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Published in final edited form as: Int J Parasitol. 2023 May 16;53(8):451–458. doi: 10.1016/j.ijpara.2023.05.001

An efficient method for viable cryopreservation and recovery of hookworms and other gastrointestinal nematodes in the laboratory

Hanchen Li a, David Gazzola a, Yan Hu b, Raffi V Aroian a,*
PMCID: PMC10330584  NIHMSID: NIHMS1902607  PMID: 37201563

Abstract

Hookworms (genera Ancylostoma and Necator) are amongst the most prevalent and important parasites of humans globally. These intestinal parasites ingest blood, resulting in anemia, growth stunting, malnutrition, and adverse pregnancy outcomes. They are also critical parasites of dogs and other animals. In addition, hookworms and hookworm products are being explored for their use in treatment of autoimmune and inflammatory diseases. There is thus a significant and growing interest in these mammalian host-obligate parasites. Laboratory research is hampered by the lack of good means of cryopreservation and recovery of parasites. Here, we describe a robust method for long-term (≥3 year) cryopreservation and recovery of both Ancylostoma and Necator hookworms that is also applicable to two other intestinal parasites that passage through the infective L3 stage, Strongyloides ratti and Heligmosomoides polygyrus bakeri. The key is a revised recovery method, in which cryopreserved L1s are thawed and raised to the infective L3 stage using activated charcoal mixed with uninfected feces from a permissive host. This technique will greatly facilitate research on and availability of gastrointestinal parasitic nematodes with great importance to global health, companion animal health, and autoimmune/inflammatory disease therapies.

Keywords: Hookworms, Cryopreservation, Ancylostoma, Necator, Heligmosomoides, Strongyloides, Gastrointestinal nematodes, Freezing

Graphical Abstract

graphic file with name nihms-1902607-f0001.jpg

1. Introduction

Hookworms (Necator americanus, Ancylostoma ceylanicum, and Ancylostoma duodenale) are blood-feeding helminth parasites of humans that live in the small intestine and are responsible for >4 million disability-adjusted life years (DALY) and $100 billion of economic losses annually (Pullan et al., 2014; Bartsch et al., 2016; Loukas et al., 2016; Jourdan et al., 2018; Hamory et al., 2021; Umbrello et al., 2021). Hookworm infection has had a significant influence on human history and is a major cause of morbidity in infants, children, and women of child-bearing age in the tropics and subtropics leading to, amongst other things, malnutrition, permanent growth stunting, cognitive impairment, adverse pregnancy outcomes, decreased worker productivity, and decreased educational status and future earnings. The closely related hookworm Ancylostoma caninum is the most prevalent and important intestinal nematode parasite of dogs in the United States of America (Jimenez Castro et al., 2020, 2021). Its prevalence is rapidly spreading, together with a multidrug-resistant phenotype, making it increasingly recalcitrant to treatment (Jimenez Castro et al, 2020, 2021). Development of new and better therapies, and a protective hookworm vaccine, are considered important goals for global human and animal health (Hu et al., 2018; Shepherd et al., 2018; Noon et al., 2019; Haldeman et al., 2020; Loukas et al., 2021; Mourão Dias Magalhães et al., 2021).

Hookworms and other helminth parasites are also major drivers in the evolution of the vertebrate and human immune systems, in particular in shaping the Th2 immune response (Maizels et al., 2009; Fumagalli et al., 2010; Loukas et al., 2016). The pressure imposed by parasitic worms on human genes has been hypothesized to be stronger than that of viral, protozoal, or bacterial agents (Fumagalli et al., 2011). Due to their strong immunomodulatory characteristic, hookworms, most notably N. americanus, and their secreted products are garnering great interest for treatment of autoimmune and autoinflammatory diseases such as celiac disease, asthma, metabolic syndrome, multiple sclerosis, and inflammatory bowel diseases (Loukas et al., 2016, 2021; Ryan et al., 2020; Chapman et al., 2021; Montaño et al., 2021; Mourão Dias Magalhães et al., 2021).

Studies on human hookworms are therefore of enormous and growing interest vis-a-vis global health, drug development, vaccine development, immunology, and autoimmune research and therapies. Currently, Necator and/or Ancylostoma hookworms can be maintained and studied in the laboratory using hamsters, dogs, and even humans as hosts (Loukas et al., 2016; Chapman et al., 2021; Montaño et al., 2021). There are, however, significant challenges in working with these obligate mammalian parasites in the laboratory that are exacerbated by the lack of reliable cryopreservation/recovery methods (see section 4). For example, laboratories working with these parasites are under constant pressure to maintain their hookworm lines or else permanently lose their invaluable cultures. Methods for long-term preservation of hookworms have thus been deemed critically important for continued human experimental infection and therapy (Chapman et al., 2021). Although such methods have been described before, these approaches have limitations in terms of efficiency and duration (Nolan et al., 1994; Duarte et al., 2003; also, see below). Here, we describe methods for the long-term (≥3 year) cryopreservation and recovery of viable Necator and Ancylostoma hookworms as well as other gastrointestinal parasitic nematodes that infect hosts via infective L3s (iL3s).

