Ancylostoma caninum: the finger cell neurons mediate thermotactic behavior by infective larvae of the dog hookworm.

Bhopale, V. M., Kupprion, E. K., Ashton, F. T., Boston, R., and Schad, G. A. 2001. Ancylostoma caninum: The finger cell neurons mediate thermotactic behavior by infective larvae of the dog hookworm. Experimental Parasitology 97, 70-76. In the amphids (anteriorly positioned, paired sensilla) of the free-living nematode Caenorhabditis elegans, the so-called finger cells (AFD), a pair of neurons, each of which ends in a cluster of microvilli-like projections, are known to be the primary thermoreceptors. A similar neuron pair in the amphids of the parasitic nematode Haemonchus contortus is also known to be thermoreceptive. The hookworm of dogs, Ancylostoma caninum, has apparent structural homologs of finger cells in its amphids. The neuroanatomy of the amphids of A. caninum and H. contortus is strikingly similar, and the amphidial cell bodies in the lateral ganglia of the latter nematode have been identified and mapped. When the lateral ganglia of first-stage larvae (L1) of A. caninum are examined with differential interference contrast microscopy, positional homologs of the recognized amphidial cell bodies in the lateral ganglia of H. contortus L1 are readily identified in A. caninum. The amphidial neurons in A. caninum were consequently given the same names as those of their apparent homologs in H. contortus. It was hypothesized that the finger cell neurons (AFD) might mediate thermotaxis by the skin-penetrating infective larvae (L3) of A. caninum. Laser microbeam ablation experiments with A. caninum were conducted, using the H. contortus L1 neuronal map as a guide. A. caninum L1 were anesthetized and the paired AFD class neurons were ablated. The larvae were then cultured to L3 and assayed for thermotaxis on a thermal gradient. L3 with ablated AFD-class neuron pairs showed significantly reduced thermotaxis compared to control groups. The thermoreceptive function of the AFD-class neurons associates this neuron pair with the host-finding process of the A. caninum infective larva and shows functional homology with the neurons of class AFD in C. elegans and in H. contortus.

Strongyloides stercoralis: oral transfer of parasitic adult worms produces infection in mice and infection with subsequent autoinfection in gerbils.

Oral transfer of parasitic adult Strongyloides stercoralis produced patent infections in gerbils, C57BL/6J and SCID mice. In gerbils receiving adult worms, 7.3% of the transferred worms established and autoinfective L3 were found beginning on day 5 post-transfer, with peak numbers seen on days 6 and 7 post-transfer and few seen by 9 days post-transfer. These results suggest that development of autoinfective L3 in the gerbil is limited by the immune response of the host. When given orally to mice, between 7.2% (C57BL/6J) and 19.5% (SCID) of the adult worms established. These levels are higher than those previously obtained by the subcutaneous infection of SCID mice with infective larvae. No autoinfective larvae were found in infected mice and the ratio of L1/adult worms was small compared with that seen in gerbils. Thus, mice infected orally can be used as a model to study the interaction between the adult worm and the host, and since autoinfection has not been seen in the murine model, as developed to date, orally infected mice may be useful as a model to study mechanisms preventing autoinfection.

Hyperinfective strongyloidiasis: Strongyloides stercoralis undergoes an autoinfective burst in neonatal gerbils.

One of the unusual aspect of the life cycle of Strongyloides stercoralis is the occurrence of autoinfective third-stage larvae (L3a). These are the causative agents of severe hyperinfective strongyloidiasis. When 6-wk-old gerbils are infected with 1,000 infective third-stage larvae (L3i), no L3a are seen during the course of the infection. However, in neonatal gerbils (1-13 days of age) infected with 1,000 L3i, a burst of autoinfection takes place between 15 and 30 days postinfection (PI). Only occasional L3a can be found in neonatally infected gerbils after 4 wk PI. This autoinfective burst is not seen in neonatal gerbils infected with 200 L3i.

Developmental switching in the parasitic nematode Strongyloides stercoralis is controlled by the ASF and ASI amphidial neurons.

Parasitic nematodes of the genus Strongyloides are remarkable for their ability to switch between alternative free-living developmental pathways in response to changing internal environmental conditions. After exiting the host, soil-dwelling larval stages may develop either to infectivity via 2 microbiverous stages (homogonic development) or to free-living adulthood via 4 microbiverous larval stages (heterogonic development). The progeny of these adults then give rise to the infective stage. In the latter case, free-living existence is extended in time and the number of infective larvae is greatly amplified. Anterior chemosensory neurons (amphidial neurons) are thought to respond to environmental cues and via signal transduction pathways control the direction of larval development. We now demonstrate by laser microbeam ablation that 2 classes of amphidial neurons (ASF and ASI), acting together, control the direction of free-living larval development. Larvae in which the neurons were killed developed to infectivity via the homogonic route rather than to adulthood via the otherwise predominant heterogonic route. These neurons are probable homologues of neurons ADF (=ASF) and ASI in Caenorhabditis elegans, suggesting the control of development at the cellular level is conserved among divergent taxa of nematodes. These observations also have important implications for the evolution of nematode parasitism and the design of new prophylactic measures against parasitic nematodes of medical and veterinary medical importance.

