Serum-stimulated feeding in vitro by third-stage infective larvae of the canine hookworm Ancylostoma caninum

Developmentally arrested nonfeeding infective larvae of hookworms resume development after entry into the host, presumably in response to a signal encountered during invasion. Logically, an initial step in the resumption of development might be the resumption of feeding. An in vitro assay for feeding is described for the third-stage larvae of the canine hookworm Ancylostoma caninum. Populations of larvae incubated under hostlike conditions in the presence of 10% canine serum resume feeding within 6 hr, as evidenced by the uptake of fluorescein-labeled bovine serum albumin. Feeding is dependent on the presence of canine serum, and peaks by 24 hr incubation. Maximal feeding levels occur at temperatures above 34 C with a gas phase of 5% CO2/95% air, whereas culture medium and pH are unimportant for feeding. Serum concentrations between 0.1% and 1.0% (v/v) initiate feeding, and the response peaks at approximately 8.0% serum. Serum triggers feeding within 6 hr and is not required for feeding to continue once initiated. The saturation effect and the trigger phenomenon suggest that the initiation of feeding is a receptor-mediated response.     

Albumin and a dialyzable serum factor stimulate feeding in vitro by third-stage larvae of the canine hookworm Ancylostoma caninum.

Previous studies demonstrated that third-stage, developmentally arrested larvae of the canine hookworm Ancylostoma caninum resume feeding in vitro in response to canine serum and hostlike temperature. Experiments to determine the identity of the serum stimulus are described. Serum from several nonhost species stimulated feeding, but to levels lower than canine serum. Heating the serum to 57 C had no effect on its stimulatory ability. Dialysis reduced serum stimulatory activity by 50%, and ultrafiltration through 10- and 30-kDa molecular weight cut-off membranes decreased activity in both the filtrates and retentates similarly. Recombination of the filtrates and retentates restored activity to whole serum control levels. Commercial canine and bovine albumin stimulated feeding to serum control levels at 10 and 50 mg/ml, respectively. These results suggest that albumin and an unidentified low molecular weight compound(s) are capable of inducing in vitro feeding by A. caninum L3.     

Heligmosomoides polygyrus: excretory/secretory antigens released in vitro by exsheathed third-stage larvae

The excretory/secretory antigens released during in vitro culture of infective third-stage Heligmosomoides polygyrus larvae were analyzed by enzyme-linked immunosorbent assay and immunoblotting using sera from repeatedly infected mice. During the first 8-10 hr of culture at 37 C, freshly exsheathed larvae released only one antigen that cosedimented with trypsin (24 kDa) upon ultracentrifugation and was composed of a single 23-kDa polypeptide chain. After 10 hr of culture, the larvae released additional antigens identified by bands equivalent to polypeptides of approximately 18, 25, 26, 32, 58, and 76 kDa on nonreduced Western blots. The release of these molecules was maintained for up to 60 hr. Their staining intensity on blots was in the order 23 much greater than 25 greater than 76 greater than 18 greater than or equal to 58 greater than or equal to 32 greater than or equal to 26 kDa. Velocity sedimentation analysis showed that the 76-kDa component exists as a monomeric 76-kDa native antigen. The 32-, 58-, and 76-kDa antigens were specifically adsorbed by concanavalin A (Con A)-Sepharose and the 76-kDa molecule was detected on blots incubated with alkaline phosphatase-conjugated Con A, indicating the presence of mannose-like residues on these molecules. The 18-, 23-, 25-, and 26-kDa antigens did not bind to Con A-Sepharose. Hyperimmune antisera raised against lyophilized larvae had negligible antibody activity against the larval ES antigens, suggesting that the ES antigens are released soon after synthesis rather than being stored in significant quantities within the larvae.     