2. Materials and methods

2.1. Medium and reagents

Cryopreservation solution was made, similar to that previously described (Duarte et al., 2003): 70% RPMI 1640, 10% dimethyl sulfoxide, 10% dextran T10, and 10% fetal bovine serum. Dexamethasone (DEX) was used to immune suppress the hamsters for N. americanus-related experiments (Hu et al., 2018). S Medium was prepared as described previously (Sulston and Hodgkin, 1988). Images were taken with an Olympus SZ-CTV dissecting microscope fitted with an Infinity1 camera. GraphPad Prism v. 9 was used for all graphs and analyses.

2.2. Animals and parasites

Ancylostoma ceylanicum and N. americanus hookworms were maintained as previously reported (Hu et al., 2012, 2018; Li et al., 2021). Three to 4 week old male Golden Syrian hamsters (HsdHan:AURA) were purchased from Envigo (U.S.A) and were infected at approximately 4–5 weeks of age with either ~130 A. ceylanicum iL3s orally or ~300 N. americanus iL3s subcutaneously. Hamsters were provided with food and water (ad libitum). For isolation of iL3s for direct freezing, infected hamster feces were cultured on activated charcoal for 7 days at 28°C and harvested using the Baermann technique.

Strongyloides ratti was maintained in 6 week old male Wistar rats by subcutaneous injection with 500–1000 iL3s. Infected feces were cultured on activated charcoal for 5–7 days at 22°C to isolate iL3s. Heligmosomoides polygyrus bakeri was maintained in 6 week old male Swiss Webster mice by oral gavage with 200 iL3s as previously reported (Hu et al., 2010). Infected feces were cultured on activated charcoal for 7 days at 22°C to isolate iL3s.

All animal experiments were carried out under protocols approved by the University of Massachusetts Chan Medical School, U.S.A. Institutional Care and Use Committee (IACUC; protocols 202100090, 202000071, 202000044). All housing and care of laboratory animals used in this study conform to the National Institutes of Health (N.I.H., U.S.A.) Guide for the Care and Use of Laboratory Animals in Research (see 18-F22) and all requirements and all regulations issued by the United States Department of Agriculture (U.S.D.A.), including regulations implementing the Animal Welfare Act (P.L. 89–544) as amended (see 18-F23). Fecal egg counts and small intestinal worm burdens were determined as previously described (Hu et al., 2010, 2012, 2018). For S. ratti, fecal egg counts were taken 7 days post-inoculation.

2.3. Freezing of L1s, recovery, and growth to iL3 stage

2.3.1. Isolation of eggs from feces and surface sterilization

The same procedure was carried out for all parasites and all rodent hosts. Feces from nematode-infected rodents were collected and eggs isolated as previously described (Mes et al., 2007) with slight modifications. Basically, feces were collected and soaked in a beaker with 13% NaCl solution (2 mL per gram of feces) for 30 min at room temperature. The feces were then homogenized with a spatula and the resultant solution was poured through a stainless steel mesh strainer into a 50 mL conical tube to filter out large material. The tube was then spun at 2000 g at room temperature for 5 min, with the eggs ending up in the supernatant. The supernatant was decanted into a beaker into which an equal volume of distilled water was added. The resultant egg suspension was split into 50 mL conical tubes and spun as above, with the eggs ending up in the pellet. After the supernatant was discarded, the pellets were combined and resuspended in 10 mL of 17% sucrose solution and transferred to a 15 mL conical tube. The suspension was spun as above, with the eggs ending up in the supernatant. The supernatant was split into two 15 mL conical tubes, filled to the top with distilled water, and spun as above (eggs would be in the pellet). The pellets were resuspended in 4.5 mL of distilled water to which was added 0.5 mL of 6% hypochlorite solution (Fisher Scientific Cat# SS290-1) and gently rocked for 1 min followed by centrifugation (800 g for 2 min) and washed three times with sterile double distilled water. After the final wash with water, the eggs were resuspended in 25 mL of S Medium and filtered with a 70 μm cell strainer to remove the small debris.

2.3.2. Hatching of eggs to the L1 stage and subsequent freezing

The isolated and surface sterilized eggs were transferred to a 50 cm2 tissue culture flask and incubated at 28°C for 42 h to hatch into L1s. The hatched L1s were spun down at 800 g for 2 min at room temperature, resuspended in the cryopreservation solution at a density of approximately 2 ×104 worms/ml, incubated at room temperature for 1 h, and then transferred to a 2 mL cryovial. The cryovials were then placed into the Nalgene Cryo 1°C Freezing Container (Nalgene Thermo Scientific, USA, Mr. Frosty, Cat# NL-51000001), which was stored at −80°C overnight. The next day, all the cryovials were moved to liquid nitrogen for long-term storage.

