Predisposition to hookworm infection in humans.

Frequency distributions of parasitic helminths within human communities are invariably highly aggregated, the majority of worms occurring in relatively small fractions of the host populations. It has been suggested that the heavily infected individuals are predisposed to this state, not by chance, but by as yet undefined genetic, ecological, behavioral, or social factors. Analyses of individual post-treatment patterns of hookworm reinfection among 112 villagers in an endemic area of West Bengal provide quantitative evidence of predisposition to heavy infection. This observation has implications for the design of control programs based on chemotherapy because of the potential economic advantage of selective or targeted treatment as opposed to mass or blanket treatment.

Arrested development in human hookworm infections: an adaptation to a seasonally unfavorable external environment.

Contrary to general belief, larvae of Ancylostoma duodenale do not always develop directly to adulthood upon invasion of man. In West Bengal, India, arrested development appears to be a seasonal phenomenon which results in (i) reduction of egg output wasted in seeding an inhospitable environment and (ii) a marked increase in eggs entering the environment just before the monsoon begins.

Sensory neuroanatomy of a passively ingested nematode parasite, Haemonchus contortus: amphidial neurons of the first stage larva.

When infective larvae of Haemonchus contortus (a highly pathogenic, economically important, gastric parasite of ruminants) are ingested by grazing hosts, they are exposed to environmental changes in the rumen, which stimulate resumption of development. Presumably, resumption is controlled by sensory neurons in sensilla known as amphids. Neuronal function can be determined by ablation of specifically recognized neurons in hatchling larvae (L1) in which neuronal cell bodies are easily visualized using differential interference microscopy. Using three-dimensional reconstructions from electron micrographs of serial transverse sections, amphidial structure of the L1 is described. Each amphid of H. contortus is innervated by 12 neurons. The ciliated dendritic processes of 10 neurons lie in the amphidial channel. Three of these end in double processes, resulting in 13 sensory cilia in the channel. One process, that of the so-called finger cell, ends in a number of digitiform projections. Another specialized dendrite enters the amphidial channel, but leaves it to end within the sheath cell, a hollow, flask-shaped cell that forms the base of the amphidial channel. Although not flattened, this process is otherwise similar to the wing cells in Caenorhabditis elegans; we consider it AWC of this group. Two other neurons, ASA and ADB, appear to be homologs of wing cells AWA and AWB in C. elegans, although they end as ciliated processes in the amphidial channel, rather than as flattened endings seen in C. elegans. Each of the 12 amphidial neurons was traced to its cell body in the lateral ganglion, posterior to the worm's nerve ring. The positions of these bodies were similar to their counterparts in C. elegans; they were named accordingly. A map for identifying the amphidial cell bodies in the living L1 was prepared, so that laser microbeam ablation studies can be conducted. These will determine which neurons are involved in the infective process, as well as others important in establishing the host-parasite relationship.

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.

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.

Thermotaxis and thermosensory neurons in infective larvae of Haemonchus contortus, a passively ingested nematode parasite.

As a basis for studies of thermal behavior of infective larvae (L3) of Haemonchus contortus resulting from ablation of amphidial neurons, the locations of the amphidial cell bodies in the hatchling larva (L1) were compared with their locations in the L3. We sought to verify that killing each targeted cell body in L1 destroys the putative corresponding dendrite of the L3. These comparisons confirmed the predicted cell body-to-dendrite connections, as well as similarities in the general amphidial structure of the two stages. We then conducted a series of studies using laser microbeam ablation of amphidial cell bodies in the L1 to determine the role of specific neurons in the thermal behavior of the L3. In a thermal gradient, normal L3 of H. contortus migrate to the temperature at which they were cultured and/or maintained. Larvae grown at 16 degrees or 26 degrees C migrate appropriately to either of these temperatures. Larvae grown to the L3 stage at 16 degrees C and then moved to 26 degrees C become acclimated to this temperature and thereafter migrate to it. However, when the putative thermosensory neurons, the finger cell neurons (AFD), were ablated in hatchling larvae with a laser microbeam, and these were grown to the L3 stage and tested on a radial thermal gradient, they failed to migrate to their culture temperature. Instead, they moved actively and continuously over much of the assay plate surface, with no obviously oriented cryo- or thermotactic movement. Ablation-control larvae, those in which putatively chemosensory neuron classes ASE or AWC were killed, migrated normally to their culture temperature. When the RIA interneurons (identified by positional homology with those of Caenorhabditis elegans) were ablated, the operated larvae moved actively, but circled near the initial placement point; control larvae, in which other nonamphidial neurons were killed, migrated normally. These results indicate that the finger cell neurons (AFD) are the primary thermosensory class in H. contortus. The RIA-class neurons integrate thermal responses in H. contortus, as do their putative structural homologs in C. elegans, but the behavior of H. contortus subsequent to RIA ablation is strikingly different.

