Strongyloides stercoralis: a model for translational research on parasitic nematode biology^*

All progeny of free-living S. stercoralis adults are fated to become iL3, which must infect a host in order to develop further (see Figure 1). Consequently, worms of this species are capable of undergoing only one generation of development outside the host and continuous laboratory rearing requires maintenance of host animals. S. stercoralis is a parasite of primates (including humans) and dogs (Schad, 1989). We maintain the strain of S. stercoralis designated UPD (University of Pennsylvania, Dog) as a subclinical infection in purpose-bred, mix-breed laboratory dogs. We typically, infect dogs by inoculating them subcutaneously with 3000 infective third-stage larvae (iL3).

Their ability to tolerate chronic infection with larger numbers of adult worms and their higher output of fecal larvae make dogs the most practical hosts for laboratory maintenance of S. stercoralis. However, the use of such a species for experiments requiring many replicate host infections or the maintenance of numerous parasite strains may be prohibitively expensive and a source of ethical concern. While attempts to infect rats and mice with S. stercoralis have failed, gerbils, Meriones unquiculatus, have been found to be susceptible to both infection and autoinfection (see Figure 1, "aL3") by this parasite. The factors that promote autoinfection in the natural canine or human hosts, immunological immaturity or corticosteroid treatment, do so in the gerbil as well, making this small mammal a valuable model in which to study host-parasite interactions that govern the autoinfective process (Nolan et al., 1993; Kerlin et al., 1995; Nolan and Schad, 1996; Nolan et al., 1999).

The iL3 of S. stercoralis invade the host percutaneously. Convention holds that they migrate via the microvasculature to the lungs where they break out of the capillary bed, enter the alveoli and then proceed to the intestine via the trachea. Work by Schad and colleagues (Schad et al., 1989), however, suggests that the majority of larvae may actually migrate via a variety of other somatic tissues. In any case, the larvae then undergo two molts to the parasitic female, which invades crypts of the intestinal mucosa. The primary symptom of uncomplicated human strongyloidiasis is abdominal pain, mimicking that associated with peptic ulcer, and acute infections are marked by the presence of first stage larvae in fresh fecal specimens and by eosinophilia. As indicated above, S. stercoralis is unique among the parasitic nematodes in its capacity to undertake autoinfection, and immunosuppressed individuals should refrain from working with active cultures of this parasite.

With regard to safety of animal husbandry staff, it is important to reinforce that 1.) S. stercoralis iL3 infect percutaneously, 2.) larvae found in feces in the first 24 hours (L1) are not infectious and 3.) 24 hours at normal room temperature are required for L1 passed in the feces to reach the infective stage via the homogonic route (see Figure 1). Therefore, provided a schedule of daily, thorough cage cleaning is maintained (standard operating procedure in most institutional vivaria), the risk of transmission should be minimal. Staff should wear protective clothing consisting at minimum of disposable gloves, gowns, shoe covers and caps, as well as eye and face protection when cleaning cages. In general, these will not constitute special precautions for maintenance of S. stercoralis, but rather standard procedure for laboratory animal husbandry staff at most institutional laboratory animal operations.

As with animal husbandry operations, laboratory safety measures mandated at most institutions are adequate to protect against infection with S. stercoralis. Although the presence of infective larvae may be predicted with some precision based on the age of the culture and the incubation temperature (Table 1), it must be borne in mind that at least a small proportion of post-parasitic L1 in most S. stercoralis strains develop directly to the iL3 within 24-48 hours of deposition (ie. the homogonic route). Since the exact deposition time of fecal samples is usually uncertain, it is therefore good practice to regard all S. stercoralis cultures, regardless of age as potentially containing infectious parasites. All disposable culture waste should be accumulated in clearly marked biohazard bags and autoclaved prior to discard. Non-disposable glassware, Baermann funnel components (see Protocol 2 below) and other implements contaminated with free-living larvae and adults of S. stercoralis may be decontaminated with a 20% solution of a commercial surgical scrub such as ChlorhexiDerm (2% Chlorhexidine, DVM Pharmaceuticals, Miami, Florida). Surgical scrub should be added to Baermann funnel effluent waste at a similar concentration to kill free-living larvae and adults prior to disposal. Because of their highly resistant cuticles, iL3 are impervious to Chlorhexidine and other conventional surgical scrubs even after several hours of exposure. Implements and solutions containing iL3 can be chemically decontaminated with Lugol's Iodine (10 mg/ml KI + 10 mg/ml I₂ in distilled water).