Barren female Strongyloides stercoralis from occult chronic infections are rejuvenated by transfer to parasite-naive recipient hosts and give rise to an autoinfective burst.

It is widely assumed that barren Strongyloides stercoralis occurring in chronically infected carriers can become fecund when immunity wanes. Evidence for this involves corticosteroid treatment of hosts harboring occult infections that subsequently return to patency. However, nematodes have ecdysteroid receptors, and it has been suggested that corticosteroids act directly on the parasite, inducing autoinfective development, rather than indirectly by suppressing host immunity. To test these competing concepts, barren females were recovered from donor dogs when the dogs' fecal examinations turned negative. Groups of 100 active barren worms were surgically transplanted into the small intestines of each of 6 naive canine recipients. Three were examined at necropsy at 4-5 days postinfection (PI), before autoinfection could amplify the number of successfully transferred parasites. The remaining recipients were examined 21-22 days PI when, if autoinfection had occurred, the worm populations should have increased. At 4-5 days, gravid worms occurred in each of the recipients (19 +/- 6 worms/dog). By 21-22 days, a remarkable population increase had occurred (522.6 +/- 296 worms/dog). Worms from chronically infected donors were stunted, and electron microscopy revealed damage to the intestine and ovaries. Successfully transplanted worms recovered at days 4-5 PI were ovigerous and less stunted and showed repair of intestinal and ovarian tissues.

Passive transfer of immune serum reduces the L1/adult ratio of worms recovered from the intestines of Strongyloides stercoralis-infected gerbils.

In Strongyloides stercoralis-infected gerbils it had been observed previously that the ratio of first-stage larvae to adult worms decreases after 3 wk of infection. Serum obtained from gerbils after this decrease in fecundity, but before worms were expelled, was transferred passively to other infected gerbils during the peak of worm fecundity. The immune serum caused a significant decrease in the L1/adult ratio but had no effect on the number of uterine eggs, the length, or the intestinal and ovarian ultrastructure of adult worms. Apparently a factor(s) in the immune serum either killed eggs, after oviposition, or the hatched larvae, without damaging the adult worms.

Sensory neuroanatomy of a skin-penetrating nematode parasite Strongyloides stercoralis. II. Labial and cephalic neurons.

Host recognition, contact, and skin-penetration by Strongyloides stercoralis infective larvae are crucially important behavioral functions mediating transition from free-living to parasitic life. The sensilla of the worm's anterior tip presumably play an important role in these processes. Besides the main chemosensilla, the amphids, which are of central importance, the larva has 16 putative mechanosensilla. There are six inner labial sensilla: two dorsal, two ventral, and two lateral. The two dorsal and ventral pairs are each innervated by two neurons, whereas each lateral sensillum is singly innervated. The six outer labial and four cephalic sensilla are all singly innervated. All of these have the characteristics of mechanoreceptors: they are closed to the external environment, and closely associated with the overlying cuticle. Distally, their dendritic processes contain granular material and associated microtubules. With two exceptions, the relevant neuronal cell bodies lie in lateral ganglia adjacent to the nerve ring, their positions remarkably similar to those of their homologues in the free-living nematode, Caenorhabditis elegans. Cell bodies of two neuronal pairs, one of two dorsal inner labial neurons and one of two ventral inner labial neurons per side, are however, found far anterior to the remaining cell bodies. All labial and cephalic sensilla are apparently mechanoreceptors, complementing the well-developed chemosensilla. Presumably infective larvae require touch and stretch receptors, not only to initiate skin penetration by finding irregularities as points of access, but also to bore through tissue to reach their ultimate enteral destination.

Immunity to a challenge infection of Strongyloides stercoralis third-stage larvae in the jird.

Jirds support the entire life-cycle of Strongyloides stercoralis. We therefore used this host as a model to define the mechanism of the immune response to a challenge infection, as well as the parasite stage effected by the response. Jirds given a primary infection of S. stercoralis are resistant to re-infection. The use of implanted diffusion chambers containing larvae showed that the immune response killed the third-stage larvae, and this was confirmed by subcutaneous infections. The larvae of a challenge infection are killed within 48 h, a time period too short to allow for the development of L4 and adult worms. The immune response is dependent on both a serum factor and cells, suggestive of an ADCC type response.

Sensory neuroanatomy of a skin-penetrating nematode parasite: Strongyloides stercoralis. I. Amphidial neurons.

