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.     

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.     

Changes in the structure of Nippostrongylus brasiliensis intestinal cells during development from the free-living to the parasitic stages

The ultrastructure of Nippostrongylus brasiliensis intestinal cells was examined in free-living, feeding second-stage larvae, infective, nonfeeding third-stage larvae, and parasitic, feeding third-stage larvae. The intestinal cells of second-stage larvae were characterized by a well-developed microvillar border, large numbers of ribosomes, Golgi complexes, rough endoplasmic reticulum, and nuclei with prominent nucleoli. The intestinal cells of infective, third-stage larvae had very few microvilli and the cells were extremely narrow. Few ribosomes, Golgi complexes, and little rough endoplasmic reticulum were present. Nuclei did not contain nucleoli. When worms were introduced into an in vitro culture system, development of intestinal cells began. By 36 hr, microvilli were well differentiated and the cysoplasm contained numerous ribosomes and Golgi complexes, and rough endoplasmic reticulum, mitochondria, and nucleoli were prominent. These morphological changes were related to changes in the physiology of Nippostrongylus brasiliensis which occur during development from a free-living to parasitic form.     

Changes in lipid contents of infective third-stage larvae of Necator americanus during desiccation and revival

Changes in lipid content of infective third-stage larvae of Necator americanus were investigated after short periods of induced desiccation and revival. A fall in lipid reserve from an outset level of 86% to 74% was recorded in the first 2 h of desiccation. With increased desiccation, lipid reserves did not show significant decline, probably as a result of decreased lipid metabolism in the desiccated larvae. During revival, there was a drastic fall in lipid reserves as a result of increased lipid utilisation by the reviving larvae. The results showed that desiccated larvae with lipid levels less than 10% did not revive. The presence of lipid did not appear to prevent desiccation but was an essential factor for revival. The ecological significance of these findings in field larvae is discussed.     

Cultivation of free-living stages of Trichostrongylus colubriformis in media without bacteria, animal tissue extract, or serum

Trichostrongylus colubriformis was cultured from hatched first-stage to third-stage larvae in bacteria-free media in the absence of animal tissue extract or serum. This was achieved for the first time with a nematode, parasitic in vertebrates, whose rhabditiform larvae are food-dependent. The best media contained enzymatic hydrolysed casein (amino nitrogen:total nitrogen ratio 0.39), yeast extract, phosphatidylcholine from soybean, and a number of chemically defined ingredients, which include a salt solution, a sterol, and an iron porphyrin. The yield of third-stage larvae obtained was up to 17% of all the living larval stages present after incubation. When casein hydrolysate with AN:TN ratio of 0.39 was replaced by casein hydrolysate with AN:TN ratio of 0.53, little or no development to third-stage larvae occurred. Development to infective larvae was shown to be possible in media with soy peptone instead of casein hydrolysate, although to a very limited extent. It is proposed that the free-living stages of the parasite require peptides, whose molecular weights all lie within a narrow range.     

Skin penetration by ensheathed third-stage infective larvae of Necator americanus, and the host's immune response to larval antigens.

In vitro experiments were conducted to assess skin penetration by ensheathed third-stage infective larvae (L3) of Necator americanus. The fact that only a small proportion of larval sheaths was recoverable from the outer skin surface suggested that some larvae penetrate mouse skin without undergoing exsheathment. Penetration by ensheathed larvae was confirmed visually using a novel fluorescein isothiocyanate (FITC)-labelling technique in which viable ensheathed larvae were fluoresceinated, applied onto intact mouse skin, and their progress monitored in frozen skin sections. This direct observation that the L2-derived sheath can present antigens to the host's immune system was also monitored by immunoassay to provide confirmatory information regarding skin penetration by ensheathed larvae. Sera from humans infected with Necator americanus were shown to react in ELISA against antigens stripped by detergent (cetyltrimethylammonium bromide) from the sheath surface, and with antigens contained in L3-exsheathing fluid. These data suggest that the host's immune response, as a result of antigenic stimulation by the cast sheath and exsheathing fluid, could in fact be diverted away from the potentially vulnerable L3 stage.     

Heligmosomoides polygyrus: Simple recovery of post-infective larvae from mouse intestines

Mice infected orally with third-stage larvae of Heligmosomoides polygyrus were killed at various times after infection. Their small intestines were removed, tied at each end and incubated at 37 C in dilute culture medium. When intestines were taken from mice infected for a period of between 1 and 7 days, a number of developing larvae comprising up to 20% of the infective dose emerged within 60 min through the intestinal wall into the medium. The recovery of emergent larvae was highest using intestines from mice infected 36 to 120 hr previously. The proportion of parasites emerging from the intestines of 48-hr-infected mice was similar for doses of 100 to 2400 larvae. Significantly fewer larvae emerged from the intestines of mice resistant to reinfection and challenged with third-stage larvae 36–72 hr before necropsy.     

