Progress 01/01/03 to 12/31/03
Outputs
Infective larvae (L3i) of Strongyloides stercoralis use signals from the environment and from potential hosts to position themselves to increase chances of successful infection. These larvae are, thus, attracted to warmth, an aqueous extract of canine skin, and 0.5 M sodium chloride. When placed on a sodium chloride gradient, L3s of S. stercoralis are attracted to a concentration of 0.05M NaCl. If placed on a NaCl gradient at the lowest concentration, they migrate toward higher concentrations, but turn back when reaching 1.2M. Upon reaching that region of the gradient where the concentration is 0.05M, they continue crawling in circles, as this is their "preferred" NaCl concentration Based on the studies of C. elegans, we hypothesized that the attraction to lower concentrations of NaCl might be mediated by the neuron class ASE in S. stercoralis. Larvae in which the ASE neuron pair was ablated were not attracted to the "preferred" NaCl concentration. Instead, 79.2 +/--
5.9% of the operated larvae migrated randomly on the gradient. Using a quadrant-assay system, we have confirmed that L3s of S. stercoralis have a preference for sodium chloride (0.05M) in comparison to other chloride salts. A similar preference for other sodium salts indicates that the attractive response to sodium chloride (0.05M) is due to the presence of sodium ions. When S. stercoralis L3 are placed on a sodium chloride gradient at a concentration of 2.4M, the larvae are repelled by this concentration, migrating rapidly and directly to that part of the gradient where the concentration is 0.05M. Operated larvae migrated randomly when placed on the salt gradient. It was suggested that neurons ASE and ASH mediate attraction to and repulsion from sodium chloride specifically The infective larva of parasitic nematodes is widely considered to be the life-stage equivalent of the dauer larva of C. elegans. Thus, homologs of the ASJ neuron pair in S. stercoralis and/or the dog hookworm
Ancylostoma caninum might mediate the resumption of larval development of either parasite upon its entry into a suitable host. These neurons would also mediate the resumption of feeding under in vitro host-like conditions which initiate parasitic development. Ablation of the ASJ neurons in S. stercoralis causes a small, but statistically significant, reduction in this feeding response, but fails to cause any reduction in feeding by A. caninum infective larvae. On entering a host, infective larvae of skin-penetrating nematode parasites are exposed to a temperature change, and a new chemical environment. Consequently, in S. stercoralis L1s, we ablated the lamellar cell neurons, known to be thermoreceptive and possibly chemoreceptive, as well, along with the ASJ pair. When these two neuron classes were ablated, approximately half of the operated larvae failed to feed. We have been unable so far to identify those neurons involved in this process in the dog hookworm, A. caninum.
Impacts
The treatment of most infections by parasitic nematodes is curative rather than preventive. The threadworm Strongyloides stercoralis is a medically important skin penetrating intestinal parasite infecting canines and primates including man. The parasite is capable of reproducing within the host, causing long-enduring low-grade infections, which may be exacerbated by immunosupressive therapy or poor host condition. Our aim is to investigate the infective process, with the long-term goal of developing prophylactic procedures that will prevent infection.
Publications
- Ligas, J.A., Kerepesi, L.A., Galioto, A.M., Lustigman, S., Nolan, T.J., Schad, G.A. and Abraham, D. 2003. Specificity and Mechanism of Immunoglobulin M (IgM)- and IgG-Dependent Protective Immunity to larval Strongyloides stercoralis in Mice. Infection and Immunity. 71: 6835-6843.