The challenge in designing preparative culture methods for free-living larvae and adults of S. stercoralis is to simulate the complex nutritional milieu of moist, fecally contaminated soil while preventing anaerobia. The charcoal coproculture method described below (see Protocol 1) accomplishes this and, assuming adequate output of fecal larvae by the host, provides excellent yields of worms. Methods for isolation of S. stercoralis and related parasites from such a chemically and microbiologically complex environment (e.g., the Baermann funnel technique, Protocol 2) generally exploit the natural migratory behaviors of the worms, as do some methods for further purification (the agarose migration technique, Protocol 3). Otherwise, by virtue of their similarity to other soil-dwelling nematodes, many standard cultures methods may be adapted from C. elegans methodology for small-scale analytical culture.

The Baermann funnel is commonly used to isolate small parasitic nematodes from solid materials such as feces, soil or animal tissue. It exploits the tendency of these worms to migrate from a solid into a surrounding liquid medium when stimulated by slightly elevated temperature and then to settle to the substratum (Bowman, 1995). By using an ordinary laboratory funnel to contain the incubating sample, and taking some simple filtration measures, it is possible to collect and concentrate large numbers of worms in small volumes of medium, relatively free of fecal debris.

For applications requiring observation and analysis of smaller populations of worms, such as screening of phenotypes or manual selection of genetic transformants, the free-living stages of S. stercoralis may be cultured on standard NGM/OP50 plates (see Maintenance of C. elegans; Lewis and Fleming, 1995). Since the worms used to inoculate such cultures will be derived from the septic environment of a coproculture, significant carryover of fecal bacteria onto the NGM/OP50 plate is likely. Contamination due to bacteria and fungi external to the worms may be minimized, but not eliminated by either of two approaches: allowing the worms to migrate through a semi-solid matrix of low gelling temperature agar (see Protocol 3) or subjecting them to density gradient centrifugation (see Protocol 4). Since the worms carry fecal bacteria within their guts, neither of these procedures will result in a sterile preparation. To date we have been unscuccessful at adapting the standard alkaline sodium hypochlorite technique used for axenization of C. elegans eggs (see Maintenance of C. elegans; Lewis and Fleming, 1995) to yield viable S. stercoralis eggs in quantity.

The size, transparency and body plan of a free-living S. stercoralis female is so similar to that of a C. elegans hermaphrodite that published techniques for delivery of DNA constructs into C. elegans by gonadal microinjection (see Transformation and microinjection; Mello and Fire, 1995) may be used for this parasite virtually without modification (Li et al., 2006; Lok and Massey, 2002). Persons experienced with gonadal microinjection of C. elegans will succeed almost immediately with S. stercoralis but will notice two relatively minor differences in the two worms. First, the reproductive tracts of free-living S. stercoralis females and C. elegans hermaphrodites are highly similar, both being in the form of a double recurved tube with the vulva placed at mid-body. However, as pictured in Figure 4, the syncytial ovary of S. stercoralis, corresponding to the usual point of injection in C. elegans, is generally narrower and less transparent than that of C. elegans, and individual oocyte nuclei are less visible by DIC microscopy. Second, due perhaps to relative fragility of its cuticle or to a higher internal hydrostatic pressure, S. stercoralis free-living females are somewhat more susceptible than C. elegans to injury by injection of an excessive fluid volume or by large or improperly placed needles. The most frequent manifestation of this is prolapse of the gonad from the injection wound. This problem may be overcome by using needles with an outside tip diameter no greater than 5μm and by keeping the injection pressure under 20 pounds per square inch.