Strongyloides stercoralis: histopathology of uncomplicated and hyperinfective strongyloidiasis in the Mongolian gerbil, a rodent model for human strongyloidiasis [corrected].

Tissues from corticosteroid-treated gerbils hyperinfected with Strongyloides stercoralis were compared grossly and microscopically to similar tissues from animals with uncomplicated strongyloidiasis. Gerbils with hyperinfection developed severe pulmonary alveolar haemorrhage with a variable degree of subacute eosinophilic interstitial pneumonia associated with numerous alveolar, vascular and interstitial larvae. Hyperinfection induced by corticosteroids, given either before inoculation of S. stercoralis larvae or after a chronic Strongyloides infection was established, produced similar lesions. In contrast, lungs from gerbils with uncomplicated Strongyloides infection had severe eosinophilic perivasculitis and vasculitis with very little haemorrhage, no pneumonia and no larvae. Sections of adult worms were present in the proximal part of the intestinal tract, lodged in spaces between mucosal epithelial cells. Adult worms were not associated with inflammation and were more common in the corticosteroid-treated gerbils. In corticosteroid-treated gerbils only, there were numerous larvae in the distal intestinal tract, throughout the intestinal wall and adjacent mesentery, within interstitial tissues and in lymphatic vessels. Significant inflammation with associated larvae was only present in the caecum and mesenteric lymph nodes, suggesting that the caecum was the main site for initiation of parenteral migration with subsequent invasion of the lymphatic system and lungs. The lesions in these gerbils were similar to those found in humans. Infection of gerbils with S. stercoralis is the best rodent model of human strongyloidiasis.

The neurons of class ALD mediate thermotaxis in the parasitic nematode, Strongyloides stercoralis.

Strongyloides stercoralis, a skin-penetrating nematode parasite of homeotherms, migrates to warmth. In nematodes, the amphids, anteriorly positioned, paired sensilla, each contain a bundle of sensory neurons. In the amphids of the free-living nematode Caenorhabditis elegans, a pair of neurons, each of which ends in a cluster of microvilli-like projections, are known to be the primary thermoreceptors, and have been named the finger cells (class AFD). A similar neuron pair in the amphids of the parasite Haemonchus contortus is also known to be thermosensory. Strongyloides stercoralis lacks finger cells but, in its amphids, it has a pair of neurons whose dendrites end in a multi-layered complex of lamellae, the so-called lamellar cells (class ALD). Consequently, it was hypothesised that these lamellar cells might mediate thermotaxis by the skin-penetrating infective larva of this species. To investigate this, first stage S. stercoralis larvae were anaesthetised and the paired ALD class neurons were ablated with a laser microbeam. The larvae were then cultured to the infective third stage (L3) and assayed for thermotaxis on a thermal gradient. L3 with ablated ALD class neuron pairs showed significantly reduced thermotaxis compared with control groups. The thermoreceptive function of the ALD class neurons (i) associates this neuron pair with the host-finding process of S. stercoralis and (ii) demonstrates a functional similarity with the neurons of class AFD in C. elegans. The structural and positional characteristics of the ALD neurons suggest that these neurons may, in fact, be homologous with one pair of flattened dendritic processes known as wing cells (AWC) in C. elegans, while their florid development and thermosensory function suggest homology with the finger cells (AFD) of that nematode.

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.

Amphidial neurons ADL and ASH initiate sodium dodecyl sulphate avoidance responses in the infective larva of the dog hookworm Anclyostoma caninum.

Ablations of specific amphidial neuron pairs with a laser microbeam were conducted to understand better the neurological basis of the behaviours of larval parasitic nematodes. To date, the functions of the amphidial neurons of Caenorhabditis elegans and their counterparts in parasitic nematodes have been found to be remarkably conserved allowing the possibility to predict the relationships between neurons and their functions. Therefore, we anticipated that ablation of neuron pairs ASH and ASK would abrogate avoidance of sodium dodecyl sulphate (SDS) by infective larvae (L3i) of Anclyostoma caninum. Instead, we have found that laser microbeam ablation of these neuron pairs did not eliminate SDS avoidance in A. caninum, but that neuron pairs ASH and ADL are the amphidial neurons responsible for SDS repulsion. When a droplet of the repellent is placed in the direct path of a normal A. caninum L3i, a strong backward avoidance response is triggered. However, when the ASH and ADL neurons are ablated, the nematodes demonstrate the opposite reaction, increasing their movement in a forward direction.