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Progress 01/01/02 to 12/31/02
Outputs
Infective larvae (L3) of Strongyloides stercoralis use signals from the environment and potential hosts to position themselves to increase chances of successful infection. These larvae appear to show negative geotactic behavior, as do L3s of the dog hookworm, Ancylostoma caninum. S. stercoralis L3s are stimulated by low concentrations of CO2, and are attracted to warmth, and to an aqueous extract of canine skin. In studies to identify the active component of this skin, we are employing anion-exchange chromatography. Chromatography shows that the active material has a weak negative charge, while gel filtration indicates that the molecular weight is less than 1000 daltons. The material is highly polar, as indicated by its insolubility in nonaqueous solvents and minimal retention on reverse-phase HPLC. These characteristics are consistent with (though not limited to) carbohydrates, glycosylated amino acids, or small glycopeptides. We are continuing to fractionate the
material by normal-phase chromatography. We will characterize the purified material both by spectroscopy and by enzymatic digestion to determine the structural basis for its biological activity. When placed on a sodium chloride gradient, L3s of S. stercoralis are attracted to a concentration of 0.5M NaCl, but strongly avoid a concentration of 2.4M. Based on the studies of the free-living nematode Caenorhabditis elegans, we hypothesize that the attraction to lower concentrations of NaCl might be mediated by the neuron class ASE, and the avoidance of higher concentrations by neuron class ASH, thus providing an assay system to elucidate the function of these neurons. When we ablated the neuron pair ASH with a laser microbeam, the avoidance reaction to the high concentrations of NaCl was abolished, thus strongly suggesting that the function of the ASH neurons in S. stercoralis is similar to that of their homologs in C. elegans, namely chemorepulsion. The infective larva of parasitic
nematodes is widely considered to be the life-stage equivalent of the dauer larva of C. elegans. Under this hypothesis, homologs of the ASJ neuron pair in S. stercoralis and/or A. caninum might mediate the resumption of development of either parasite upon entry into a suitable host. They would also mediate the feeding response under in vitro conditions which signal resumption of development.. We have ablated the ASJ neuron pair, in combination with chemosensory or thermosensory neurons in S. stercoralis L3s, causing a small, but statistically significant, reduction in this feeding response, but, so far, have failed to inhibit feeding in L3s of A. caninum.
Impacts
(N/A)
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
Infective larvae (L3) of Strongyloides stercoralis, being parasites of warm blooded hosts, are attracted to warmth, and are stimulated by the concentration of CO2 (3 - 4%) found in the breath of homeotherms. Higher concentrations (5%), however, immobilize these larvae. Infective larvae of Ancylostoma caninum show similar behavior. When placed on a sodium chloride gradient, L3s of S. stercoralis are attracted to a concentration of 0.5M NaCl, and strongly avoid a concentration of 2.4M, the larvae migrating directly to that part of the gradient where the concentration is 0.5M. If the larvae are placed on a NaCl gradient at the lowest concentration, they migrate toward higher concentrations, but turn back when reaching a concentration of 1.2M. Upon reaching that region of the gradient where the concentration is 0.5M, they continue crawling in circles, as this is their "preferred" concentration of NaCl. wWe hypothesize that the attraction to lower concentrations of NaCl
might be mediated by the neuron class ASE, and the avoidance of higher concentrations by neuron class ASH, thus providing an assay system to elucidate the function of these neurons. S. stercoralis L3s, and those of A. caninum, as well, will migrate up an agarose cone. Conversely, these larvae will not migrate down the side of a hollow agarose cone, if the temperatures at the top and bottom of the cone are the same. If, however, the temperature at the bottom of the cone is only slightly higher, larvae will migrate down. Thermotactic behavior overrides geotactic behavior. It is known that only a few pairs of chemosensory and thermosensory neurons control important developmental processes in the life cycle of this nematode. When conditions become adverse, larvae of C. elegans enter an environmentally resistant form known as the dauer larva. A specific neuron pair, known as ASJ, mediates resumption of development when conditions again become favorable. The infective larva of parasitic
nematodes is considered by some to be the life-stage equivalent of the dauer larva of C. elegans. Homologs of the ASJ neuron pair in S. stercoralis might mediate the resumption of development of the parasite upon entry into a suitable host. They would also mediate the feeding response under in vitro conditions which signal resumption of development. Ablation of the ASJ neuron pair causes a small, but significant, reduction in this feeding response. On entering a host, infective larvae of skin-penetrating nematode parasites are exposed to a temperature change, and a new chemical environment. For these reasons, we ablated the so-called lamellar cell neurons, known to be thermoreceptors, and possibly chemoreceptors as well, along with the ASJ pair. When these two neuron classes are ablated approximately half of the operated larvae fail to feed. If the lamellar cells, the thermosensory neurons, are ablated, larvae raised at 36C develop to adults, as they would at 26C. If larvae are held
at 26C for 4 - 6 hours, then placed at 36C, they develop to adults, as their direction of development is already determined.