The Strongyloides stercoralis infective larva resumes feeding and development on receipt of signals, presumably chemical, from a host. Only two of the anterior sense organs of this larva are open to the external environment. These large, paired goblet-shaped sensilla, known as amphids, are presumably, therefore, the only chemoreceptors. Using three-dimensional reconstructions made from serial electron micrographs, amphidial structure was investigated. In each amphid, cilialike dendritic processes of 11 neurons extend nearly to the amphidial pore; a twelfth terminates at the base of the amphidial channel, behind an array of lateral projections on the other processes. A specialized dendritic process leaves the amphidial channel and forms a complex of lamellae that interdigitate with lamellae of the amphidial sheath cell. This "lamellar cell" is similar to one of the "wing cells" or possibly the "finger cell" of Caenorhabditis elegans. Each of the 13 amphidial neurons was traced to its cell body. Ten neurons, including the lamellar cell, connect to cell bodies in the lateral ganglion, posterior to the nerve ring. The positions of these cell bodies were similar to those of the amphidial cell bodies in C.elegans. Therefore, they were named by using C. elegans nomenclature. Three other amphidial processes connect to cell bodies anterior to the nerve ring; these have no homologs in C. elegans. A map allowing identification of the amphidial cell bodies in the living worm was prepared. Consequently, laser ablation studies can be conducted to determine which neurons are involved in the infective process.

Cryopreservation of human hookworms.

First-stage larvae (L1) of the human hookworms Ancylostoma duodenale and Necator americanus were frozen over liquid nitrogen after a 1-hr incubation in a cryoprotectant (10% DMSO and 10% dextran). Thawed larvae developed to the infective third stage (L3) on agar plates. In the case of A. duodenale, the larvae were infective to dogs. The infectivity of N. americanus L3 was not tested.

Strongyloides stercoralis: the first rodent model for uncomplicated and hyperinfective strongyloidiasis, the Mongolian gerbil (Meriones unguiculatus).

Strongyloidiasis is the most common endemic helminthiasis in several of the world's industrialized nations, yet relatively little is known about its basic biology and immunobiology because a practical rodent model for the investigation of this clinically important parasitism is lacking. This study reports such a model for use in the investigation of Strongyloides stercoralis infection. Normal male gerbils infected subcutaneously with 1000 infective filariform larvae harbored moderate numbers (83.6 +/- 27.6) of adult worms at 35 days after infection, and a low-grade infection persisted for at least 131 days mimicking the chronicity of human infections. Gerbils treated weekly with 2 mg of methylprednisolone acetate developed hyperinfective strongyloidiasis with up to 8000 autoinfective larvae occurring in these animals at postinfection day 21. Autoinfection never occurred in normal (untreated) gerbils.

Strongyloides stercoralis: an initial autoinfective burst amplifies primary infection.

Compartmental analysis of Strongloides stercoralis burdens in experimentally infected, serially necropsied dogs was used to test an autoinfective burst hypothesis. The hypothesis states that in well-established, active infections and in chronic infections as well, the rate of larval development is down-regulated so that most larvae do not attain infectivity internally. The majority pass in the feces as preinfective, rhabditiform larvae, but a few (those with the most rapid developmental rate) attain infectivity internally, and therefore are positioned for autoinfectivity. In contrast, in immunologically naive hosts, larval development proceeds without host hindrance and many larvae, proceeding at the most rapid rate of a spectrum of normal intrinsic developmental rates, attain infectivity internally. For a brief period, hyperinfection occurs, during which the adult worm population increases sharply. Gut-level resistance soon occurs, larval development is retarded, and an increasing proportion of larvae are discharged as preinfective rhabditiform larvae. With fewer larvae developing to infectivity internally, recruitment into the adult population decreases, with an attendant increase in the mean age and a gradual decrease in the size of the adult population. The data and the attendant model strongly support this autoinfective burst hypothesis.

Developmental biology and migration of Strongyloides ratti in the rat.

Most recent authors suggest that larvae of the threadworm, Strongyloides ratti, migrate from a cutaneous infection site to the small intestine via the naso-frontal region of the head. However, the proportion of larvae that successfully reach the intestine having followed this pathway had not been determined. Using compartmental analysis we have obtained a comprehensive quantitative description of larval migration during a primary infection of rats with S. ratti. Mean residence times in organs through which larvae migrate were calculated and the proportion of the larvae arriving in the small intestine via the head was estimated as 0.5-0.86, depending on the mathematical model used to generate the estimate. With 50% or more of successful larvae using this pathway it is now reasonable to recognize the "head route" as an important migratory pathway for larvae traveling to their intestinal predilection site. During the first 12 hr in the host, larvae were difficult to recover from the skin and subjacent muscles of the infection site. Apparently, upon invading a host, many larvae enter a quiescent phase during which Baermannization, an active recovery technique, fails to recover them. Infections were short-lived, with the daily finite mortality rate for adult worms attaining 50-80% on day 16 of infection.