Sex determination in the parasitic nematode Strongyloides ratti

The parasitic nematode Strongyloides ratti reproduces by both parthenogenesis and sexual reproduction, but its genetics are poorly understood. Cytological evidence suggests that sex determination is an XX/XO system. To investigate this genetically, we isolated a number of sex-linked DNA markers. One of these markers, Sr-mvP1, was shown to be single copy and present at a higher dose in free-living females than in free-living males. The inheritance of two alleles of Sr-mvP1 by RFLP analysis was consistent with XX female and XO male genotypes. Analysis of the results of sexual reproduction demonstrated that all progeny inherit the single paternal X chromosome and one of the two maternal X chromosomes. Therefore, all stages of the S. ratti life cycle, with the exception of the free-living males, are XX and genetically female. These findings are considered in relation to previous analyses of S. ratti and to other known sex determination systems.     

Functional consequences of genetic diversity in Strongyloides ratti infections

Parasitic nematodes show levels of genetic diversity comparable to other taxa, but the functional consequences of this are not understood. Thus, a large body of theoretical work highlights the potential consequences of parasite genetic diversity for the epidemiology of parasite infections and its possible implications for the evolution of host and parasite populations. However, few relevant empirical data are available from parasites in general and none from parasitic nematodes in particular. Here, we test two hypotheses. First, that different parasitic nematode genotypes vary in life-history traits, such as survivorship and fecundity, which may cause variation in infection dynamics. Second, that different parasitic nematode genotypes interact within the host (either directly or via the host immune system) to increase the mean reproductive output of mixed-genotype infections compared with single-genotype infections. We test these hypotheses in laboratory infections using genetically homogeneous lines of Strongyloides ratti. We find that nematode genotypes do vary in their survivorship and fecundity and, consequently, in their dynamics of infection. However, we find little evidence of interactions between genotypes within hosts under a variety of trickle- and single-infected infection regimes.     

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.     

Density-dependent immune responses against the gastrointestinal nematode Strongyloides ratti

Negative density-dependent effects on the fitness of parasite populations are an important force in their population dynamics. For the parasitic nematode Strongyloides ratti, density-dependent fitness effects require the rat host immune response. By analysis of both measurements of components of parasite fitness and of the host immune response to different doses of S. ratti infection, we have identified specific parts of the host immune response underlying the negative density-dependent effects on the fitness of S. ratti. The host immune response changes both qualitatively from an inflammatory Th1- to a Th2-type immune profile and the Th2-type response increases quantitatively, as the density of S. ratti infection increases. Parasite survivorship was significantly negatively related to the concentration of parasite-specific IgG(1) and IgA, whereas parasite fecundity was significantly negatively related to the concentration of IgA only.     

Strongyloides ratti and Trichinella spiralis: net charge of epicuticle

The intact epicuticles of Strongyloides ratti stage-3 larvae and Trichinella spiralis stage-1 larvae were found to have a surface net negative charge. Ultrastructural studies on S. ratti using cationized ferritin and ruthenium red showed the negative charge to be dense and uniformly distributed over the epicuticular surface. Staining with acetic acid-ferric oxide hydrosol occurred at pH 1.65 and suggests that amino acid carboxyl groups were not responsible for the negative charge property. Alcian blue staining occurred at pH 0.5 and at a critical electrolyte concentration (CEC) of 0.9 M MgCl2, a property similar to that of highly sulfated mucopolysaccharides such as the proteoglycan keratan sulfate. In contrast, T. spiralis larvae failed to stain with alcian blue below pH 5.0 or at a CEC of 0.1 M, suggesting its negative charge is associated with dissociated amino acid carboxyl groups. Attempts to remove the negative charge-bearing components in the epicuticle of S. ratti by detergents, organic solvents, denaturing agents, proteases, uronidases, neuraminidases, and lipases were unsuccessful. The presence of elastin in the S. ratti larval outer cortical layer was indicated by its vulnerability to elastase and its reaction to aldehyde fuchsin-alcian blue stain. These results show that the epicuticle of S. ratti is not a typical cell membrane, although it appears to have ultrastructural similarities. It is suggested that the association of highly sulfated mucopolysaccharides with the epicuticular surface of free-living nematodes such as S. ratti L3 may reflect a greater need to protect against surface desiccation. It is also postulated that the highly negatively charged surface may have anticomplementary and anticoagulation effects.     

