was set to 10% considering that CO 2 pressure (pCO 2 ) is higher in the rumen (380 mmHg), where H. contortus L 3 can remain for approximately 12 h after ingestion before moving to the abomasum (50 mmHg pCO 2 ) (Sommerville, 1966), and considering the beneficial effect of 40% CO 2 injected into the media (Sommerville, 1977).1) were DMEM (Sigma D5648) with or without 44 mM bicarbonate (Sigma S5761), EBSS (Sigma E7510) without bicarbonate, and Medium 199 (Sigma M5017) with 26.2 mM bicarbonate.For prolonged culture, media were variously supplemented with 1% antibiotics-antimycotic (100 units penicillin, 0.1 mg streptomycin, and 0.25 μg amphotericin B; Sigma A5955), 10% fetal calf serum (FCS; Cripion), 1 mM sodium pyruvate (Sigma P4562), 1% non-essential amino acids (MEM; Sigma M7145; Supplementary Table 1), 2 mM L-glutamine (Sigma G3126), and 10 mM HEPES (Sigma H4034) (Table 1).Every 7 days for up to 42 days, all media were removed by centrifugation and replaced with freshly prepared media.Each culture condition was tested once, and all combinations tested for prolonged culture are shown in Table 1.

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Haemonchus contortus in vitro culture For co-cultures, a fresh 33-passage Caco-2 cell line obtained from Banco de Células do Rio de Janeiro (BCRJ 0059, lot 001783) was incubated in DMEM with bicarbonate, FCS, amino acids, pyruvate, L-glutamine, and HEPES at 37°C under 5% CO 2 , which are optimal conditions for culturing Caco-2 cells.Subcultures were performed when the cells reached 80% confluency.H. contortus larvae were co-cultured with confluent Caco-2 cells from passages 37-39 in the same conditions used for Caco-2 cell cultures.

Larvae development assessment and statistical analysis
Every 7 days, simultaneously to the media replacement after centrifugation, an aliquot of 50-100 µL from the pellet was removed, and all larvae recovered in the aliquot (varying from 14 to 140 larvae) were assessed under an optical microscope.Progression to L 4 and sexual differentiation were evaluated according to the morphological classification by Veglia (1915).Mouth development and asymmetrical tail with dorsal curvature indicated progression to L 4 .Furthermore, in early differentiated L 4 , thick posterior end with the tail being short, conical, smooth, and slightly curved posteriorly, with no detection of bursa, was observed in males, while longer tail tapered slightly and bent dorsally was present in females.From the total number of recovered larvae, the number of larvae in the L 4 stage was counted (developmental rates).Additionally, on Days 21, 28, 35, and 42, the number of L 4 larvae showing sexual differentiation (differentiation rates) and the number of degenerated L 3 -and L 4 -stage larvae (degeneration rates) were counted from the total number of recovered larvae.a,b,c,d,e Values with different superscripts in the same column differ at a significance level of 0.05.

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Haemonchus contortus in vitro culture The purpose was to achieve an in vitro culture protocol resulting in higher developmental and differentiation rates with lower degeneration rates.Thus, larvae development, differentiation, and degeneration rates were compared by chi-square test at a significance level of 5%.

H. contortus larvae recovery and in vitro culture
Most larvae recovered from fecal cultures were L 3 (Figure 1B); however, L 2 were also retrieved (Figure 1A).After in vitro culture, L 4 were observed, based on mouthpart development and asymmetrical tail with dorsal curvature (Figure 1C).
No differences were observed in cultures using 25 or 75 cm 2 flasks with 1,000-5,000 larvae; however, using more than 5,000 L 3 /10 mL resulted in frequent culture contamination, observed through changes in media color and turbidity and larvae death.
Results of prolonged culture protocols for up to 21 days are presented in Table 1.Due to the large number of degenerated and dead larvae recovered on Days 28, 35, and 42, these results are described in the text and not included in Table 1.

An acid pH and the use of a steroid hormone supplement promotes molting of exsheathed L 3 to L 4 in vitro
For the first three to six days in culture, two different media without bicarbonate supplemented with 2.3 μM Δ4-DA and antibiotics-antimycotic were tested: EBSS (pH 4.6) and DMEM (pH 6.1).These resulted in similar (p>0.05)L 4 development: 26.3% on Day 3 and 60-70% on Day 6.As DMEM was the media employed for Caco-2 cell cultures and there were no significant differences in L 4 development, DMEM was selected for use in further experiments to allow comparisons between cell-free and co-cultures, while EBSS was not included in prolonged culture experiments.In addition, it was observed that the Δ4-DA steroid hormone, and not only the Δ7-DA reported in the literature (Ma et al., 2019;Marks et al., 2019), can be used for in vitro culture supplementation leading to increased H. contortus L 4 development.

