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.     

Strongyloides stercoralis: antigenic analysis of infective larvae and adult worms

The protein composition of Strongyloides stercoralis infective larvae and adult worms solubilized sequentially in water, sodium deoxycholate and sodium dodecyl sulphate (SDS) and their excretory/secretory products were analysed by one- and two-dimensional SDS-polyacrylamide electrophoresis. These extracts were demonstrated to be complex mixtures containing many proteins, some of which were common and others which were stage-specific. Western blot analysis of these antigens with infected human sera showed most sero-reactivity against larval antigens, whilst normal human sera were unreactive. These data identify immunogenic antigens which may be available for detection in an antigen assay.     

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: 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.     

Cryopreservation of Dirofilaria immitis microfilariae and third-stage larvae

Microfilariae of Dirofilaria immitis retained their infectivity for susceptible mosquitoes after cooling to -196 degrees C in the presence of 5% dimethylsulphoxide (Me2SO) using a two-step cooling sequence. Motility and in vitro development of cryopreserved microfilariae also compared favourably with unfrozen controls. Third-stage larvae frozen by the same cooling sequence in the presence of either 5% Me2SO or 16% hydroxyethyl starch were motile upon thawing. Thawed larvae completed the third- to fourth-stage moult in vitro at a frequency approximately 5 to 10% of that seen in unfrozen controls.     

Chemokinetic behavior of the infective third-stage larvae of Strongyloides ratti on a sodium chloride gradient.

The movements of the infective third-stage larvae (L3) of a rodent parasitic nematode Strongyloides ratti were examined on a sodium chloride (NaCl) gradient set up on agarose plates. The movements of larvae were followed by observing their tracks on the surface of the agarose. The direction of movement depended on the NaCl concentration at the point of their initial placement on the gradient. Larvae placed at between 230 and 370 mM NaCl tended to migrate towards areas of lower concentration. On the other hand, when placed at concentrations less than 20 mM NaCl, larvae tended to migrate initially towards higher concentrations but did not linger in areas where the concentration was over approximately 80 mM NaCl. It seems that S. ratti L3, tested in vitro, prefer regions with a concentration of NaCl below 80 mM NaCl. Two typical chemokinetic behaviors are seen; a unidirectional avoidance movement when initially placed in unfavorable environmental conditions and a random dispersal movement when placed within an area of favorable conditions. Track patterns were straight in the avoidance movement but included multiple changes of direction and loops in the dispersal movement. This study introduces an assay system suitable for studying chemokinetic behavior of larvae of Strongyloides ratti.     

Strongyloides stercoralis: identification of a protease that facilitates penetration of skin by the infective larvae

Host invasion and tissue migration of several helminths have been linked to the expression and release of parasite-derived proteases. One of the most remarkable examples of tissue migration is that of larvae of the nematode parasite Strongyloides stercoralis, which can move through tissue at speeds of up to 10 cm per hour. We have shown the Strongyloides L3 larvae secrete a potent histolytic metalloprotease to facilitate their rapid migration. This protease has elastase activity and catalyzes the degradation of a model of dermal extracellular matrix. The importance of this enzyme in the pathogenesis of strongyloidiasis is underscored by the observation that invasion by larvae of skin in vitro is prevented by metalloprotease inhibitors. These results substantiate the role of proteases as virulence factors in strongyloidiasis, as well as other related parasitic infections, and suggest new approaches to therapy.     

Cryopreservation of the first-stage larvae of trichostrongylid nematode parasites

First stage (L1) larvae of Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta can be cryopreserved in the presence of DMSO using a two-step freezing protocol involving an initial period at −80°C prior to transfer to liquid nitrogen. Thawed L1 larvae continue development in vitro producing third stage (L3) larvae that are infective to sheep when dosed per os. Establishment rates for L3 larvae grown from thawed L1 larvae were 40 and 80% for H. contortus and T. colubriformis, respectively. There was no difference in survival or infectivity between benzimidazole (BZ)-susceptible and BZ-resistant H. contortus isolates and cryopreservation caused no shift in their BZ-resistance status as indicated in an in vitro larval development assay. Cryopreservation also had no effect on the sensitivity of these isolates to the avermectins or levamisole in vitro. High survival rates (60–70%), good levels of establishment and the stability of anthelmintic resistance status of isolates indicate that little if any selection occurs during the cryopreservation process. L1 larvae of all 3 species have been successfully recovered after 16 months storage in liquid nitrogen, cultured to the L3 stage and established in sheep.     

Strongyloides stercoralis.

Strongyloides stercoralis, a nematode not well known to many Canadian physicians, infects 35% of some tropical populations. Larvae can be isolated from the stools in 25% of cases and from duodenal aspirates in 95%. Treatment is with thiabendazole given twice daily in a dose of 25 mg/kg up to a maximum of 1.5 g/d. Frenquently an individual with a previously asymptomatic infection presents with hyperinfection and death rapidly ensues, but usually classical symptoms are present. Such a case is described. Immunosuppression is frequently associated with the hyperinfective state.     