Early attempts to transform S. stercoralis involved a series of constructs in which various C. elegans and S. stercoralis promoters were fused to the coding sequence of gfp in standard C. elegans vectors from the vector kit maintained previously by A. Fire and colleagues (Stanford University). These constructs incorporated either of two commonly used C. elegans 3′ UTRs, that of unc-54 or of let-858. Invariably, transgene constructs incorporating C. elegans 3′ UTRs were expressed in apparently unregulated fashion in degenerating embryos of S. stercoralis and never in normally developing F1 larvae (Li et al., 2006; Lok and Massey, 2002). Regulated, cell- and tissue-specific transgene expression was not achieved until both 5′ and 3′ regulatory sequences derived from the parasite were incorporated. For example, as with all such constructs, gfp expression under the control of the S. stercoralis era-1 promoter in a standard C. elegans vector background (pPD 95.81) is always of the unregulated type described above. In contrast to this, substitution of the endogenous Ss era-1 3′ UTR for the C. elegans unc-54 3′ UTR in the original vector is sufficient to bring about tissue-specific expression in normally developing F1 larvae (Li et al., 2006). Sequence comparison reveals that the endogenous Ss era-1 3′ UTR, which gives regulated expression, contains three canonical polyadenylation signals whereas the previously used C. elegans unc-54 element, which gives unregulated expression, contains only one (Li et al., 2006). This signal alone may be insufficient to properly terminate message transcription in S. stercoralis. The Ss era-1 3′ UTR is also sufficient to give regulated expression of gene constructs incorporating other S. stercoralis promoters (see Table 2). In combination with this element, the promoter from the cellular actin gene Ss act-2 gives expression primarily in body wall muscle of F1 larvae while that of the Gα protein-encoding gene Ss gpa-3 gives a pattern of expression in sensory neurons remarkably similar to that of its C. elegans ortholog (Jansen et al., 1999). Our group has developed a "toolkit" of S. stercoralis vectors for distribution, which are designed to accept promoters in all reading frames upstream of the gfpS65C coding sequence with artificial introns and the Ss era-1 3′ UTR. This toolkit is available from the author upon request.

For experiments involving smaller numbers of worms, we typically cultivate microinjected free-living female worms along with 1-2 males per female, and their F1 progeny on standard NGM agar plates spread with OP50 bacteria. We have been able to rear isolated P0 transformant females (plus males) and their broods in this manner (Li et al., 2006) as well as pools of up to 30 transformant females and an equal number of males. Carryover of fecal bacteria to the NGM/OP50 plates may be problematic and can be minimized by washing free-living adults from Baermann isolations with two changes of M9 buffer.

For larger cohorts of P0 transformants (100-200 individuals), as would be required for host passage of transgenics, we temporarily pool females as they are microinjected, along with a roughly equal number of males, on NGM/OP50 plates. At the end of a day's injection session, microinjected females and males are washed from the plate with a small volume of M9 buffer and transferred as a pool to a charcoal coproculture dish (see section 4.2.3) made with feces from a non-infected dog. These plates are incubated at 22°C for 5-6 days to allow development to the iL3, which are collected by the Baermann funnel protocol (see section 4.3).

Behavioral or selectable transformation markers have not been developed as yet for S. stercoralis. However, gfp expression under the control of strong promoters such as that of Ss act-2 constitutes a workable visual marker for transformation. Ss act-2p::gfp transformants are easily hand-selected under a stereomicroscope equipped with epifluorescence.

Although reliable protocols for derivation of stable transgenic lines of S. stercoralis are not yet in hand, we have used experimental infections in gerbils to accomplish the following required steps in this process. First, we have shown that relatively small cohorts of transgenic S. stercoralis (i.e., inocula containing 100-200 F1 transformant iL3) can establish patent infections in gerbils and that construct DNA can be maintained in transgenic populations through two host passages with intervening free-living generations in culture to the F5 generation (Li et al., 2006). This initial finding was made with a non-expressed construct, the C. elegans-based pTG96_2, which contains the sur-5 promoter fused to gfp and the unc-54 3′ UTR (Gu et al., 1998), and so host passages had to be carried out without selection. More recent attempts to effect host passage of F1 larvae showing regulated expression of the Strongyloides-based Ss act-2p::gfp construct, pAJ08, have yielded gfp-expressing parasitic females. However no gfp-expressing individuals have been observed among the progeny of these transgenic parasitic females and, thus, we have not yet succeeded in establishing a stable gfp-expressing line.

The tendency of gonadal microinjection to result in the incorporation of transgenes into multi-copy episomal arrays has led to some interest in biolistic DNA transfer as an alternate method of gene delivery facilitating the isolation of stable lines of C. elegans with low-copy chromosomal integrations or homologous gene replacements. Microparticle bombardment has also been used for transient transfection of the adult filariae such as Litomosoides carinii (Jackstadt et al., 1999) and Brugia malayi (Higazi et al., 2002). One of the major impediments to the use of biolistics for heritable transgenesis in parasitic nematodes is the low frequency of germline transformation by this method relative to the number of adult or L4 parasites that may be practicably obtained. The most efficient systems for biolistic transformation of C. elegans allow derivation of one stable transformant line per 20,000 individuals bombarded, and typical bombardments involve 10⁴-10⁵ animals (Praitis et al., 2001). The ease of large-scale C. elegans culture and the availability of powerful, selectable co-transformation markers such as the unc-119 mutant rescue make it relatively straightforward to amass the numbers of worms required for bombardment and to select integrated lines (see Transformation and microinjection). It has been possible for us to prepare cohorts of 5-7 x 10⁴ free-living S. stercoralis adults using the fecal output of two infected dogs and scaled up versions of the charcoal coproculture and Baermann isolation techniques described above (see Protocols 1 and 2). Initial problems with overgrowth of these large cultures by fecal bacteria can be overcome using the percoll gradient method (see Protocol 4) to decontaminate the worms. However, using the PDS/1000 He bombardment apparatus we have been unable, thus far, to detect F1 transformants expressing the Ss act-2p::gfp construct, pAJ08 (Table 2), which has proven so effective in producing F1 transformants by microinjection. A selectable transformation marker, possibly one encoding resistance to an anthelmintic drug, would greatly facilitate the detection of transformants in large populations of bombarded S. stercoralis, and our future efforts in this area will concentrate on this approach.