Prolonged culture in weak acidic media is detrimental to larvae in vitro
Incubation in acidic media for periods longer than 6 days was detrimental to larval development and resulted in the death of all larvae by Day 14.Exposure to more acidic conditions (pH 2) for 5 min resulted in the death of H. contortus larvae (Sommerville, 1977).Thus, contrary to natural acidic environment in the abomasum, prolonged culture under low pH conditions was deleterious for H. contortus larval development in vitro.Haemonchus contortus in vitro culture Subsequently, acidic DMEM media used for 3 or 6 days, supplemented with Δ4-DA for all 6 days, followed by culture in DMEM pH 7.4 without Fildes' reagent resulted in 74.4-77.8%L 4 on Day 14 and 95.2-98.4% on Day 21 (Table 1).Similar percentages (p>0.05) were observed following larval culture in DMEM pH 7.4 supplemented with Fildes' reagent (86.8-88.4% on Day 14 and 85.7-97.0%on Day 21) (Table 1).On Day 21, in all culture conditions at 39.6ºC and 10% CO 2 , differentiation rates of 1.5-8.1% in L 4 were observed (Table 1), and distinct morphological patterns suggested the development of males (Figure 2A) and females (Figure 2B-2C).However, incubation in acidic conditions for 3 or 6 days resulted in high rates (p<0.05) of degeneration (48.6-69.6%) on Day 21 (Table 1) and by Day 35 almost all larvae were degenerated, immobile, or dead.Morphological alterations observed in degenerated larvae on Day 21 consisted of granule or vacuole formation in intestinal cells (Figure 3A), cuticle wrinkling (Figure 3B), cuticle detachment (Figure 3C), or cuticle rupture and evisceration (Figure 3D).These changes were similar to those observed in larvae exposure to anthelmintics, indicating death or imminent death (Acevedo-Ramírez et al., 2019;Nguyen et al., 2019).In addition, there was no progression to adulthood (L 5 ) for up to 35 days in culture.Using additional supplementation of media promotes development, with the use of Caco-2 cells found to be of increased benefit to larval development than Fildes' reagent In Trichinella spiralis, Caco-2 cell monolayers supported L 1 molting, ecdysis, adult development, and reproduction of 50% larvae in vitro cultured for 11 days (Gagliardo et al., 2002).Therefore, H. contortus co-cultures with Caco-2 cells were evaluated and compared with supplementing a cell-free DMEM culture with Fildes' reagent.In this experiment, Δ4-DA was not used and not initial period in acidic media was provided.Since Caco-2 cells remained viable only at 37°C in DMEM without Fildes' reagent, the incubation temperature for these experiments was reduced to 37°C.At 5% CO 2 , co-cultures with Caco-2 cells in DMEM resulted in higher (p<0.05)developmental rates on Days 7, 14, and 21 (42.9%,92.6%, and 97.2%, respectively), compared to DMEM supplemented with Fildes (0.0%, 0.0%, and 32.2%, respectively) and Medium 199 supplemented with Fildes (0.0%, 6.1%, and 14.6%, respectively) (Table 1).Medium 199 contains more vitamins and amino acids than DMEM supplemented with non-essential amino acids (Supplementary Table 1).However, higher (p<0.05)rates of L 4 development were observed on Day 21 following culture in DMEM with supplements.In addition, co-culture with Caco-2 enhanced (p<0.05)L 4 development compared to supplementation with Fildes' reagent (Table 1).This is the first report comparing Caco-2 cells to Fildes' reagent supplementation in prolonged cultures.In the literature, H. contortus culture with Caco-2 cells for up to 3 weeks resulted in higher rates of development from L 3 to L 4 , but no development to adults (Britton et al., 2016).Then, we tested the use of Caco-2 in co-culture for prolonged periods (up to 42 days or 6 weeks), but still no adults were obtained.
In vitro co-culture of H. contortus larvae with Caco-2 cells at 37°C and 5% CO 2 resulted in similar (p>0.05)rates of L 4 development on Days 6-7, 14, and 21 to cell-free conditions using acidic media at 39.6°C and 10% CO 2 , but with Haemonchus contortus in vitro culture higher (p<0.05)differentiation rates (25.0%; Figure 4A) and inferior (p<0.05)degeneration rates on Day 21 (Table 1).In addition, in Caco-2 cell co-culture, morphologically viable larvae were recovered on Day 28 (at a degeneration rate of 31.9%), and differentiated larvae were still observed on Day 35 (Figure 4B), however most larvae were degenerated (presence of granules or vacuoles and reduced movement) or dead on Days 35 and 42.Degeneration of 50% of larvae on Day 28 in culture was reported by Stringfellow (1984), which was higher than that observed here on Day 28 in co-cultures.Thus, co-culture with Caco-2 cells resulted in viable H. contortus L 4 for up to 28 days in vitro, but no progression beyond the L 4 stage was observed.
In the literature, the development of adult H. contortus following in vitro culture has been reported by Stringfellow (1984Stringfellow ( , 1986)), using API-I medium (Douvres & Malakatis, 1977; Supplementary Table 1), resulting in the development of 0.1% males on Day 28 and 0.07% females on Day 36 (Stringfellow, 1986).Stringfellow (1986) used API-I medium supplemented with Fildes' reagent and sheep gastric contents, with the pH adjusted to 6.4 for 1 week and then to pH 6.8, at 39°C under 85% N 2 , 5% O 2 , and 10% CO 2 .In the present study, culture under acidic pH beyond Day 6 was detrimental to larval development, whereas incubation in an atmosphere with nitrogen delayed and reduced larval development in co-culture with Caco-2 cells.Considering L 4 development, the rates observed in the present study using the best protocol (co-culture with Caco-2 cells at 5% CO 2 ) were similar but delayed (42.9% on Days 6-7 and 92.6% on Day 14) compared to those reported previously for short-term cultures.Sommerville (1966) achieved 90% L 4 in 3 days following culture in saline solution pH 6 at 40°C ± 0.5°C under 40% CO 2 and 10% O 2 .Preston et al. (2015) reported 80% L 4 after 5 days of culture in DMEM with L-glutamine, antibiotics, and antimycotics at 40°C and 10% CO 2 .Nguyen et al. (2019) obtained more than 80% L 4 after 7 days using Luria-Bertani medium supplemented with antibiotics and antimycotic at 38°C and 10% CO 2 .Marks et al. (2019) obtained more than 65% L 4 on Day 3 following culture in EBSS pH 5 with antibiotics, antimycotics, and 2.5 μM Δ7-DA at 37°C and 5% CO 2 .