Impacts
(N/A)
Publications
- Chun-chi Wang, Thomas J. Nolan, Gerhard A. Schad, and David Abraham. 2001. Infection of mice with the helminth Strongyloides stercoralis suppresses pulmonary allergic responses to Ovalbumin. Clinical and Experimental Allergy 31 (3): 495-503.
- Alejandro J. Krolewiecki, Shalom Leon, Phillip A. Scott, Thomas J. Nolan, Gerhard A. Schad, and David Abraham. 2001. Effect of chronic ethanol consumption on protective T-Helper 1 and T- Helper 2 immune responses against the parasites Leishmania major and Strongyloides stercoralis in mice. Alcoholism: Clinical and Experimental Research 25 (4):571-578.
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Progress 01/01/98 to 12/31/98
Outputs
Neuronal control of development: Neurons ADF, and ASI of Caenorhabditis elegans (C.e.) control development to either a dauer larva (=homogonic development) or to an adult (=heterogonic development). Using a map of the neuronal cell bodies, DIC-microscopy, and a laser microbeam, homologs of these neurons in Strongyloides stercoralis (S.s.) were killed in attempts to switch larvae from heterogonic to homogonic development. Hatchling larvae and embryonated eggs were obtained from donor dogs via jejunostomies. When both neuron pairs ASF and ASI were killed with the laser microbeam in these larvae, 60 - 80% developed directly to the infective stage, while if only one pair was ablated, only 12% became infective larvae, not significantly different from the number in the control larvae.Infective third stage larvae (L3) migrate to an aqueous extract of dog skin., A 3000 MW fraction of this extract was used to test ablation of ASE; with neuron ASF as a control. In several
experiments, many of the ASE-ablated larvae did not migrate to the extract, while others did so slowly. The ASF-ablated worms, as well as the unoperated controls, migrated to the attractant rapidly, as expected. We are attempting to fractionate this attractant to determine the active component. S. s. third stage infective larvae will migrate rapidly to the warm end of a thermal gradient, as would be expected for a parasite of warm blooded hosts. In C. e., the finger cell neuron is the primary thermosensor. While S. s. has no finger cell, it does have a neuron which ends in a complex of lamellae. Preliminary experiments indicate that this lamellar cell (ALD)is, indeed, a thermosensor. These experiments are continuing. We are also testing third stage larvae of the stomach worm of sheep, Haemonchus contortus, and the hookworm of dogs, Ancylostoma caninum, both of which have true finger cells, in the thermal gradient. Our ablation work involves identification of neuron ASJ (which controls
resumption of development in C. e) and whose death in S.s. we predict will block resumption of development as judged by resumed feeding of L3 under host-like conditions. Identification of the cell body of neuron ASJ, which lies close to the esophagal bulb, has proven difficult. If this organ is damaged during ablation of the cell bodies, the operated larvae will not survive. Indeed, there has been a high mortality in larvae in which the ASJ neuronal cell bodies have been ablated. We are refining our technique. Autoinfection studies in the gerbil model: One of the questions we asked with the gerbil model was: Is the autoinfective larvae (L3a) a precociously developing homogonic larvae or a separate subset of third-stage larvae? We used a highly homogonic strain and a highly heterogonic strain of S. stercoralis and at 21 days post-infection (PI) placed the infected gerbils on Lomotil to stop gut motility (a strategy known to allow the development of autoinfection). At necropsy on day 24
PI the 2 groups of gerbils contained the same number of adult worms but the group which received the homogonic strain had significantly more L3a, suggesting that the L3a were precocious homogonic larvae.
Impacts
(N/A)
Publications
- Ashton, F.T., Bhopale, V.M., Holt, D., Smith, G., and Schad, G.A. 1998. Developmental switching in the parasitic nematode Strongyloides stercoralis is controlled by the ASF and ASI amphidial neurons. J. Parasitol. 84:691-695.
- Brigandi, R.A., Rotman, H.L., Leon, O, Nolan, T.J., Schad, G.A., and Abraham, D. 1998. Strongyloides stercoralis host-adapted third-stage larvae are the target of eosinophil-associated immune-mediated killing in mice. J. Parasitol. 84:440-445.
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