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Hookworms are blood-feeding intestinal nematodes that currently infect more than one billion people in the developing world.1 For ongoing laboratory studies of hookworm biology, pathogenesis, or vaccine development, it is necessary to maintain the parasite life cycle in a permissive animal host model. This is both labor-intensive and expensive, requiring frequent inoculation of animals and cultivation of hookworm larvae from feces.2 To address the need to maintain a secure and continuous supply of infectious third-stage hookworm larvae (L3), protocols have been developed that allow for the recovery of L3 following long-term cryopreservation in liquid nitrogen.3–5 Similar techniques have been reported for other parasitic nematodes, including Haemonchus contortus and Strongyloides stercoralis.6–8 Interestingly, it has been reported that L3 from the dog hookworm Ancylostoma caninum must first be stimulated to shed or exsheath the cuticle prior to freezing to obtain successful recovery following cryopreservation.3 Later work by Vetter and Klaver-Wessels also found exsheathment to be a requirement for recovery of A. ceylanicum, a hookworm species for which humans are permissive hosts.4 Nolan and others subsequently demonstrated that this exsheathment step was not necessary for successful cryopreservation of first-stage (L1) Ancylostoma hookworm larvae, presumably because this stage does not have a cuticle.5
To test whether exsheathment was an absolute requirement for successful recovery of frozen L3, we compared two protocols for cryopreservation of the hookworm A. ceylanicum. Approximately 20,000 A. ceylanicum L3 recovered from the feces of infected hamsters using the Baermann technique were washed and resuspended in buffered saline.9 All animal experimentation was carried out under a protocol approved by the Yale Animal Care and Use Committee.
On day 2, half of the L3 were resuspended in an exsheathing cocktail (25mM glutathione [Sigma, St. Louis, MO], 10% fetal calf serum [FCS] [Sigma] in standard RPMI 1640 cell culture medium [Invitrogen, Carlsbad, CA])5 and incubated for 16 hours at 37°C in an atmosphere of 5% CO2. The remaining L3 were maintained in buffered saline at 25°C. After incubation, both sets of larvae were washed and resuspended in phosphate-buffered saline.
The two aliquots of L3 (exsheathed and non-exsheathed) were then resuspended in a cryoprotectant solution of 10% dimethyl sulfoxide, 10% dextran, and 10% FCS in RPMI 1640 medium at a concentration of approximately 10,000 L3/mL. After incubation for one hour at 25°C, the larvae were aliquoted into smaller tubes containing approximately 5,000 L3 each. Both sets of larvae were placed at -80°C overnight, and then transferred to liquid nitrogen.
After 13 days frozen in liquid nitrogen, one 5,000-worm aliquot from each set of L3 (exsheathed and non-exsheathed) was thawed in a 37°C water bath for three minutes. After thawing, the larvae were resuspended in 5 mL of RPMI 1640 medium and centrifuged at 150 x g for seven minutes. The volume of RPMI 1640 medium was reduced to 2 mL, and the larvae were then checked for viability. Based on examination of approximately 50 worms from each group, the percentage of motile L3 was slightly greater in the non-exsheathed group (27%) than the exsheathed group (18%), which is in contrast to the findings of previous reports.3,5 To further maximize the percentage of viable L3, a mini-filter apparatus was constructed using a small glass funnel loosely filled with folded laboratory filter paper (Kimwipes®; Kimberly Clark, Roswell, GA) and placed in a 100-mL beaker containing RPMI 1640 medium warmed to 37°C. The larvae were placed in the funnel, and after 20 minutes the fluid in the beaker was examined for L3 that had successfully migrated through the filter paper. This step dramatically increased the percentage of viable L3 because virtually all of the larvae recovered were visibly motile.
To determine whether the non-exsheathed A. ceylanicum L3 were still infectious, two 22-day-old LVG hamsters were inoculated by oral gavage with approximately 100 viable L3 per animal.10,11 On day 19 post challenge, hookworm eggs were detected in the feces of the hamsters, and L3 were successfully isolated using the Baermann technique. On day 20 post-inoculation, the two infected hamsters were killed and eight adult A. ceylanicum were removed from the intestinal mucosa. The recovered L3 from the two hamsters infected with cryopreserved larvae were then used to inoculate three additional naive animals (175 L3/hamster). On day 20-post inoculation, these animals were killed, and 57 adult worms were recovered.
To confirm the reproducibility of this cryopreservation technique, a second aliquot of frozen non-exsheathed L3 was thawed after 100 days in liquid nitrogen. These larvae were filtered as described earlier. Two naive hamsters were inoculated with approximately 50 viable L3 each, and eggs were detected in the feces 18 days later. Adult worms were detected in these animals, and viable L3 were subsequently recovered from fecal cultures as described earlier.
In conclusion, we report a reliable and straightforward protocol for successful cryopreservation of L3 of the human hookworm A. ceylanicum (Figure 1
). There are notable differences between this technique and those previously published for Ancylostoma species. First, we found that subjecting the L3 to specific conditions to stimulate exsheathment of the cuticle is not necessary for successful recovery following cryopreservation. Eliminating this step reduces the number of manipulations required for preparation of L3 prior to freezing. Second, using L3 obviates the need to isolate first-stage larvae from newly hatched eggs,5 which is less efficient and more labor-intensive than the standard Baermann technique. Third, modifying the technique to include a filtering step after thawing the L3 appears to increase the percentage of motile, and presumably viable, larvae that can be administered to a susceptible laboratory animal host. It is hoped that this modified cryopreservation protocol will provide a useful tool for maintaining a reservoir of Ancylostoma L3 for uninterrupted scientific study.
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Received June 4, 2002. Accepted for publication September 25, 2002.
Acknowledgments: We thank Peter Hotez and John Hawdon (George Washington University, Washington, DC) for providing the A. ceylanicum L3, and Richard Bungiro for his thoughtful comments and suggestions during the course of this work.
Financial support: This work was supported by National Institutes of Health grant AI-47929 (Michael Cappello) and a New Investigator Award from the Burroughs Wellcome Fund (Michael Cappello).
Reprint requests: Michael Cappello, Department of Pediatrics, Yale University School of Medicine, 464 Congress Avenue, New Haven, CT 06520, Telephone: 203-737-4320, Fax: 203-737-5972, E-mail: michael.cappello{at}yale.edu
Authors’ addresses: John Duarte, Lisa M. Harrison, and Michael Cappello, Department of Pediatrics, Yale University School of Medicine, 464 Congress Avenue, New Haven, CT 06520.
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