Strongyloides stercoralis infection in IgA-deficient dogs.

Two litters of eight-week-old, IgA-deficient pups, each including one normal littermate control, were infected with 5,000 infective third-stage Strongyloides stercoralis larvae per pup. No significant differences between the dogs deficient in serum and mucosal IgA and the normal control dogs were observed in any of the parasitologic parameters measured during the course of infection. The time required to reach patency, the maximum number of larvae shed in the feces, the number of days postinfection at which larval shedding was at its maximum, and the time required to eliminate shedding were similar in the two groups. Once larval shedding ceased, there was no recrudescence, even though serum and fecal IgA were absent or low throughout the seven-month course of the investigation. All dogs remained asymptomatic, with complete blood counts and clinical chemistries within normal ranges. In addition, serum IgM and IgG levels were within the normal range for both groups of infected dogs. All dogs from which necropsy samples were obtained harbored low numbers of adult female worms, some of which were barren. An IgA deficiency apparently does not affect the course or severity of S. stercoralis infection in the dog.

Arrested development in Ancylostoma duodenale: course of a self-induced infection in man.

The course of self-induced infection with about 100 Ancylostoma duodenale larvae was followed by twice-weekly blood and stool examinations. After a small initial increase, no further change in eosinophil counts occurred until the 33rd week of infection when they began to increase sharply. Seven weeks later, eggs were first observed in stools. Thus, in this infection, the prepatent period was five times the expected. It is suggested that a strain of A.duodenale exists which either 1) has an inherent, abnormally long prepatent period or 2) has a prepatent period which is susceptible to change depending upon the environmental conditions experienced by the free-living larvae. In either case, the third stage larvae probably enter a hypobiotic state within the host which lasts for about 8 months.

Population biology of hookworms in children in rural West Bengal. I. General parasitological observations.

Hookworm infection was studied over a 22-month period in 31-49% of a population of 1,803 1- to 10-year-old children in a rural area near Calcutta where both Necator americanus and Ancylostoma duodenale were prevalent. Half of the children were infected before age 5 and 90% were infected by age 9, when the mean egg count reached 2,000 eggs/g of feces. Infection was significantly heavier and more prevalent among males than among females, and greater among Muslims than among Hindus. These differences were apparent in children less than 2 years of age. Numerical factors were devised to adjust fecal egg counts for both the smaller fecal output of children and the increased dilution of eggs in watery feces as compared to formed feces. It is proposed that egg counts from 1- to 3-year-olds be multiplied by 0.3, those from 4- to 6-year-olds by 0.5, those from 7- to 9-year-olds by 0.6, and those from 10 to 12-year-olds by 0.7; differences in mean egg density among various fecal consistencies produced factors of 1, 1.5, 2, 3, and 3.5 by which the egg counts in formed, mushy-formed, mushy, mushy-diarrheic, and diarrheic feces should be increased.

Population biology of hookworms in children in rural West Bengal. II. Acquisition and loss of hookworms.

From changes in hookworm egg counts in feces of children in Gangetic West Bengal we estimate that, on the average, each child lost about 11.1 female hookworms during the 1969 post-monsoon dry period and gained about 10.3 females during the subsequent premonsoon and monsoon seasons, resulting in a net loss of worms over the 22-month study period. However, the mean fecal egg count for each 1-year age group of children from 3-11 years suggests an average net gain of 2.7 female worms/year. From seasonal changes in the relative abundance of larvae recovered from fecal cultures, it is estimated that at least 82% of the yearly gain in Ancylostoma duodenale females is a result of the maturation of hypobiotic (latent) larvae acquired during the previous wet season. A duodenale was present about equally with Necator americanus but it was significantly more common among males than among females, and more common among Muslims than among Hindus. Using damp gauze pads, we sampled the soil surface around freshly-passed feces for infective hookworm larvae and estimated that 252 larvae were contacted by each child durint the transmission season. Efficiency of penetration and maturation of each hookworm species was calculated from estimated larval densities on the soil surface, from seasonal changes in fecal egg counts, and from variations in the number of larvae recovered from fecal cultures by season and age of the host; it is estimated that N. americanus is 17.5% efficient and A. duodenale 5.3%. Children negative for hookworm became positive at all times of the year and at a significantly higher rate in 1970 than in 1969; incidence averaged 4.2% per 2-month period and mean initial fecal egg count suggested infection with about 2.0 female worms. The average life span for N. americanus is calculated to be 3-4 years and that of A. duodenale about 1 year under the conditions of reinfection found in the study area.

Gamma camera scintigraphy for direct visualization of larval migration in Strongyloides stercoralis-infected dogs.