Conclusion
Despite no progression to the adult L 5 stage, co-culture with Caco-2 cells at 37ºC and 5% CO 2 was sufficient to stimulate transition to L 4 and assured maintenance of morphologically viable and differentiated L 4 for 28 days.Thus, it was considered the best protocol for in vitro culture of H. contortus from L 3 recovered from fecal cultures using commercial media.

Figure 2 .
Figure 2. Haemonchus contortus L 4 with morphological differentiation of posterior end after 21 days in cell-free in vitro culture.A) Suggestive pattern of male development with thick and curved tail (arrow).B) Suggestive pattern of female development with longer and taped tail (arrow).A and B) Culture in acidic DMEM for 3 days with Δ4-DA for 6 days followed by incubation in DMEM without Fildes' reagent.C) Cuticle inflation or potential knob-shaped vulva (arrow) in differentiated female, following culture in acidic DMEM for 3 days with Δ4-DA for 6 days followed by incubation in DMEM supplemented with Fildes' reagent.*Rectum and anus.Optical magnification 10× and digital zoom.

Figure 3 .
Figure 3. Morphological alterations in Haemonchus contortus L 4 on Day 21 after in vitro culture in acidic media for the first 3 or 6 days.A) Granule or vacuole formation.B) Cuticle wrinkling.C) Cuticle detachment.D) Cuticle rupture and evisceration.Optical magnification 10× and digital zoom.

Figure 4 .
Figure 4. Progression of Haemonchus contortus L 4 differentiation after in vitro co-culture with Caco-2 cells.A) L 4 with developed mouth (*) and posterior end thickening (arrow) on Day 21.B) L 4 with posterior end differentiation, suggestive of male development on Day 35.Optical magnification 10× and digital zoom.