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The study of parasitic nematodes often requires maintenance of the life cycle by sequential laboratory passage in permissive hosts. In the case of hookworms, as well as other intestinal nematodes, eggs are cultured from the feces to infectious third (L3) stage larvae, which are used to infect subsequent mammalian hosts.1–3 Standard methods of culturing hookworm larvae include the Baermann and Harada-Mori methods, each of which has been modified in various ways to enhance yield or ease of maintenance.4–7 An important advantage of the Baermann method, which involves culturing feces mixed with charcoal for 5–15 days, includes the high yield of viable larvae that are relatively free of debris. Limitations of this method include the fact that each individual sample requires a separate apparatus, which is generally used for repeated cultures, making it difficult to adapt for use in large-scale laboratory or epidemiologic studies. In contrast, the Harada-Mori technique uses filter paper strips coated with feces and stored upright in tubes containing a small volume of water to maintain moisture. Advantages of this method are that it does not require additional organic material (i.e., charcoal) and that individual sample tubes can be incubated together in large numbers. A disadvantage of the Harada-Mori technique is that the fecal cultures must be monitored closely to prevent desiccation caused by evaporation. Importantly, neither of these methods, because they both involve culturing larvae from feces, allow for the biochemical, molecular, or nutritional characterization of hookworm development under experimentally defined conditions.
To determine conditions necessary for successful development of the hookworm Ancylostoma ceylanicum, we compared the growth kinetics and infectivity of larvae reared using the Baermann, Harada-Mori, or a recently developed solid phase agar plate (AP) method. The A. ceylanicum life cycle was maintained in male Syrian hamsters of the Lak: LVG(SYR)BR outbred strain (Harlan) as previously described,8 and all animal experimentation was carried out using protocols approved by the Yale Animal Care and Use Committee. Baermann cultures were prepared by mixing feces from laboratory-infected hamsters with bone charcoal (Ebonex, Melvindale, MI) and incubating in covered dishes for 7–14 days at 26–28°C. At that time, the mixtures were placed in a plugged funnel apparatus filled with warm (37–40°C) water.9–11 The third-stage (L3) larvae were allowed to migrate out of the fecal mixture, into the water, and were collected at the bottom of the funnel. The Harada-Mori12 cultures were prepared by applying 0.5-g aliquots of infected hamster feces in a thick smear to individual 1-cm-wide strips of filter paper and placed into open 15-mL conical tubes. Sterile water was added to the bottom of each tube so that the meniscus was ~0.5 cm below the fecal mass. The volume of liquid in each tube was monitored daily and adjusted as needed, and larvae were collected from the bottom of the tubes on Day 7.
The third technique consisted of a modified AP method similar to that used to maintain laboratory strains of the free-living nematode Caenorhabiditis elegans and recently adapted to the animal nematode parasite Parastrongyloides trichosuri.13–15 Feces were collected from A. ceylanicum–infected hamsters, and eggs were concentrated using a protocol modified from that reported by Kotze and others.16 Briefly, 10–30 g of feces were suspended in 100 mL 0.9% NaCl and filtered through gauze. After centrifugation at 500g, the pellet containing eggs was washed in 40 mL of detergent (0.015% brij-35 in dH2O; Sigma, St Louis, MO), followed by a second wash in 40 mL sodium nitrate (2 mol/L). After centrifugation at 500g, the first 10 mL of supernatant containing the hookworm eggs were decanted into 200 mL of distilled water. The egg suspension was further clarified by sequential passage through 80- and 20-µm nylon mesh membranes (Millipore, Billerica, MA). Agar plates containing nematode growth media (NGM)17 (US Biologicals, Swampscott, MA) supplemented with carbenicillin (50 µg/mL) were prepared and allowed to dry. Liquid cultures of the HT115-DE3 strain of E. coli containing a plasmid conferring resistance to carbenicillin were maintained at 37°C under selective conditions in lysogeny broth (LB) with shaking until log phase growth (OD600, 0.4–0.6). Approximately 200 A. ceylanicum eggs were added to the surface of the NGM plates with 0.5 mL of the live bacterial culture and incubated at 27°C. Similar plates were also prepared using heat-killed (HK) bacteria or LB broth alone.
