Immunization with infective larvae of Strongyloides ratti (Nematoda) exposed to microwave radiation

Microwaves have not been tested previously for possible application in producing immunogenic preparations of parasites. This study examines the immunizing capacity of microwave-irradiated, infective larvae of Strongyloides ratti in rats. Rats were inoculated subcutaneously with untreated, microwaved, or microwaved and homogenized larvae, or distilled water, and challenged with untreated larvae. Data were collected on egg production and worm number/rat during primary infections and on egg production, worm number/rat, worm size, and eggs in utero/worm following challenge. Our results demonstrated that microwaved, infective larvae (intact or homogenized) of S. ratti were immunogenic for rats, even though they were incapable of reaching the intestine and maturing to adult worms. The immunity elicited by exposure to microwaved larvae was characterized on challenge by a significant reduction in the number of eggs produced/worm, by the formation of perioral plugs, and by reductions in worm numbers and size. These results suggest that microwave radiation may provide a valuable new tool for parasitic vaccine production. In addition, we have demonstrated the occurrence of a feature of the immune response of rats to S. ratti that may have been overlooked previously; i.e., a gut-level response that was elicited by larvae, but manifested against adult worms in the intestine.     

Shedding of antibody complexes by Strongyloides ratti (Nematoda) larvae

Antigens on the epicuticular surface of Strongyloides ratti infective third-stage larvae (L3) could be demonstrated by an indirect fluorescent antibody technique under certain conditions. Infective L3 shed anti-antibody complexes at room temperature, but not at 4 C or in the presence of sodium azide or colchicine. Shedding of antibody did not appear to involve epicuticular antigens, and only occurred when anti-rat IgG was complexed to rat anti-larval antibody. However, parasitic L3 removed from rats did not exhibit this shedding reaction, suggesting that an important developmental change in cuticle physiology occurs during the transition from a free-living existence to a parasitic mode. The ability to shed foreign objects from the epicuticle of free-living infective L3 may be a defensive or protective response to soil microorganisms.     

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.     

Experimental evolution of parasite life-history traits in Strongyloides ratti (Nematoda)

Evolutionary ecology predicts that parasite life-history traits, including a parasite's survivorship and fecundity within a host, will evolve in response to selection and that their evolution will be constrained by trade-offs between traits. Here, we test these predictions using a nematode parasite of rats, Strongyloides ratti, as a model. We performed a selection experiment by passage of parasite progeny from either early in an infection ('fast' lines) or late in an infection ('slow' lines). We found that parasite fecundity responded to selection but that parasite survivorship did not. We found a trade-off mediated via conspecific density-dependent constraints; namely, that fast lines exhibit higher density-independent fecundity than slow lines, but fast lines suffered greater reduction in fecundity in the presence of density-dependent constraints than slow lines. We also found that slow lines both stimulate a higher level of IgG1, which is a marker for a Th2-type immune response, and show less of a reduction in fecundity in response to IgG1 levels than for fast lines. Our results confirm the general prediction that parasite life-history traits can evolve in response to selection and indicate that such evolutionary responses may have significant implications for the epidemiology of infectious disease.     

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.     

Electron microscopical studies of Strongyloides ratti infective larvae: loss of the surface coat during skin penetration

Previous indications using radiolabelled larvae that Strongyloides ratti free-living infective larvae lose a surface coat during penetration of the skin were further investigated by transmission electron microscopy of the cuticle of S. ratti infective larvae in the free-living stage, after penetration of mouse skin, and after migration to the lungs. These studies demonstrated the presence of a faint electron-dense surface coat external to the epicuticle on free-living worms which was absent from larvae recovered from the skin and lungs. When free-living infective larvae were incubated in 10% CO2 at 37 C and then examined with phase-contrast microscopy, worms were observed in the process of losing this coat. These observations confirm the hypothesis that S. ratti infective larvae lose a surface coat during penetration of the skin.     

Strongyloides spearei n. sp. (Nematoda: Strongyloididae) from the common wombatVombatus ursinus (Marsupialia: Vombatidae)

Strongyloides spearei n. sp. is described from the small intestine of the common wombatVombatus ursinus from Healesville, Victoria. The new species is distinguished from all known congeners by: the triangular shape of the stoma and the length of the parasitic female; the blunt spicules in the free-living male; and the presence of eggs in the faeces of the host.S. spearei andS. thylacis Mackerras, 1959 form a separate group withinStrongyloides based on both species infecting marsupials, having directly recurrent ovaries in the parasitic female and having blunt spicules in the free-living male. The histological localisation ofS. spearei is predominantly within the crypts of the small intestine.     

Tracking radioactive larvae of Strongyloides ratti in the host

Infective larvae of homogonic Strongyloides ratti grown in faecal culture with 32P or 75Se acquired a significant amount of radioactivity which was firmly attached to them. Heating removed most of the 32P but left 75Se in place. Subcutaneous injection of virgin and nursing mother rats with living and heat-killed radioactive larvae resulted in a pattern of labelling in the small intestine of injected animals and, in the case of 75Se, those of suckling pups, which can only be explained if labelled worms follow the natural migratory routes. The use of this tool in migratory studies is discussed, with precautions to allow for flaws in the technique.     

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.     

The response of intact Strongyloides ratti infective (L3) larvae to substrates and inhibitors of respiratory electron transport.