Acquired hookworm immunity in the golden hamster (Mesocricetus auratus) elicited by living Necator americanus third-stage infective larvae

The aim of the study is to demonstrate and understand the acquired immunity in golden hamsters (Mesocricetus auratus) elicited by primary Necator americanus infective third-stage larvae (L3) infection. Hamsters infected with 150 L3 for 1, 2, 3, 6 and 10 weeks, were challenged with the same number of L3 and sacrificed 25 days post challenge. The primarily infected hamsters exhibited 99-100% protection against subsequent L3 challenge compared to un-infected naive hamsters. The acquired immunity was developed as early as 1 week post L3 infection and lasted up to 10 weeks. Similar protective immunity was obtained in hamsters infected with N. americanus L3 and then treated orally with a single of 100mg/kg albendazole, followed by challenge with N. americanus L3 4 and 8 weeks post-treatment. The infected hamsters exhibited a rise in IgG antibodies against L3 and juvenile adult worm antigens. Histological examination showed that challenging L3 were trapped in the skin of primarily infected hamsters and surrounded or infiltrated by different inflammatory cells. The trapped L3 were damaged and dead followed by the formation of granulomas encasing dead worms. The results demonstrate that hamsters primarily infected with N. americanus L3 develop acquired immunity against re-infection.     

Angiostrongylus cantonensis: in vitro cultivation from the first-stage to infective third-stage larvae

The first-stage larvae of Angiostrongylus cantonensis were cultured in various media at 27 degrees C. The most suitable medium for the development was Chernin's balanced salt solution supplemented with 10% L-15, 10% tryptose phosphate broth, 20% fetal calf serum, and 26 mM sodium bicarbonate. Addition of sodium bicarbonate to the medium facilitated early development of the first-stage larvae. When the first-stage larvae were cultured in the medium under 5% CO2 in air, the worms developed gradually to become quiescent and showed the C shape. Thereafter, the larvae developed to the second stage, retaining their first sheath. About 23 days later, the larvae began to develop to the third stage, being enclosed within the sheaths of the first and second molts. Under these conditions, the larvae developed uniformly and 82% of the larvae reached the third stage 50 days later. About 70% of the third-stage larvae discarded their two sheaths, showing almost the same size as those obtained in vivo. When these exsheathed larvae were inoculated into rats, they developed into adult worms and deposited numerous first-stage larvae.     

Strongyloides ratti: migration study of third-stage larvae in rats by whole-body autoradiography after 35S-methionine labeling.

In order to clarify the migration pathway of Strongyloides ratti, Wistar rats were given 5,000 35S-labeled infective larvae subcutaneously and killed at 10, 15, 20, 25, 30, 40, and 50 hr postinfection. Prior to inoculation, the specific radioactivity level was assessed in the labeled larvae using a scintillation counter. The frozen rat specimens were sectioned at 50 microm, and the sections were freeze-dried and mounted on X-ray film in darkness. The labeled larvae appeared as dark spots on the film after 14 days of exposure. The infected larvae remained at the inoculated site (lower abdomen) until 10 hr after infection. Some larvae were found in the head portion, whereas others existed sporadically in the skin, liver, and lungs at 15 hr. After 20 and 25 hr, the majority of larvae had accumulated in the head portion. Many larvae appeared in the cranial and nasal cavities; however, no larvae were found in any other organs or tissues. At 30 hr, most larvae had begun to accumulate in the ethmoid region again. At 40 and 50 hr, some larvae were recognized in the ethmoid region, and most had already reached the small intestine. This suggests that the larvae directly move to the nasofrontal portion through the subcutis, rather than migrating to the head through either the viscera, ascending vessels, or the foramen occipital magnum.     