2.3.3. Recovery of frozen L1s and culturing to the iL3 stage

For recovery and growth of frozen L1s, 5–8 g of fresh feces from relevant hosts (hamsters for hookworms, rats for S. ratti, mice for H. p. bakeri) were collected and soaked in an equal volume of tap water for 10–20 min in a beaker. The feces were mixed using a spatula with an equal amount of activated charcoal (Sigma-Aldrich, USA, Cat#: C2889) and transferred to a 100×25 mm deep petri dish (USA Scientific Catalog #8609-0625). The frozen L1s were thawed by plunging the cryovial into a 50°C water bath and shaking the tube slowly. The larvae in solution were transferred to a 15 mL conical tube, to which 13 mL of distilled water were added. The larvae were pelleted at 800 g for 2 min, resuspended in 10 mL of tap water, incubated at 28°C for 1 h, checked for viability, recentrifuged, and then resuspended in 1 mL of tap water. The L1s were then loaded onto the top of the fecal-charcoal mixture and incubated at 28°C for 7 days for hookworms and 22°C for 7 days for S. ratti and H. p. bakeri, at which point the iL3s were collected by the Baermann technique and washed with sterile double distilled water three times. After the last wash, the iL3 worms were resuspend with BU saline (Hawdon and Schad, 1991) and stored in a 25 cm2 tissue culture flask (Genesee Scientific cat#25-207) at room temperature until they were used to infect host rodents as per standard protocols. For the images of thawed L1s, frozen L1s were thawed quickly in a 55°C water bath, brought up to 50 mL volume with sterile water, spun down, resuspended in sterile water, allowed to recover for 1–2 h at 28°C, and then imaged.

For L1s, the health of the larvae was determined mostly based on motility. For L1s, a drop of larvae was added to a watch glass, and each individual larva was scored for movement or not (this was independently repeated 3–5 times). Morphology was also informative, as moving larvae are invariably curved but dead larvae are rigid and straight. For iL3s, only live worms were recovered from the Baermann funnel. The classification as iL3 was based on morphology as they are thin, long, and have a waxy cuticle, which is lacking in other larvae. Confirmation of iL3s is shown in Supplementary Fig. S1.

2.4. Infection efficacy studies

2.4.1. Ancylostoma ceylanicum

Male hamsters were infected per os with 130 live iL3s for infection efficacy experiments. The hamsters were sacrificed on day 22 post-inoculation (PI) and intestinal parasite burdens and fecal egg counts were determined as described previously (Hu et al., 2012, 2013, 2018).

2.4.2. Necator americanus

Male hamsters were infected subcutaneously with 300 live iL3s for infection efficacy experiments. The hamsters were sacrificed on day 55 PI and intestinal parasite burdens and fecal egg counts were determined as described previously (Hu et al., 2018; Li et al., 2021).

2.4.3. Strongyloides ratti

Male rats were subcutaneously inoculated with 500 iL3s, and fecal egg counts were taken on day 7 PI.

2.4.4. Heligmosomoides polygyrus bakeri

Male mice were inoculated per os with 200 iL3s, and fecal egg counts were taken on day 16 PI as described previously (Hu et al., 2010).

3. Results

3.1. Cryopreservation results with published protocols

An earlier publication noted the freezing of A. duodenale hookworm L1s, recovery of live larvae after 6 months, growth to the iL3 stage on sterile agar plates using exogenous Escherichia coli (Aikens and Schad, 1989), and infection of a single immunosuppressed dog (Nolan et al., 1994). Although L1s from N. americanus hookworms were also frozen in this study, the ability of iL3s derived from these L1s grown on sterile on agar plates with E. coli to successfully infect a dog were not tested (Nolan et al., 1994). To test this, we similarly froze N. americanus L1s, thawed and grew them in S medium with exogenous E. coli for 7 days at 28° C until they reached the iL3 stage (Hu et al., 2018), and infected two immunosuppressed hamsters with 300 iL3s each (Hu et al., 2018; Li et al., 2021). Neither infection was successful, despite the apparent health (based on morphology and motility) of the iL3s. This result suggested to us that this protocol might not be optimal for Necator hookworms.

A different approach involving the direct freezing of iL3s was more recently reported for cryopreservation of exsheathed and non-exsheathed A. ceylanicum (Duarte et al., 2003). Direct freezing of iL3s is also used for cryopreservation of the sheep gastrointestinal nematode parasite, Haemonchus contortus (Chylinski et al., 2015). We used the A. ceylanicum cryopreservation protocol (non-exsheathed iL3s) to freeze A. ceylanicum and N. americanus iL3s (Fig. 1). When harvested from fresh cultures prior to freezing, virtually all iL3s of A. ceyalnicum (Fig. 1A) and N. americanus (Fig. 1B) were curved, active, and alive. However, following freezing, very few (6/376) A. ceylanicum iL3s appeared alive based on morphology and motility (Fig. 1C) and no N. americanus iL3s appeared alive based on morphology and motility (Fig. 1D). These results suggested that this freezing protocol was not efficient for Ancylostoma and that Necator hookworms were even more difficult to successfully cryopreserve (as was also suggested in the study above).