Immunological responses elicited by different infection regimes with Strongyloides ratti

Nematode infections are a ubiquitous feature of vertebrate life. In nature, such nematode infections are acquired by continued exposure to infective stages over a prolonged period of time. By contrast, experimental laboratory infections are typically induced by the administration of a single (and often large) dose of infective stages. Previous work has shown that the size of an infection dose can have significant effects on anti-nematode immune responses. Here we investigated the effect of different infection regimes of Strongyloides ratti, comparing single and repeated dose infections, on the host immune response that was elicited. We considered and compared infections of the same size, but administered in different ways. We considered infection size in two ways: the maximum dose of worms administered and the cumulative worm exposure time. We found that both infection regimes resulted in Th2-type immune response, characterised by IL4 and IL13 produced by S. ratti stimulated mesenteric lymph node cells, anti-S. ratti IgG(1) and intestinal rat mast cell protease II (RMCPII) production. We observed some small quantitative immunological differences between different infection regimes, in which the concentration of IL4, IL13, anti-S. ratti IgG(1) and IgG(2a) and RMCPII were affected. However, these differences were quantitatively relatively modest compared with the temporal dynamics of the anti-S. ratti immune response as a whole.     

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: in vitro and in vivo activity of tribendimidine

Background

Strongyloidiasis is a truly neglected tropical disease, but its public health significance is far from being negligible. At present, only a few drugs are available for the treatment and control of strongyloidiasis.

Methodology/Principal Findings

We investigated the activity of tribendimidine against third-stage larvae (L₃) of Strongyloides ratti in vitro and against juvenile and adult stages of the parasite in vivo. S. ratti larvae incubated in PBS buffer containing 10–100 µg/ml tribendimidine died within 24 hours. A single 50 mg/kg oral dose of tribendimidine administered to rats infected with 1-day-old S. ratti showed no effect. The same dose administered to rats harboring a 2-day-old infection showed a moderate reduction of the intestinal parasite load. Three days post-exposure a significant reduction of the immature worm burden was found. Administration of tribendimidine at doses of 50 mg/kg and above to rats harboring mature S. ratti resulted in a complete elimination of the larval and adult worm burden. For comparison, we also administered ivermectin at a single 0.5 mg/kg oral dose to rats infected with adult S. ratti and found a 90% reduction of larvae and a 100% reduction of adult worms.

Conclusion/Significance

Tribendimidine exhibits activity against S. ratti in vitro and in vivo. The effect of tribendimidine in humans infected with S. stercoralis should be assessed.     

Dissociation of the protective immune response in the mouse to Strongyloides ratti

The generation of protective immunity by various stages in the life-cycle of Strongyloides ratti and the phases against which resistance is directed has been examined in murine strongyloidiasis. Mice were exposed to natural, complete infections, were treated with thiabendazole (which largely resembles the natural infection), were treated with cambendazole (which restricts infection to the larval stage), or infected directly by oral transfer of adult worms. Mice that were infected with infective larvae alone did not become resistant to infective larvae or the complete infection but were resistant to adult worms implanted directly into the gut. Mice exposed to adult worms alone were resistant to natural infections and adults worms implanted directly but were not resistant to infective larvae. On the other hand, mice that had received prior natural infections showed evidence of resistance to infective larvae, adult worms, and natural, complete infections. It is concluded that there is immunological cross-reactivity between infective larvae and adult worms but that under certain circumstances the infective larvae are able to evade the host's protective immune response.     

The control of morph development in the parasitic nematode Strongyloides ratti.

The parasitic nematode Strongyloides ratti has a complex life cycle. The progeny of the parasitic females can develop into three distinct morphs, namely directly developing infective third-stage larvae (iL3s), free-living adult males and free-living adult females. We have analysed of the effect of host immune status (an intra-host factor), environmental temperature (an extra-host factor) and their interaction on the proportion of larvae that develop into these three morphs. The results are consistent with the developmental decision of larvae being controlled by at least two discrete developmental switches. One is a sex-determination event that is affected by host immune status and the other is a switch between alternative female morphs that is affected by both host immune status and environmental temperature. These findings clarify the basis of the life cycle of S. ratti and demonstrate how such complex life cycles can result from a combination of simple developmental switches.     

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 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.