Variable infectivity of third-stage larvae of Brugia malayi

Infectivity of third-stage larvae of Brugia malayi was assessed following intraperitoneal inoculation into jirds, Meriones unguiculatus. Larvae were of two ages and were derived from two sites in Aedes aegypti mosquitoes, i.e., specimens collected from the thorax 11 days after infection and from the head on day 14. Larvae from the thorax had just completed the second moult and measured 990 to 1100 micron in length. Only 6% of these specimens developed to adult worms in jirds. Larvae that migrated to the head were 1400 to 1700 micron long on day 14 and, in contrast, 23% of the inocula developed to adult worms. This study establishes that all third-stage larvae, regardless of their age or location in the arthropod host, are potentially infective. However, pronounced physical maturation does seem to be accompanied by a marked increase in infectivity.     

Observations on Toxocara pteropodis infections in mice

Infection in mice with Toxocara pteropodis was investigated. In mice fed infective eggs, third-stage larvae hatched out and penetrated the mucosa, predominantly that of the lower intestine. They travelled via the portal vein to the liver, where they remained at least 14 months. They grew in length from 430 +/- 15 micron, at three days post infection (p.i.), to 600 +/- 50 micron, at six to nine weeks p.i., after which time growth ceased. Blood eosinophilia appeared at 28 days p.i., and eosinophil levels continued to rise gradually beyond this time. In female mice the larvae did not migrate from the liver in response to pregnancy or lactation. When infective eggs were inoculated subcutaneously or intra-peritoneally, larvae hatched out and ultimately appeared in the liver in larger numbers than seen with oral infections.     

Energy cost of locomotory activity in larval hookworms

Lipid changes in the infective third-stage larvae of Ancylostoma tubaeforme following measured periods of activity have been quantified. ‘Excessive’ activity (more than 16 activity régimes, or 8 h in neostigmine bromide) resulted in a significant (> 10%) depletion of the lipid reserves, especially from the anterior region of the intestine.     

Field method for isolation of trichostrongyle larvae from vegetation of natural pastures of Arctic ruminants

The extent to which wild ruminant populations are exposed to infective helminth larvae on their natural pastures is relatively undetermined. In the present study, a modified method for sampling of herbage and isolation of trichostrongyle infective third-stage larvae from natural pastures was used successfully in a muskox habitat in low-Arctic Greenland. The method, a revision of the Macro-Baermann method, is particularly aimed at fieldwork under primitive conditions.     

Effect of vaccinations with recombinant fusion proteins on Ancylostoma caninum habitat selection in the canine intestine

Laboratory dogs were vaccinated subcutaneously with 3 different recombinant fusion proteins, each precipitated with alum or calcium phosphate. The vaccinated dogs were then challenged orally with 400 third-stage infective larvae (L3) of the canine hookworm, Ancylostoma caninum. The 3 A. caninum antigens selected were Ac-TMP, an adult-specific secreted tissue inhibitor of metalloproteases; Ac-AP, an adult-specific secreted factor Xa serine protease inhibitor anticoagulant; and Ac-ARR-1, a cathepsin D-like aspartic protease. Each of the 3 groups comprised 6 male beagles (8 +/- 1 wk of age). A fourth group comprised control dogs injected with alum. All of the dogs vaccinated with Ac-TMP or Ac-APR-1 exhibited a vigorous antigen-specific antibody response, whereas only a single dog vaccinated with Ac-AP developed an antibody response. Dogs with circulating antibody responses exhibited 4.5-18% reduction in the numbers of adult hookworms recovered from the small intestines at necropsy, relative to alum-injected dogs. In contrast, there was a concomitant increase in the number of adult hookworms recovered from the colon. The increase in colonic hookworms was as high as 500%, relative to alum-injected dogs. Female adult hookworms were more likely to migrate into the colon than were males. Anti-enzyme and anti-enzyme inhibitor antibodies correlated with an alteration in adult hookworm habitat selection in the canine gastroinntestinal tract.     

Experimental infection routes of Angiostrongylus cantonensis in mice.

Stomach intubation is the most common method used in the experimental infection of animals with Angiostrongylus cantonensis. In order to compare the effectiveness of other possible transmission methods, groups of BALB/c mice were given infective third-stage larvae of A. cantonensis by different routes including intraperitoneal or subcutaneous injections, and penetration of anal mucosa, vaginal mucosa, conjunctival mucosa, lacerated skin, unabraded skin, foot pad and tail skin, while stomach intubation was used as control. Recovery of fifth-stage larvae was higher in mice inoculated with third-stage larvae subcutaneously. Successful infections were established through all experimental transmission routes except tail skin penetration. This study suggests that oral infection may not be the only route for the transmission of human angiostrongyliasis, and subcutaneous infection may be a better method for experimental infection.     