Fig. 1.

Fig. 1.

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Morphology and survival of infective L3s (iL3s) from two hookworm species before and after freezing. Morphology of Ancylostoma ceylanicum (A) and Necator americanus (B) iL3s within 1 week of harvesting from charcoal culturing of infected hamster feces. The iL3 are curved, active, and alive. Morphology of thawed (C) A. ceylanicum and (D) N. americanus iL3 frozen for 2 weeks. Almost all (~99%) of the A. ceylanicum iL3s and all of the N. americanus iL3s are rigid rods— immotile and dead. Scale bar is the same in all panels.

We nonetheless tested infectivity of these cryopreserved A. ceylanicum iL3 hookworms. Hamsters were infected with live iL3s from either fresh charcoal cultures or recovered from cryopreserved iL3s frozen for 2 weeks or 3 months. Based both on intestinal hookworm burdens and fecal egg counts, infectivity of cryopreserved iL3s was very poor compared with infection with iL3s from fresh fecal cultures even when equal numbers of live iL3s were used for the infection (Fig. 2). When infecting with iL3s frozen for 2 weeks or 3 months, A. ceylanicum hookworm burdens were reduced 92% and 96%, respectively, relative to levels found when infecting with fresh iL3s; fecal egg counts were reduced 90% and 94%, respectively. Because we did not recover any live Necator hookworms, we did not perform similar experiments with this parasite.

Fig. 2.

Fig. 2.

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Infection efficacy of thawed Ancylostoma ceylanicum previously frozen at the infective L3 (iL3) stage. Average small intestinal burdens (A) and parasite fecal egg counts (B) of hamsters infected with iL3s either harvested from a fresh infection or from frozen stocks held at −80°C for the time indicated. Each dot represents an individual hamster (n=6/group). Error bars are S.E.M.

3.2. Development of a method for recovering cryopreserved L1s to iL3s

Because the literature suggested cryopreservation of L1s might be superior to that of iL3s (Nolan et al., 1994), we decided to adapt the L1 method, changing the way the L1s were allowed to progress to the iL3 stage. We hypothesized that growth of larvae on E. coli in culture might be less than ideal and that growth under conditions more similar to those used to normally give rise to iL3s in the laboratory might yield superior results.

Hookworm eggs (A. ceylanicum and N. americanus) were purified from an overnight fecal collection of infected hamsters using standard techniques (see section 2; (Mes, Eysker, and Ploeger 2007)). After the final wash, the eggs were allowed to hatch as L1s in Caenorhabditis elegans S Medium (Sulston and Hodgkin, 1988) at 28°C for 42 h. The larvae (L1s) were then resuspended in previously established cryopreservation solution (Nolan et al., 1994; Duarte et al., 2003) in cryovials. The cryovials were then placed in a −80°C incubator overnight and allowed to cool at a rate of 1°C per min using a freezing container. The following day, the cryovials were transferred to liquid nitrogen. This freezing protocol worked well, based on a comparison of freshly isolated L1s from feces (Figs. 3A, B, respectively, A. ceylanicum and N. americanus) with freeze-thawed L1s (Figs. 3C, D, respectively, A. ceylanicum and N. americanus). One to 3 years after storage at −80° C, a high percentage of viable L1s can still be recovered for both parasites with this protocol (Fig. 3E; 48% and 46%, respectively, for A. ceylanicum and N. americanus).

Fig. 3.

Fig. 3.

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Morphology and survival of L1s and survival of infective L3s (iL3s) from two hookworm species before and after freezing. Morphology of Ancylostoma ceylanicum (A) and Necator americanus (B) L1s 42–48 h following hatching off in media of parasite eggs freshly isolated from infected feces. Many of the L1s are curved, active, and alive. Morphology of thawed (C) A. ceylanicum and (D) N. americanus L1s frozen for 15 and 36 months, respectively. Scale bar is the same in all panels. (E) The percentage of thawed L1s that were alive are graphed for each hookworm species frozen for the times indicated in C and D. An aliquot of L1s was taken (~100 worms per aliquot) and the motility and morphology of all the worms was noted. Each individual point is an independent thaw from independent frozen stocks (each stock was sampled three times and an average was taken). (F) Percentage of iL3s recovered from frozen L1s. Approximately 10,000 thawed L1s (without discriminating live versus dead) were pipetted onto charcoal-uninfected hamster fecal mixture, incubated at 28°C, harvested using a Baermann funnel, and then the total number of iL3s was counted. Each data point is from an independent frozen sample. The A. ceylanicum samples were frozen for a minimum of 1 year. The N. americanus samples were frozen for a minimum of 2 years. Error bars are S.E.M.