Strongyloides ratti: chemokinesis of glycolytic enzyme- and lectin-treated third-stage infective larvae in vitro

The infective third-stage larvae (L3s) of Strongyloides ratti, a parasitic nematode in rodents, showed two types of chemokinesis on a gradient of sodium chloride (NaCl) in an in vitro agarose tracking assay. The types were a consistent directional avoidance behavior under unfavorable environmental conditions and a reduced avoidance behavior under favorable conditions. We examined the effects of treatments with glycolytic enzymes and lectins by analyzing the avoidance behavior. L-Fucose dehydrogenase, hyaluronidase, beta-glucosidase, alpha-mannosidase, beta-galactosidase, concanavalin A, wheat germ agglutinin and soybean agglutinin exhibited inhibitory or enhancive effects on chemokinesis. We also confirmed the sites of the amphids of L3s aside from the mouth at the anterior end by scanning electron microscopy, and that concanavalin A-binding sites existed in the vicinity of the amphids using lectin-histochemistry. The carbohydrate moieties in the amphids of S. ratti L3s may play an important role as chemosensors in perceiving environmental cues.     

A comparative in vitro study of antibody binding to different stages of the hookworm Ancylostoma caninum.

The Indirect Fluorescent Antibody Technique (IFAT) and the Indirect Immuno Peroxidase Technique (IIPT) have been applied to cryostat sections and intact stages of the hookworm species Ancylostoma caninum with sera from infected dogs. Especially the role of the body surface (= cuticle (cortex, matrix, basal layer) and hypodermis) in immunity was studied. Using cryostat sections and dead intact stages as the antigen, specific antibody binding was demonstrated round the ovum membrane and the cuticle of all stages of this hookworm species. Cryostat sections of adult worms showed, that it probably is not the cuticle itself that is antigenic, but that the specific reaction that is observed consisted of a layer, covering the cortex of the cuticle. Infective and parasitic living stages, however, showed no antibody binding in contrast to the free-living stages in which specific antibody binding was demonstrated.     

Electron and light microscopy of neutrophil responses in mice vaccinated and challenged with third-stage infective hookworm (Ancylostoma caninum) larvae.

The role of neutrophils in mediating host inflammation was examined in mice vaccinated with living third-stage infective hookworm larvae (L₃). Mice were vaccinated by oral immunization with 500 L₃ (Ancylostoma caninum) once every 2 weeks for a total of three immunizations. The vaccinated mice were then challenged intraperitoneally with 2000 L_3, 1 week after the final immunization. To stimulate peritoneal production of neutrophils, 2 ml of 2% glycogen were injected intraperitoneally at 16 h prior to the challenge infection. Neutrophils were found to comprise 85% of the peritoneal cell population. L₃ from the challenge infection were collected and then examined at timed intervals by inverted light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Greater than a fivefold increase in the total numbers of peritoneal cells was noted in the vaccinated mice as compared to unvaccinated mice. In the peritoneal cavity of vaccinated mice, the neutrophils adhered to the L₃ within 2 h, and over 55% of the L₃ were surround by clusters of neutrophils to form a sausage-like sheath 4 h later. At 24–72 h after challenge, almost all of the L₃ recovered from the vaccinated mice were covered with thick clusters of cells. Both SEM and TEM demonstrated extensive ultrastructural damage to the L_3. In contrast, the L₃ recovered from the unvaccinated mice appeared to be unaffected by neutrophils. These studies suggest that neutrophils, like macrophages, can have an important role as effector cells in L₃-vaccinated mice.     

In vitro cultivation of Dipetalonema viteae third-stage larvae: effect of the gas phase

The effect of the gas phase on the in vitro growth and development of Dipetalonema viteae (Nematoda: Filarioidea) third-stage larvae obtained from the tick vector and 3 day infections of jirds was examined. Measurements of the oxygen (pO2) and carbon dioxide (pCO2) tensions and the pH in the medium were made for each gas phase. In cultures gassed with 5% carbon dioxide in nitrogen the pO2 was between 32 and 50 mm Hg, the pCO2 ranged from 25 to 40 mm Hg and the pH was between 7.2 and 7.4. This gas phase resulted in the best growth and development of third-stage larvae to the fourth-stage. Survival and development of larvae were decreased in cultures with oxygen tensions less than 20 mm Hg and greater than 50 mm Hg.     