Cryopreservation of infective larvae of Dipetalonema viteae.

infective larvae of Dipetalonema viteae produced infections in Mongolian jirds (Meriones unguiculatus) after storage of infected ticks (Ornithodoros tartakovskyi) in the presence of dimethyl sulfoxide (DMSO, 5%) for 7 or 595 days in liquid nitrogen (-196 C). Infectivity of these larvae was only partially impaired. Microfilaremias of test jirds were generally lower than those of control jirds given nonfrozen larvae; however, the majority of test jirds developed microfilarial counts suitable for use in infecting ticks. In contradistinction, larvae frozen free of the tick failed to retain infectivity. Apparently the tick, in conjunction with DMSO, protects the larvae during freezing and thawing.     

Strongyloides stercoralis: Amphidial neuron pair ASJ triggers significant resumption of development by infective larvae under host-mimicking in vitro conditions

Resumption of development by infective larvae (L3i) of parasitic nematodes upon entering a host is a critical first step in establishing a parasitic relationship with a definitive host. It is also considered equivalent to exit from the dauer stage by the free-living nematode Caenorhabditis elegans. Initiation of feeding, an early event in this process, is induced in vitro in L3i of Strongyloides stercoralis, a parasite of humans, other primates and dogs, by culturing the larvae in DMEM with 10% canine serum and 5mM glutathione at 37 degrees C with 5% CO(2). Based on the developmental neurobiology of C. elegans, resumption of development by S. stercoralis L3i should be mediated, in part at least, by neurons homologous to the ASJ pair of C. elegans. To test this hypothesis, the ASJ neurons in S. stercoralis first-stage larvae (L1) were ablated with a laser microbeam. This resulted in a statistically significant (33%) reduction in the number of L3i that resumed feeding in culture. In a second expanded investigation, the thermosensitive ALD neurons, along with the ASJ neurons, were ablated, but there was no further decrease in the initiation of feeding by these worms compared to those in which only the ASJ pair was ablated.     

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.     

Strongyloides ratti: chemotactic responses of third-stage larvae to selected serum proteins and albumins

Experiments were carried out in vitro to investigate whether the sera of several animals as well as albumins and peptides might act as attractants for larvae of Strongyloides ratti. Samples of sera from several mammal species were dialysed and the aliquots were further centrifuged using ultrafiltration cartridges to remove any remaining small molecules. Additional test substances included commercially obtained ovalbumin, rat and bovine serum albumins, polypeptides such as peptone, tryptone and tryptose, amino nitrogens, monosaccharides, and reduced glutathione (triaminopeptide). Larvae were strongly attracted to the dialysed mammalian sera, which mainly consisted of serum albumin and globulins. Ov- and serum albumins, and polypeptides also acted as attractants. On the other hand, reduced glutathione, 16 kinds of amino acids and four kinds of monosaccharides did not attract this nematode.     

The effect of fatty acids on the developmental direction of Strongyloides ratti first-stage larvae

The effect of fatty acids was studied on the developmental direction of Strongyloides ratti first-stage larvae (L1). The proportion of third-stage infective larvae increased markedly when L1 were cultured in faeces with added fatty acids such as palmitic (C16), stearic (C18), oleic (C18:1) and linoleic (C18:2) acids. Unsaturated fatty acids were more effective than saturated ones. Moreover, the proportion of infective larvae increased with quantity of linoleic acid but the triacylglycerols of any fatty acid had no effect. These results suggest that these free fatty acids cause physiological changes that determine the developmental course of L1 of S. ratti in nature.     

Determinants of the longevity of third-stage infective larvae of Ancylostoma tubaeforme.

The effects of some extrinsic factors on the lipid (energy) reserves and longevity of third-stage larvae of the cat hookworm Ancylostoma tubaeforme, were investigated under controlled laboratory conditions. In nonstressful microenvironmental conditions, larval longevity was directly related to the rate of utilisation of the lipid reserves. The effects of the various environmental stresses on longevity could also be explained largely on the basis of their deleterious effects on the lipid metabolism of the larvae.     

Strongyloides stercoralis and the immune response

The immune system is a highly evolved network of cells and molecules that can distinguish between invading pathogens and the body's own cells. But helminths, in their complex forms, are capable of down-regulating host immunity, protecting them from being eliminated and also minimizing severe pathology in the host. This review focuses on Strongyloides stercoralis and the immune responses in immunocompetent and/or immunocompromised individuals. It also highlights the implications for diagnosis/treatment and draws attention to an emerging public health disease. The solution to reducing the prevalence of strongyloidiasis remains on the effectiveness of pre-emptive measures in endemic communities, increased awareness, prompt early diagnosis as well as timely treatment.