To mimic a more natural progression to the iL3 stage than previous protocols, we thawed frozen L1s and plated them on a fecal-charcoal mixture typical for normal life cycle maintenance in the laboratory. However, unlike normal laboratory maintenance whereby feces from infected hamsters are mixed with activated charcoal and allowed to develop, here we mixed thawed L1s and added them to activated charcoal pre-mixed with uninfected hamster feces, providing the thawed L1s with a robust, more natural fecal environment in which to develop. After 7 days of larval development on activated charcoal, the iL3s were recovered by the Baermann technique. Using this technique we found that for A. ceylanicum and N. americanus, respectively, 31% and 16% of the total L1s (independent of whether alive or dead) plated on charcoal developed to the iL3 stage (Fig. 3F; Supplementary Fig. S1). Given that ~50% of the L1s appeared alive (Fig. 3E), ~30–60% of the live L1s were then able to develop to the iL3 stage using these culture conditions.

3.3. iL3s recovered from L1s frozen for up to 3 years are able to regenerate the hookworm life cycle

Ancylostoma ceylanicum iL3s recovered using fecal-charcoal plates mentioned above were tested for their ability to complete the life cycle in hamsters (Fig. 4). The infectivity of iL3s from an active infection (non-frozen), iL3s recovered from L1s frozen for 15 months, and iL3s recovered from L1s frozen for 36 months, were used to infect hamsters. Based on intestinal hookworm burdens, there was no difference in the infectivity of iL3s from fresh L1 cultures, from 15 month frozen L1 cultures, or from 36 month frozen L1 cultures (Fig. 4A; P=0.77 and 0.27, respectively, using one-way ANOVA and Dunnett’s post-test comparing each frozen group with fresh iL3s). Similarly, based on fecal egg counts, there was no difference whether infecting with iL3s from fresh L1 cultures, from 15 month frozen L1 cultures, or from 36 month frozen L1 cultures (Fig. 4B; P=0.76 and 0.20, respectively, using one-way ANOVA and Dunnett’s post-test comparing each frozen group with fresh iL3s).

Fig. 4.

Fig. 4.

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Infectivity of Ancylostoma ceylanicum infective L3s (iL3s) recovered from frozen L1 samples. (A) Total intestinal A. ceylanicum hookworm burdens of hamsters infected with iL3s recovered from charcoal plates seeded with feces from ongoing infection (fresh) or from charcoal-fecal plates seeded with cryopreseved L1s frozen for the length of time indicated. The average worm burdens were 24.2, 21.5, and 17.8 for fresh iL3s, 15-month frozen iL3s, and 36-month frozen iL3s, respectively. (B) Eggs per gram of feces from the same animals in A. The average eggs per gram of feces were 2467, 2158, and 1642 for fresh iL3s, 15-month frozen iL3s, and 36-month frozen iL3s, respectively. Error bars are S.E.M.

We also compared the infectivity of N. americanus iL3s from fresh L1s (active infection) and from L1s frozen for 36 months. In both cases, hookworm adults were found in the small intestinal tract of all hamsters, although there were noticeably fewer hookworms when infecting with iL3s from frozen L1 cultures (Fig. 5A; 65% reduction; P=0.014 using student’s t-test). In both cases, parasite eggs were also found in the feces of all hamsters, although there were noticeably fewer hookworm eggs when infecting with iL3s from frozen L1 cultures (Fig. 5B; 72% reduction; P=0.0038 using student’s t-test). Nonetheless, the infection using iL3s from L1s frozen for 3 years was robust.

Fig. 5.

Fig. 5.

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Infectivity of Necator americanus infective L3s (iL3s) recovered from frozen L1 samples. (A) Total intestinal N. americanus hookworm burdens of hamsters infected with iL3s recovered from charcoal plates seeded with feces from ongoing infection (fresh), from charcoal-fecal plates were seeded with cryopreseved L1s frozen for 36 months, or from charcoal plates seeded with feces from the first generation following recovery from cryopreservation. The experiment on the right side of the graph was done at a separate time from the other two. The average worm burdens from left to right were 15.7, 6.4, and 17.0. (B) Eggs per gram of feces from the same animals in A. The average eggs per gram of feces were from left to right 3407, 940, and 3286. Error bars are S.E.M.

We harvested parasite eggs from hamsters infected with iL3s generated from frozen L1s. Strikingly, when these first-generation eggs were allowed to progress to the iL3 stage and used to infect hamsters, infectivity was normal (Fig. 5A, B). Thus, we were able to fully resuscitate a healthy N. americanus lifecycle even after 3 years of cryopreservation.

3.4. The protocol can be generalized to S. ratti and H. p. bakeri

We maintain S. ratti in laboratory rats. The S. ratti lifecycle in rats is similar to that of N. americanus in hamsters. Feces from infected rodent hosts containing parasite eggs can be cultured on activated charcoal until the iL3 stage, at which point they can be administered subcutaneously into naive rodent hosts, allowing completion of the life cycle. Although S. ratti and N. americanus are phylogenetically distant and appear in different clades of the phylum Nematoda (clade IV and clade V, respectively; (Parkinson et al., 2004)), we tested whether or not the cryopreservation protocol could be applied to S. ratti. Strongyloides ratti L1s were similarly frozen as for hookworms and thawed after 2 weeks. Compared with L1s freshly isolated from feces (Fig. 6A), L1s recovered from frozen culture appeared healthy (Fig. 6B). Upon thawing, L1s were grown on activated charcoal mixed with uninfected rat feces until the iL3 stage. Compared with iL3s from a fresh infection cycle (Fig. 6C), iL3s derived from frozen L1s were also very healthy (Fig. 6D). On average, 73±5% of thawed L1s appeared alive (based on morphology and motility) and 24±4% of L1s plated on fecal-charcoal mixture developed to iL3s. When introduced into rats, the infectivity of iL3s cultured from frozen L1s was similar to that from iL3s cultured from fresh L1s, based on fecal egg counts (Fig. 6E; P=0.78).