Gamma camera scintigraphy of dogs infected with radiolabeled third-stage larvae of Strongyloides stercoralis was successful for visualizing larval migration in a live host. Ten-day-old pups were infected subcutaneously in a lateral inguinal site with 100,000 75Se-selenomethionine-labeled third-stage S. stercoralis and imaged serially using a clinical gamma camera during the prepatent period. Radioactivity corresponding to the larval inoculum was visualized at the infection site immediately after injection. Radioactivity spread diffusely, corresponding to radial dispersal of larvae from the infection site. The majority of the radioactivity concentrated over or through the abdomen from 48 to 144 hr after infection. Larvae arrived in the small intestine in numbers adequate for visualization beginning at 114 hr after infection. At no time was there concentration of radioactivity associated with the lungs of an infected pup. A control pup injected only with 75Se-selenomethionine showed uniform activity diffusely throughout the entire body at all times. We concluded that the majority of larvae moved to the gut by means other than the pulmonary route.

Chronicity in Strongyloides stercoralis infections: dichotomy of the protective immune response to infective and autoinfective larvae in a mouse model.

Strongyloidiasis is an intestinal disease that can last for decades due to the occurrence of autoinfective larvae (L3a) in an infected person, which contribute to the maintenance of the population of adult worms in the intestine. The goal of the present study was to determine if L3a are susceptible to the protective immunity that targets the infective stage of the worm, the third-stage larvae (L3). Mice immunized and challenged with Strongyloides stercoralis L3 kill more than 90% of challenge larvae contained within diffusion chambers. The L3 do not remain antigenically static in mice, however, but undergo some degree of antigenic change before they are killed, becoming host-activated larvae (L3+). The L3/L3+ are killed in this model system by the combined effects of both parasite-specific IgM and eosinophils. Mice immunized with L3 were able to kill L3/L3+, but did not kill L3a, in challenge infections. Eosinophils were, however, present in diffusion chambers containing L3a, and IgM bound to the surface of L3a. We hypothesized that differential IgM recognition of soluble L3a, L3, and L3+ antigens is the reason why the immune response generated against L3 could not kill L3a. Many common antigens on L3, L3+, and L3a were recognized by serum from mice immunized with L3, as determined by immunoblotting. However, several unique L3, L3+, and L3a antigens were also recognized by immune serum, thus indicating that antigen recognition with IgM antibodies is different between the L3, L3+, and L3a stages. This difference in antigen recognition could explain why L3a are able to evade the immune response that targets L3/L3+ in chronically infected hosts.

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.

The clinical and immunologic responses of normal human volunteers to low dose hookworm (Necator americanus) infection.

Five normal human volunteers were exposed to approximately 50 infective larvae of Necator americanus and were observed for the development of clinical signs or symptoms and for changes in blood eosinophil levels, IgG antibody titers, total and parasite-specific IgE, and lymphocyte blastogenic responses for 6-10 weeks. Bronchoalveolar lavage was performed on four subjects prior to infection and at times when larval migration through the pulmonary tree was likely. Eggs were demonstrated in the stools of four volunteers who remained untreated for more than 6 weeks; one volunteer had to be treated at day 40 because of severe gastrointestinal symptoms. All others also complained of abdominal pain and flatulence between days 35-40. All volunteers developed marked blood eosinophilia which peaked between days 38-64 and ranged from 1,350-3,828 eosinophils/mm3. Small increases in total and parasite-specific IgE and IgG were noted in some volunteers. One volunteer showed a significant lymphocyte blastogenic response. With the exception of mucosal erythema, bronchoalveolar lavage results were unremarkable. Our data indicate that a single small inoculum of hookworm larvae is capable of producing significant transient gastrointestinal morbidity and marked blood eosinophilia but does not induce other prominent T cell- and B cell-dependent immune responses.

Strongyloides stercoralis: maintenance of exceedingly chronic infections.

Two hypotheses were tested to identify the mechanism(s) by which chronic Strongyloides stercoralis infections are maintained in experimental dogs as a model to explain delayed onset recrudescence in humans. Investigations tested the hypotheses that chronic infections result from 1) periodic reactivation of third-stage larvae from a reservoir of dormant parasites outside the gastrointestinal tract or 2) the periodic rejuvenation of postreproductive female worms remaining from a previous infection, lodged in the mucosal crypts. Populations of parenteral larvae survived in mature experimentally infected female dogs for 66 days; individual worms survived for 88 days, but there was no evidence that these larvae re-established patent, adult worm infections. Late in these infections, female worms were present in greater than predicted numbers with no evidence that autoinfection had occurred, suggesting that some postreproductive worms were long-lived. In separate trials, long-lived spent females were once again capable of producing viable larvae when the host was treated with corticosteroids.