The three hookworm culture systems were maintained at 27°C for 7 days. For those larvae cultured using the AP method, daily worm counts were recorded, and the mean lengths of representative samples of larvae (mean, 11 worms/group) were measured by digital light microscopy. After 7 days of incubation, the larvae were collected from the Harada-Mori and Baermann cultures and counted, and the lengths of larvae were recorded. Using analysis of variance (ANOVA; see Figure 1
legend for statistical methods), we identified a statistically significant interaction between culture condition and time as factors affecting worm length (P < 0.0001). As shown in Figure 1A, larvae maintained on NGM plates in the presence of live HT115-DE3 E. coli grew from a mean length of 144 ± 5 µm on Day 1 to 604 ± 7 µm by Day 7 of incubation, a statistically significant 320% increase in size (P < 0.01). Notably, after a plateau in the growth from Days 4 to 6, the mean larval length increased significantly from Day 6 to Day 7 (P < 0.001), which coincides with a molt from the L2 to L3 stage.
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The differences in mean length at Day 7 between larvae cultured with live HT115-DE3 and those cultured with either HK bacteria or no bacteria were both statistically significant (P < 0.001; Figure 1B
). However, there was no significant difference in larval length between worms cultured using the AP method with live HT115 (604 ± 7 µm) and those harvested from Baermann (621 ± 10 µm) or Harada-Mori (582 ± 16 µm) fecal cultures. Larvae grown using the Baermann, Harada Mori, and AP techniques were also indistinguishable on inspection by light microscopy (Figure 2). Furthermore, the yield of each method was determined by dividing the total number of living larvae recovered at Day 7 by the estimated number of eggs added to the original culture. Although the three methods studied resulted in the harvest of viable A. ceylanicum L3, there were differences noted in the yields of each method. The AP and Baermann techniques of culture were comparable, with yields of 47% (850 larvae from 1,820 eggs) and 50% (33,750 larvae from 67,500 eggs), respectively. In contrast, the Harada-Mori method was found to have the lowest yield (100 larvae from 4,800 eggs; 2.1% yield) of the three techniques studied. These data suggest that the AP method is comparable in yield to Baermann but has the advantage of allowing for more precise manipulation of culture conditions, either through modifications of NGM components or introduction of alternative bacterial strains.
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It is not clear whether this difference in yield or efficiency of infection is caused by reduced fitness from physiologic stress of larvae cultured using the AP method. Of note, there was no statistically significant difference in the sex ratio (male/female) of adult worms harvested from infected animals in either larval culture group, nor were gross morphologic differences noted between adult worms harvested from the two groups, based on assessment by light microscopy (data not shown). Viable and infectious larvae were also obtained from the Day 20 PI culture of feces from animals infected with AP-reared L3. These data showed that L3 cultured by the AP method establish patent infections that can be maintained for at least two generations in a normally permissive host.
Although agar plate–based methods have been described for use with parasitic nematodes, these techniques have been evaluated primarily for diagnosis of infection or testing of anthelminthics.16,18,19 The AP method described here complements and extends this work by showing the feasibility of plate-based culture techniques in defining aspects of the development and pathogenesis of a hookworm species for which humans are permissive hosts.20–22 An additional advantage of this method is that it allows for analysis of larval development of human parasitic nematodes under more defined conditions than are currently feasible using standard coproculture techniques. In addition, the AP method also allows for periodic sampling of larvae for analysis of growth and the developmentally regulated expression of specific genes or proteins. Because developing larvae require live bacteria as a food source, the AP method described here also establishes the potential to use genetically defined or modified E. coli as a vector for delivering specific proteins or other factors (e.g., dsRNA), to evaluate effects on parasite development. This method also allows for the screening of conditions or factors that are essential to development, especially those that might be derived from the environment or host23 and represent potential drug or vaccine targets.24,25 Last, because this method yields viable and infectious L3, targeting specific biologic phenomena (e.g., gene expression/silencing) during development can be assessed in terms of the impact on infection and disease pathogenesis in vivo. We anticipate that the AP method will complement currently available techniques for studying the biology of hookworms and other globally important parasitic nematodes.
Received May 7, 2007. Accepted for publication August 15, 2007.
Acknowledgments: The authors thank Lisa DiFedele, Keke Fairfax, and George Porter for technical assistance and Michael Stern for providing NGM plates.
Financial support: This work was supported by NIH Grants AI58980, AI47929, and AI57855.
* Address correspondence to Michael Cappello, Yale Child Health Research Center, 464 Congress Avenue, New Haven, CT 06520. E-mail: michael.cappello{at}yale.edu 
Authors’ addresses: Daniel Reiss, Lisa Harrison, Richard Bungiro, and Michael Cappello, 464 Congress Avenue, New Haven, CT 06520. Tel: 203-737-4320, Fax: 203-737-5972, E-mail: Michael.cappello{at}yale.edu.
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