Fig. 6.

Fig. 6.

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Successful cryopreservation of Strongyloides ratti and Heligmosomoides polygyrus bakeri. (A) Morphology of S. ratti L1s following hatching off in S medium of parasite eggs freshly isolated from infected feces. (B) Morphology of S. ratti L1s thawed from stocks frozen for 2 weeks. Many of the L1s are curved, motile, and alive. (C) Morphology of S. ratti infective L3s (iL3s) harvested from charcoal culture seeded with feces from an active infection. (D) Morphology of S. ratti iL3s cultured from L1s frozen for 2 weeks on charcoal seeded with rat feces. Scale bar is the same in all panels. (E) Infectivity of S. ratti iL3s isolated from charcoal culture plates seeded with fresh L1s (iL3) or frozen L1s based on fecal egg counts. The average fecal egg counts were 1475 and 1342 eggs per gram of feces, respectively. (F) Infectivity of H. polygyrus bakeri iL3s isolated from charcoal culture plates seeded with fresh L1s (iL3) or frozen L1s based on fecal egg counts. The average fecal egg counts were 6840 and 3770 eggs per gram of feces, respectively. Statistically there is no difference between the two conditions (P=0.22 Student’s t-test). Error bars are S.E.M.

We also maintain H. p. bakeri in the laboratory. This natural parasite of mice is perhaps the most common model for immunological studies of gastrointestinal nematode parasites – mammalian host interaction (Reynolds et al., 2012). Similar to S. ratti, we were able to successfully infect with H. p. bakeri iL3s derived from L1s frozen for 2 weeks and cultured on activated charcoal mixed with uninfected mouse feces (Fig. 6F).

4. Discussion

We have demonstrated a robust method for cryopreservation of iL3s from both general human hookworms (A. ceylanicum, N. americanus), the threadworm S. ratti, and the model gastrointestinal nematode parasite H. p. bakeri. For hookworms, successful recovery and reintroduction of the parasite into host hamsters was demonstrated for at least 3 years after freezing. To our knowledge, this is the first successful demonstration of freezing and recovery for this length of time and for these nematodes.

The key to this technique is the culturing of thawed (previously frozen) L1 parasites in charcoal-fecal mixture in which the feces come from uninfected hosts (hamsters for hookworms, rats for S. ratti, and mice for H. p. bakeri). The growth of L1s to iL3s under these conditions is more robust and closer to the natural cycle than, for example, growth on a monoculture of bacteria (e.g., Nolan et al., 1994). Conversely, our attempts at infection with frozen iL3s resulted in poor infectivity for A. ceylanicum and no recovery of live hookworms for N. americanus.

Successful cryopreservation and recovery of gastrointestinal nematode parasites should provide a significant boost to the field of gastrointestinal nematode studies. There are significant challenges associated with studies of these parasites. Currently, most of these parasites have to be continually maintained in mammalian hosts, which is a significant burden. This burden can be additionally increased due to external events, e.g., as many laboratories discovered during COVID. Frozen stocks provide an important sense of security and obviate the need to continually maintain cultures during times when that is difficult.

Helminths parasites such as hookworms can also show great strain variability at the genomic level, whereas the populations maintained in the laboratory are relatively small. Thus, we have found that sometimes laboratory populations can bottleneck and become unhealthy and difficult to maintain. In these instances, frozen stocks can allow successful recovery of such lines. Genotypes can also change over time and it may be desirable to preserve a population at a current genotype, which can be returned to in the future with frozen stocks. As hookworms for immunotherapies move towards Good Manufacturing Practice (GMP) production, methods for long-term preservation of hookworms are also critically important for continued human experimental infection and therapy.

In summary, we provide here a detailed and validated methodology for long-term preservation of human hookworm lines and other gastrointestinal nematode parasites that should greatly facilitate and result in significant expansion of research in vital studies of these parasites.

Supplementary Material

1
2

Highlights.

  • We developed a new technique for recovery of cryopreserved gastrointestinal nematode larvae.

  • Successful long-term (>3 year) cryopreservation and resuscitation of human hookworm lines were achieved in vivo.

  • Our method resulted in successful cryopreservation and resuscitation of both Ancylostoma and Necator genera hookworms.

  • We also achieved successful cryopreservation and resuscitation of Strongyloides and Heligmosomoides.