Human thelaziosis--a neglected parasitic disease of the eye

The oriental eyeworm, Thelazia callipaeda (Spirurida, Thelaziidae), infects a range of definitive hosts, such as dogs, cats, foxes, rabbits, and humans. This parasite usually lives under the nictitating membrane of the eye, where the adult females release first-stage larvae into the lachrymal secretions; these larvae are subsequently ingested by the intermediate arthropod host within which they develop to the infective, third-stage larvae. The latter larvae are then deposited into the eyes of the definitive host. Recently, T. callipaeda has been reported to infect dogs, foxes, and/or cats in Europe (Italy, France, and Germany). Human thelaziosis (HT) is considered to be an underestimated parasitic disease, whose prevalence appears to have increased in poor socioeconomic settings in many Asian countries, including China. In humans, the disease can be subclinical or symptomatic, exhibiting epiphora, conjunctivitis, keratitis, excessive lachrymation, corneal opacity, and/or ulcers. Knowledge about HT is presently fragmentary and mainly limited to clinical case reports. This article provides a background on the parasite and its life cycle, reviews cases of human thelaziosis, summarizes key aspects regarding the diagnosis of thelaziosis, and proposes future research and methods of control of the disease in humans, particularly in Asia.     

Resumption of feeding in vitro by hookworm third-stage larvae: a comparative study.

Third-stage infective larvae of the canine hookworm Ancylostoma caninum resume feeding in vitro in response to several stimuli. Experiments were conducted to characterize the in vitro feeding behavior of several hookworm species. Reduced glutathione and, to a lesser extent, canine and human serum stimulated third-stage larvae of Ancylostoma duodenale to resume feeding. Glutathione-induced feeding reached a maximum by 16 hr and was concentration-dependent between 0- and 15-mM glutathione. Oxidized glutathione and the reducing agents dithiothreitol and L-cysteine failed to induce feeding, suggesting that reducing conditions alone were not stimulatory. Serum incubated with glutathione was the most efficient stimulus for Ancylostoma ceylanicum, Ancylostoma braziliense, and Ancylostoma tubaeforme larvae, whereas Uncinaria stenocephala larvae responded best to canine serum alone. Necator americanus larvae did not resume feeding in response to glutathione, serum, glutathione plus serum, or linoleic acid (0.1-10 mM). These differences in feeding behavior suggest that generalizations concerning hookworm biology must be interpreted cautiously.     

Nippostrongylus brasiliensis and Haemonchus contortus: Function of the excretory ampulla of the third-stage larva

An improved technique for measuring the water content of nematodes is described using an electronic interferometer. Changes in phase of a laser beam passing through a known pathlength of the nematode have been used to measure the refractive index and hence the water content and relative volume of the animal. Third-stage larvae of Nippostrongylus brasiliensis and Haemonchus contortus which possess an excretory ampulla, differed from second-stage larvae lacking this ampulla in requiring a greater fall in the osmolarity of artificial tap water before there was a significant increase in their water content. Increases in the pulsation frequency of the ampulla also occurred in less hypotonic solutions than those required to increase the water content of the third-stage larvae. The ampulla pulsation frequency of third-stage larvae of N. brasiliensis increased after locomotor activity in hypotonic tap water and locomotory wave frequency of third-stage larvae of N. brasiliensis was independent of the extent of hypotonicity for a range of solutions that reduced wave propogation by its second-stage larva. The results suggest that the ampulla is an adaptation to hypotonic conditions favouring a volume homeostasis that is required for optimal locomotor activity of the third-stage infective larvae of these nematodes.     

Epizootiology of Eustrongylides ignotus in Florida: transmission and development of larvae in intermediate hosts

Under laboratory conditions, 2 modes of transmission of Eustrongylides ignotus (Nematoda: Dioctophymatoidea) to fish were identified. Eastern mosquitofish (Gambusia holbrooki) became infected after ingestion of either eggs of E. ignotus containing first-stage larvae or aquatic oligochaetes (Limnodrilus hoffmeisteri) containing third-stage larvae of E. ignotus. After removal from the uterus of gravid E. ignotus females and incubation for 17-28 days, depending on temperature, it was found that parasite eggs contained first-stage larvae that were infective to fish and oligochaetes. Larvae developed to the third stage in oligochaetes and were infective to fish 35-77 days postinfection (PI) and when fed to fish, developed to the fourth stage between 127 and 184 days PI. Eggs containing first-stage larvae fed directly to fish developed to the fourth stage between 84 and 105 days PI. The amount of time for development from the undifferentiated egg to the fourth-stage larva was 78-156 days shorter when fish ingested eggs containing first-stage larvae than when fish ingested oligochaetes containing third-stage larvae. Three species of large piscivorous fish, including black crappie (Pomoxis nigromaculatus), largemouth bass (Micropterus salmoides), and warmouth (Lepomis gulosus), were fed mosquitofish containing fourth-stage larvae. At necropsy, live E. ignotus larvae were recovered from all 3 species. Several fish had multiple infections after ingesting > 1 larva, indicating that bioaccumulation of the parasite in the food chain may occur.     

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.