Gnathostoma binucleatum: excretion-secretion antigen analysis obtained from advanced third-stage larvae in in vitro culture

The paper describes an introductory characterization of antigenic stimulation of excretion-secretion products (ESP) of Gnathostoma binucleatum advanced third-stage larvae cultured in vitro and proteinases present in this products. Excretory and secretory proteins were obtained after 10 larvae were maintained in 5% CO(2) RPMI medium. The supernatant was collected each week for two months. The proteins were dialyzed, concentrated, and separated in 10% SDS-PAGE gels under reducing conditions and transferred to nitrocellulose paper for immunoblot analyses. G. binucleatum immunized mice serum was used to determine protein antigenicity. Four proteins of 40, 80, 120, and 208 kDa persisted for two months and three proteins, 80, 120, and 208 kDa were recognized for antibodies of mice. In SDS-PAGE gelatin substrate gels ESP resolved as two proteins with molecular weight of 80 and 208 kDa that were sensitive to a metalloproteinase inhibitor, and thus it may be inferred that they might be used for diagnosis of gnathostomiasis.     

Dirofilaria immitis: molting process of third-stage larvae

The objective of this study was to determine the molting process of Dirofilaria immitis third-stage larvae (L-3) to fourth-stage larvae (L-4), as it occurred in vitro. After 48 hr in vitro, the L-4 epicuticle was completely formed, and by 72 hr there was a clear separation between the L-3 and L-4 cuticles. The thickness of the newly formed L-4 cuticle was significantly less than that which has been described for larvae recovered from dogs after a similar incubation time period. If culture conditions were lacking in bovine albumin or proper temperature, larvae successfully developed the L-4 epicuticle but did not complete ecdysis. The molting process of D. immitis L-3 was thus shown to be multistepped with different factors required to induce the various developmental phases.     

Radiolabeling of infective third-stage larvae of Strongyloides stercoralis by feeding [75 Se]selenomethionine-labeled Escherichia coli to first- and second-stage larvae

A technique is described for radiolabeling Strongyloides stercoralis larvae with [75Se]selenomethionine. Cultures of an auxotrophic methionine-dependent stain of Escherichia coli were grown in a medium containing Dulbecco's modified Eagle's medium supplemented with 5% nutrient broth, amino acids, and [75Se]selenomethionine. When the 75Se-labeled bacterial populations were in the stationary phase of growth, cultures were harvested and the bacteria dispersed on agar plates to serve as food for S. stercoralis larvae. Use of nondividing bacteria is important for successful labeling because the isotope is not diluted by cell division and death of larvae attributable to overgrowth by bacteria is prevented. First-stage S. stercoralis larvae were recovered from feces of infected dogs and reared in humid air at 30 C on agar plates seeded with bacteria. After 7 days, infective third-stage larvae were harvested. The mean specific activity of 6 different batches of larvae ranged from 75 to 330 counts per min/larva with 91.8 +/- 9.5% of the population labeled sufficiently to produce an autoradiographic focus during a practicable, 6-wk period of exposure. Labeled infective larvae penetrated the skin of 10-day-old puppies and migrated to the small intestine, where the developed to adulthood.     

Protective immunity induced by irradiated third-stage larvae of the filaria Acanthocheilonema viteae is directed against challenge third-stage larvae before molting

Jirds (Meriones unguiculatus) were vaccinated with irradiated L3 third-stage larvae (L3) of Acanthocheilonema viteae, and the time required for killing of the challenge L3 was determined. The number of parasites recovered from vaccinated jirds was reduced to about 10% of the control values on the second day after challenge infection and later on. Histological studies revealed an eosinophil-rich infiltrate containing macrophages, neutrophils, and mast cells in the vicinity of the L3 on day 2 after challenge and destruction of the worms by day 4 after challenge. Ultrastructural studies confirmed these data and showed that eosinophils, macrophages, and mast cells were close to the L3 on day 2 after challenge. Flattening of the eosinophils onto the surface of the worms, degranulation of electron-dense material, and rupture of the L3 surface was observed on day 4 after challenge, followed by invasion of the inner of the worms by phagocytic cells. These data show that immune attack against the challenge L3 in vaccinated jirds is initiated between the first and the second day after challenge and that killing occurs around the fourth day after challenge, before the worms undergo their first molt.     