Acknowledgements

This work was financially supported by the National Institutes of Health (USA) - National Institute of Allergy and Infectious Diseases grants R01-AI056189, R01-AI150866, and R21-AI149037 and by the National Institutes of Health - Eunice Kennedy Shriver National Institute of Child Health & Human Development grant 1R01-HD099072 to R.V.A.

Footnotes

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References

  1. Aikens LM, Schad GA, 1989. Radiolabeling of Infective Third-Stage Larvae of Strongyloides stercoralis by Feeding [75 Se]Selenomethionine-Labeled Escherichia coli to First- and Second-Stage Larvae. J. Parasitol 75 (5), 735–39. [PubMed] [Google Scholar]
  2. Bartsch SM, Hotez PJ, Asti L, Zapf KM, Bottazzi ME, Diemert DJ, Lee BY, 2016. The Global Economic and Health Burden of Human Hookworm Infection.” PLoS Negl. Trop. Dis 10 (9), e0004922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chapman PR, Giacomin P, Loukas A, McCarthy JS, 2021. Experimental Human Hookworm Infection: A Narrative Historical Review. PLoS Negl. Trop. Dis 15 (12), e0009908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chylinski C, Cortet J, Sallé G, Jacquiet P, Cabaret J, 2015. Storage of Gastrointestinal Nematode Infective Larvae for Species Preservation and Experimental Infections. Parasitol. Res 114 (2), 715–20. [DOI] [PubMed] [Google Scholar]
  5. Duarte J, Harrison LM, Cappello M, 2003. Short Report: Ancylostoma ceylanicum: Exsheathment Is Not Required for Successful Cryopreservation of Third Stage Hookworm Larvae. Am. J. Trop. Med. Hyg 68 (1), 44–45. [PubMed] [Google Scholar]
  6. Fumagalli M, Pozzoli U, Cagliani R, Comi GP, Bresolin N, Clerici M, Sironi M, 2010. The Landscape of Human Genes Involved in the Immune Response to Parasitic Worms. BMC Evol. Biol 10 (1), 264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fumagalli M, Sironi M, Pozzoli U, Ferrer-Admetlla A, Pattini L, Nielsen R, 2011. Signatures of Environmental Genetic Adaptation Pinpoint Pathogens as the Main Selective Pressure through Human Evolution. PLoS Genet. 7 (11), e1002355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Haldeman MS, Nolan MS, Ng’habi KRN, 2020. Human Hookworm Infection: Is Effective Control Possible? A Review of Hookworm Control Efforts and Future Directions. Acta Trop. 201, 105214. [DOI] [PubMed] [Google Scholar]
  9. Hamory J, Miguel E, Walker M, Kremer M, Baird S, 2021. Twenty-Year Economic Impacts of Deworming. Proc. Nat. Acad. Sci. USA 118 (14), e2023185118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hawdon JM, Schad GA, 1991. Long Term Storage of Hookworm Infective Larvae in Buffered Saline Solution Maintains Larval Responsiveness to Host Signals. J. Helminthol. Soc. Wash 58, 140–42. [Google Scholar]
  11. Hu Y, Georghiou SB, Kelleher AJ, Aroian RV, 2010. Bacillus thuringiensis Cry5B Protein Is Highly Efficacious as a Single-Dose Therapy against an Intestinal Roundworm Infection in Mice. PLoS Negl. Trop. Dis 4 (3), e614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hu Y, Miller MM, Derman AI, Ellis BL, Gomes Monnerat R, Pogliano J, Aroian RV, 2013. Bacillus subtilis Strain Engineered for Treatment of Soil-Transmitted Helminth Diseases. Applied Environ. Microbiol 79 (18), 5527–32. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hu Y, Miller M, Zhang B, Nguyen T-T, Nielsen MK, Aroian RV, 2018. In Vivo and in Vitro Studies of Cry5B and Nicotinic Acetylcholine Receptor Agonist Anthelmintics Reveal a Powerful and Unique Combination Therapy against Intestinal Nematode Parasites. PLoS Negl. Trop. Dis 12 (5), e0006506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hu Y, Nguyen T-T, Lee ACY, Urban JF Jr, Miller MM, Zhan B, Koch DJ, Noon JB, Abraham A, Fujiwara RT,Bowman DD, Ostroff GR, Aroian RV, 2018. Bacillus thuringiensis Cry5B Protein as a New Pan-Hookworm Cure. Int. J. Parasitol. Drugs Drug Resist 8 (2), 287–94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hu Y, Zhan B, Keegan B, Yiu YY, Miller MM, Jones K, Aroian RV, 2012. Mechanistic and Single-Dose in Vivo Therapeutic Studies of Cry5B Anthelmintic Action against Hookworms. PLoS Negl. Trop. Dis 6 (11), e1900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jimenez Castro PD, Mansour A, Charles S, Hostetler J, Settje T, Kulke D, Kaplan RM, 2020. Efficacy Evaluation of Anthelmintic