Microsurgical ablation of cells and observation of resulting developmental and behavioral deficits is a standard approach to the study of cell function in whole organisms. C. elegans' transparent cuticle and the fact that few of its cells are strictly required for survival make microlaser ablation, particularly of neurons, an important part of the methodological repertoire for this model organism (Bargmann and Avery, 1995). The power of this method was recently demonstrated in the elucidation of a complete “.....behavioral circuit for navigation in C. elegans, from sensory input to motor output...” (Gray et al., 2005). As has been the case for microinjection of transgene DNA, we have been able to exploit morphological similarities between C. elegans and S. stercoralis to adapt established techniques for laser cell killing from the model organism to the parasite and have used this method to investigate the neuronal control of various behaviors and developmental events associated with the infective process (Ashton et al., 2007). The method for cell ablation in S. stercoralis detailed in Protocol 5 below is a modification of that reported for C. elegans (Bargmann and Avery, 1995).

The recent successes with transgenesis in strongyloidoid parasites (Grant et al., 2006b; Li et al., 2006) are highly encouraging. However, in the cases of both Strongyloides and Parastrongyloides, some key components of a practical transgenesis system have yet to be identified. We have succeeded in designing a number of reporter constructs S. stercoralis based upon cell- and tissue-specific promoters linked to the in vivo reporters gfp and mrfp. However, it remains for us to establish any of these in a stable transformant line through host passage, and much of our current effort is directed toward this goal. By contrast, stable transgenic lines of P. trichosuri expressing constructs incorporating the (β-GAL reporter system have been established (Grant et al., 2006b), but, to date, constructs that will express gfp or other in vivo reporters have not been reported for this parasite.

The utility of RNA interference (RNAi) as a method of investigating gene function in C. elegans is well substantiated (Fire et al., 1998; Johnson et al., 2005; Tabara et al., 1998; Tavernarakis et al., 2000; Timmons et al., 2001). Robust RNAi effects following natural uptake or electroporation of dsRNA have been observed in the filariae (Aboobaker and Blaxter, 2003; Ford et al., 2005; Hashmi et al., 2004) and the trichostrongyles (Hussein et al., 2002; Issa et al., 2005), parasites occupying Clades III and V, respectively, in the contemporary nematode phylogeny (Blaxter et al., 1998). By contrast, despite concerted efforts, none of the laboratories investigating Strongyloides or Parastrongyloides, members of nematode Clade IV, have been able to demonstrate clear RNAi effects in these parasites following application of dsRNA in various configurations via the common routes of administration. Our own efforts have shown a significant knock down of mRNA encoding the S. stercoralis homolog of unc-54 in the progeny of free-living females microinjected with homologous dsRNA. However, whereas unc-54 shows a strong paralyzed RNAi phenotype in C. elegans, no such phenotype has been seen in S. stercoralis. Among planned approaches to address this problem in the future are combinatorial experiments in which we will attempt to enhance RNAi effects in S. stercoralis by knocking down mRNAs encoding putative orthologs of molecules such as LIN-53, which modulate RNAi effects in C. elegans, simultaneously with targets of interest.

The extensive body of work by Viney and co-workers on S. ratti demonstrates that limited forward genetic approaches may be feasible with the strongyloidoid parasites. For example, they have shown that clonal populations, varying in key developmental traits, may be selected and maintained through host passage, that genetic exchange occurs during mating in the free-living generation (Viney et al., 1993) and that free living populations may be chemically mutagenized, screened for phenotypes of interest and selected for relevant mutations (Viney et al., 2002). The ability of P. trichosuri to undergo multiple free-living generations in culture make it an attractive subject with which to build upon these advancements for further development of a forward genetic system in a parasitic nematode (Grant et al., 2006a).