Oesophagostomum columbianum: gamma-irradiated third-stage larvae for immunization in lambs

40-kR gamma-irradiated third-stage larvae of Oesophagostomum columbianum were used for the immunization of Kashmir Merino lambs. Male lambs (aged from 8 to 12 weeks) were immunized in two separate experiments by two doses of irradiated larvae, given 21 days apart, and subsequently challenged with normal larvae. Judging by the establishment of worms resulting from the challenge infections in the immunized and control groups of lambs in the two experiments, a high degree of immunity was shown to develop in young lambs vaccinated with 500, followed 21 days later with 2000, 40-kR irradiated larvae. Lambs from the immunized groups showed more nodules in the intestine, a high percentage of which were positive for histotrophic stages of O. columbianum, than did controls. The importance of this finding in relation to the possible use of a vaccine for the immuno-prophylaxis of oesophagostomiasis in sheep and other animals is discussed.     

Strongyloides ratti: thermokinetic behavior of third-stage larvae on a temperature gradient

We examined the thermokinetic behaviors of infective third-stage larvae (L3) of the rodent parasitic nematode Strongyloides ratti on temperature gradients using an in vitro agarose tracking assay method. Observed behaviors included both negative and positive thermokineses, the direction of movement depending both on the gradient temperature at which larvae were initially placed and on prior experience of culture temperature. Larvae isolated from rat feces cultured at 25 degrees C and placed on a gradient at temperatures between 22 degrees and 29 degrees C tended to move toward higher temperatures. At higher placement temperatures, most larvae moved little and showed no directional response, whereas at lower placement temperatures, many migrated toward cooler temperatures. At placement temperatures of 20 degrees C or below, few or no larvae moved toward the zone of higher temperature. Larvae isolated from rat feces cultured at 20 degrees C tended to migrate to a high temperature area regardless of placed temperature. Those cultured at 30 degrees C did not respond to the temperature gradient. L3 cultured at 30 degrees C were significantly less infective to rats than those cultured at 25 degrees or 20 degrees C. Additional experiments were designed to demonstrate thermokinetic behaviors during the period after reaching the L3 stage. Larvae incubated in double distilled water (DDW) for 24 h at 37 degrees C lost their ability to respond to lower temperatures, while in those incubated in DDW at 15 degrees and 25 degrees C, responses were still apparent. The thermokinetic behavior of S. ratti L3 is affected by surrounding environmental temperatures and this may have an important role in host finding.     

Angiostrongylus costaricensis: culture of third-stage larvae to young adults in a defined medium

The third-stage larvae of Angiostrongylus costaricensis were successfully cultured to young adults in a chemically defined medium. The most suitable medium for the development was Waymouth's medium among eight defined media examined. Twenty-eight days after cultivation in this medium, 77% of the larvae developed to young adults, although these worms gradually died thereafter. When Waymouth's medium was supplemented with mouse red blood cells, these young adult worms continued their development. The mean body lengths of the worms cultivated in Waymouth's medium supplemented with RBCs were significantly larger than those of the worms in the medium without RBCs on Days 14 and 21 after cultivation. Addition of RBCs was essential for their further development. At 28 days after cultivation, the maximum body length of the worms was 2.1 mm for males and 3.3 mm for females. Additions of serum, yeast extract lactalbumin hydrolysate, and growth factors to Waymouth's medium did not provide any additional benefits for worm development.     

Inoculation of ferrets with ten third-stage larvae of Dracunculus insignis.

Infection with Dracunculus insignis was established in 7 of 10 ferrets experimentally inoculated intraperitoneally with 10 third-stage larvae (L3's). Worm recovery and infection rate were comparable to animals inoculated with 50 L3's. This study demonstrates that once infective larvae traverse the gut and pass into the peritoneal cavity, very few larvae are required to establish infection.