Products against an Infection with the Canine Hookworm (Ancylostoma caninum) Isolate Worthy 4.1F3P in Dogs. Int. J. Parasitol. Drugs Drug Resist 13, 22–27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jimenez Castro PD, Venkatesan A, Redman E, Chen R, Malatesta A, Huff H, Zuluaga Salazar DA, Avramenko R, Gilleard JS, Kaplan RM, 2021. Multiple Drug Resistance in Hookworms Infecting Greyhound Dogs in the USA. Int. J. Parasitol. Drugs Drug Resist 17, 107–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jourdan PM, Lamberton PHL, Fenwick A, Addiss DG, 2018. Soil-Transmitted Helminth Infections. Lancet 391 (10117), 252–65. [DOI] [PubMed] [Google Scholar]
  19. Li H, Abraham A, Gazzola D, Hu Y, Beamer G, Flanagan K, Soto E, Rus F, Mirza Z, Draper A, Vakalapudi S, Stockman C, Bain P, Urban JF Jr., Ostroff GR, Aroian RV, 2021. Recombinant Paraprobiotics as a New Paradigm for Treating Gastrointestinal Nematode Parasites of Humans. Antimicrob. Agents Chemother 65 (3), e01469–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Loukas A, Hotez PJ, D., Yazdanbakhsh M McCarthy JS, Correa-Oliveira R, Croese J, Bethony JM, 2016. Hookworm Infection. Nature Rev. Dis. Primers 2, 16088. [DOI] [PubMed] [Google Scholar]
  21. Loukas A, Maizels RM, Hotez PJ, 2021. The Yin and Yang of Human Soil-Transmitted Helminth Infections. Int. J. Parasitol 51 (13–14), 1243–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Maizels RM, Pearce EJ, Artis D, Yazdanbakhsh M, Wynn TA, 2009. Regulation of Pathogenesis and Immunity in Helminth Infections. J. Exp. Med 206 (10), 2059–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mes THM, Eysker M, Ploeger HW, 2007. A Simple, Robust and Semi-Automated Parasite Egg Isolation Protocol. Nature Protoc. 2 (3), 486–89. [DOI] [PubMed] [Google Scholar]
  24. Montaño KJ, Cuéllar C, Sotillo J,. 2021. Rodent Models for the Study of Soil-Transmitted Helminths: A Proteomics Approach. Front. Cell. Infect. Microbiol 11, 639573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mourão Dias Magalhães L, Silva Araújo Passos L, Fujiwara RT, Lacerda Bueno L, 2021. Immunopathology and Modulation Induced by Hookworms: From Understanding to Intervention. Parasite Immunol. 43 (2), e12798. [DOI] [PubMed] [Google Scholar]
  26. Nolan TJ, Bhopale VM, Megyeri Z, Schad GA, 1994. Cryopreservation of Human Hookworms. J. Parasitol 80 (4), 648–50. [PubMed] [Google Scholar]
  27. Noon JB, Schwarz EM, Ostroff GR, Aroian RV, 2019. A Highly Expressed Intestinal Cysteine Protease of Ancylostoma ceylanicum Protects Vaccinated Hamsters from Hookworm Infection. PLOS Negl. Trop. Dis 13(4), e0007345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Parkinson J, Mitreva M, Whitton C, Thomson M, Daub J, Martin J, Schmid R, Hall N, Barrell B, Waterston RH, McCarter JP, Blaxter ML, 2004. A Transcriptomic Analysis of the Phylum Nematoda. Nature Genet. 36 (12), 1259–67. [DOI] [PubMed] [Google Scholar]
  29. Pullan RL, Smith JL, Jasrasaria R, Brooker SJ, 2014. Global Numbers of Infection and Disease Burden of Soil Transmitted Helminth Infections in 2010. Parasites Vectors 7, 37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Reynolds LA, Filbey KJ, Maizels RM, 2012. Immunity to the Model Intestinal Helminth Parasite Heligmosomoides polygyrus. Sem. Immunopathol 34 (6), 829–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ryan SM,. Eichenberger RM, Ruscher R, Giacomin PR, Loukas Al., 2020. Harnessing Helminth-Driven Immunoregulation in the Search for Novel Therapeutic Modalities. PLoS Pathog. 16 (5), e1008508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shepherd C, Wangchuk P, Loukas A, 2018. Of Dogs and Hookworms: Man’s Best Friend and His Parasites as a Model for Translational Biomedical Research.” Parasites Vectors 11 (1), 59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sulston J, Hodgkin J, 1988. Methods. In: Wood WB, (Ed.), The Nematode Caenorhabditis Elegans, Cold Spring Harbor Laboratory Press, New York. [Google Scholar]
  34. Umbrello G, Pinzani R, Bandera A, Formenti F, Zavarise G, Arghittu M, Girelli D, Maraschini A, Muscatello A, Marchisio P, Bosis S, 2021. Hookworm Infection in Infants: A Case Report and Review of Literature. Ital. J. Pediat 47 (1), 26. [DOI] [PMC free article] [PubMed] [Google Scholar]

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