Live, intact third-stage larvae (L3s) of Strongyloides ratti in the absence of exogenous substrates consumed oxygen at a rate (E-QO₂) of 181.8 ± 12.4 ng atoms min⁻¹ mg dry weight⁻¹ at 35°C. Respiratory electron transport (RET) Complex I inhibitor rotenone (2 μ ) produced 33 ± 6.5% inhibition of the E-QO₂. Unusually the rotenone-induced inhibition was not relieved by 5 μ -succinate. The E-QO₂ of intact L3s was refractory to RET Complex III inhibitor antimycin A at 2 μ ; 4 μ -antimycin inhibited ≤ 10% of the E-QO₂. The electron donor couple ascorbate/TMPD augmented the E-QO₂ in the presence of rotenone (2 μ ) and antimycin A (4 μ ) by 110%. Azide (1 m ) stimulated the antimycin A refractory QO₂ by 36.6 ± 7.2% which was only partially inhibited by 1.0 m -KCN ( ). The data suggest the presence of classical (CPW) and alternate (APW) electron transport pathways in S. ratti L3s.     

Loss of surface coat by Strongyloides ratti infective larvae during skin penetration: evidence using larvae radiolabelled with 67gallium

The optimal conditions for labelling infective larvae of Strongyloides ratti with 67gallium citrate were determined. Radiolabelled larvae were injected s.c. into normal and previously infected rats. The distribution of radioactivity in these animals was compared with that in rats infected subcutaneously with a similar dose of free 67Ga by using a gamma camera linked to a computer system. Whereas free 67Ga was distributed throughout the body and excreted via the hepatobiliary system, the bulk of radioactivity in rats injected with radiolabelled larvae remained at the injection sites. Direct microscopical examination of these sites, however, revealed only minimal numbers of worms. When rats were infected percutaneously with radiolabelled larvae, it was found that most radioactivity remained at the surface, despite penetration of worms. When infective larvae were exposed to CO2 in vitro and examined carefully by light microscopy, loss of an outer coat was observed. It was concluded that infective larvae lose an outer coat on skin penetration.     

Strongyloides ratti: the role of interleukin-5 in protection against tissue migrating larvae and intestinal adult worms

To determine the role of interleukin-5 (IL-5) and eosinophils in protection against Strongyloides ratti, mice treated with anti-IL-5 monoclonal antibody (mAb) were infected with S. ratti larvae. Strongyloides ratti egg numbers in faeces (EPG) in mAb treated mice were higher than those in control mice on days 6 and 7 after inoculation. The numbers of migrating worms in mAb treated mice 36 h after inoculation were higher than those observed in control mice. Intestinal worm numbers in mAb treated mice 5 days after inoculation were higher than those in control mice. These results show that eosinophils effectively protected the host against S. ratti infection by mainly the larval stage in primary infections. The involvement of eosinophils in protection against secondary infection was also examined. Before secondary infection, mice were treated with anti-IL-5 mAb and infected with S. ratti. Patent infections were not observed in either mAb treated or control Ab treated mice. The numbers of migrating worms in the head and lungs of mAb treated mice increased to 60% of that in primary infected mice. Intestinal worms were not found in mAb treated mice or in control mice after oral implantation of adult worms. Eosinophils were therefore mainly involved in protection against tissue migrating worms in secondary infections.     

Cultivation of Haemonchus contortus (Nematoda: Trichostrongylidae) from infective larvae to the adult male and the egg-laying female

The parasitic nematode Haemonchus contortus was cultured in vitro to the adult male and the egg-laying female. Gastric contents from 2 cannulated calves and 2 sheep as well as extracts of stomach mucosa from both hosts were added to a mixture of API-1 culture medium and Fildes' reagent (a pepsin digest of defibrinated bovine blood) adjusted to pH 6.4 for the first week and pH 6.8 thereafter. The gas phase was 85% N2:5% O2:10% CO2. Best results were obtained when API-1 culture medium plus Fildes' reagent was supplemented with ovine gastric contents. Adult males up to 10 mm were obtained, and they produced and stored sperm at about 28 days in culture. Spermatozoa were never seen in the uteri of females grown in vitro. After 36 days in culture, female worms up to 16 mm were obtained which produced and layed eggs. Some of the eggs underwent division up to the 8-cell stage but did not develop further. No live larvae were observed at a stage earlier than the fourth. Presumably, this was because no matings were observed, although large clumps of worms did occur in the culture medium and mating might have occurred. The growth of adults from young to mature phases of this stage included: for the female, oogenesis and laying of eggs; for the male, development of paired sclerotized spicules, and spermatogenesis with the production of spermatozoa.     

No evidence for specificity between host and parasite genotypes in experimental Strongyloides ratti (Nematoda) infections

A key requirement for several theories involving the evolution of sex and sexual selection is a specificity between host and parasite genotypes, i.e. the resistance of particular host genotypes to particular parasite genotypes and the infectivity of particular parasite genotypes for particular host genotypes. Determining the scope and nature of any such specificity is also of applied relevance, since any specificity for different parasite genotypes to infect particular host genotypes may affect the level of protection afforded by vaccination, the efficacy of selective breeding of livestock for parasite resistance and the long-term evolution of parasite populations in response to these control measures. Whereas we have some evidence for the role of specificity between host and pathogen genotypes in viral and bacterial infections, its role in macroparasitic infections is seldom considered. The first empirical test of this specificity for a vertebrate–nematode system is provided here using clonal lines of parasite and inbred and congenic strains of rat that differ either across the genome or only at the major histocompatibility complex. Although significant differences between the resistance of host genotypes to infection and between the fitness of different parasite genotypes are found, there is no evidence for an interaction between host and parasite genotypes. It is concluded that a specificity between host and parasite genotypes is unlikely in this system.