Mucosal mast cells (MMC) play a crucial role in the expulsion of Strongyloides ratti adults from the small intestine of mice. We reported the large intestinal parasitism of S. ratti in rats, and there has been no report on MMC in the large intestine of the natural host. We studied kinetics of MMC, together with eosinophils, in the upper and lower small intestines, caecum and colon of infected rats. Two distinct phases of mastocytosis were revealed: one in the upper small intestine triggered by stimulation of ‘ordinary’ adults, and the other in the colon stimulated by ‘immune-resistant’ adults that started parasitizing the colon around 19 days post-infection. In all 4 intestinal sites, the MMC peaks were observed 5–7 days after the number of adult worms became the maximum and the height of MMC peaks appeared to be dependent on the number of parasitic adults, suggesting an important role played by worms themselves in the MMC buildup.

Development of exact doses (<100) of Strongyloides venezuelensis third-stage larvae in adult Wistar rats was insignificant (mean proportion of 0·076 of the dose at day 8, n = 16) compared with a homogonic strain of S. ratti (0·538, n = 6; 0·726, n = 6) and heterogonic S. ratti (0·681, n = 6). Newly-weaned Wistars allowed development of a mean proportion of S. venezuelensis of 0·298 (n = 4) compared with 0·013 (n = 4) of the same sample of larvae in adult hosts. Experiments with 75Se-labelled larvae established that S. venezuelensis effectively failed to migrate from skin to intestine in adult animals, while mean proportions of 0·141 (n = 5) and 0·138 (n = 4) of the label was found in the intestines of newly-weaned rats 72 h after skin application. Labelled larvae of homogonic S. ratti migrated equally well in both age groups of host (0·350 and 0·358 in 12- and 3-week-olds respectively). Adult S. venezuelensis transferred surgically to the intestines of previously uninfected full-grown Wistars survived over a 21-day period to the same extent as either strain of S. ratti. Resistance of Wistar rats to S. venezuelensis therefore appears to affect the migratory stage preferentially. S. venezuelensis developed better in mature PVG inbred rats (mean = 0·301, n = 20). Studies of S. ratti showed that infections of both strains initiated by exact (<100) doses in Wistar rats had decayed to insignificance between days 26 and 32. The rate of loss of adults of the heterogonic strain was significantly greater than that for the homogonic. The egg content of worms declined as infection progressed and rats were idiosyncratic in their influence on parasite reproduction from the earliest time of sampling (8 d). It was established that ‘autoinfection’ was an unlikely feature of the biology of homogonic S. ratti following the surgical transfer of 450 first-stage larvae to the intestines of 8 adult Wistar rats. No evidence of infection appeared in the guts of these animals 8 days post-transfer. The significance of these results in terms of the biology of Strongyloides spp. naturally occurring in the rat is discussed.

Immunological memory generated by infection with S. ratti was studied separately in the migratory and intestinal phases in mice. Protection against reinfection in the migratory phase was 96–98% at 2 weeks but significantly decreased to 60% at 12 weeks after the primary infection. However, protection in the intestinal phase was 96% even 12 weeks after the primary infection. Recall of immunity against the intestinal phase persists for longer than that against the migratory phase in mice.

Excretory and secretory (ES) products collected from adult worms of Strongyloides ratti stimulated interleukin-3 (IL-3) production with mesenteric lymph node cells from infected C57BL/6 mice, hut not with normal mesenteric lymph node cells. The IL-3 stimulating components were not major IgG binding antigens. Activity of the IL-3 stimulating components was stable by treatment with protease, although reduced by heating in boiling water.

The effect of linolenic acid (C18:3ω3) on the development of Strongyloides ratti first-stage larvae (L1) in culture was studied. The fatty acid composition of S. ratti free-living generations was analyzed by gas chromatography. L1 had abundant linoleic acid (C18:2ω6) but its proportion decreased with development. On the contrary, eicosapentaenoic acid (C20:5ω3) and C20:4ω3 were prominent in the filariform larva (L3). Because C20:5ω3 is generally synthesized from C18:3ω3 via C20:4ω3, the high ratio of C20:5ω3/C18:3ω3 of L3 in all the free-living generations suggested that polyunsaturated fatty acid metabolism, particularly the ω-3 series, and eicosanoids produced had important roles in the development of S. ratti L1.

The initial pathway of skin penetrating larvae of Strongyloides ratti inside the host is not systemic and could well involve local components of the lymphatic system. The experiments described were an attempt to detect an effect on immunity depending on whether larvae of a challenge infection were committed to a pathway through heavily primed or lightly primed lymph nodes. Female rats were immunised by subcutaneous injection into the right forearm of 1000 heat killed, or 1000 live, third stage larvae of S. ratti. Animals given living parasites were placed on a diet containing 0·1 or 0·2% thiabendazole 48h or 36h after ifection. Challenge infections of <100 larvae (‘exact’ doses) were applied on day 21 to either the right or left flank and, similarly, to controls that had received the anthelmintic but not the priming infection. Heat killed parasites elicited no response at all. Priming with live larvae stimulated a significant immunity (16% and 37% depression in 2 experiments), but there was no difference between rats whose challenge was on the same side as the priming dose and those which had the contralateral treatment. The significance of these results to theories of pathfinding in general, and to practical immunology, is discussed.

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 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.

Subcutaneous injection of the larvae is the almost universally adopted means of initiating experimental infections of skin-invading roundworms but, so far, the possibility that this procedure introduses artefacts of one kind or another has not been critically studied. Experiments described in this paper were used to compare the effect of (a) injection and (b) skin application, of a small, precisely counted (‘exact’) dose of larvae. Results with two strains of s. ratti shoewed that the same proportion the dose developed to adults in the intestines of rats irrespective of the method. With the same exact dose technique it has been shown that milk-borne infection of the pups of lactating rats is not an artefact produced by injectcion. Large doses (mean 4000) of larvae of the homogonic strain of s. ratti carrying a radioactive label of 75 Se were tracked in their migration to the mammary gland following injection or skin application at two different sites on the right-hand side of nursing mother rats. The broad conclusion of earlier work in this laboratory using injection. that larvae move by a local route and not a systemic one, was supported by the results. The detailed distribution of the label and of unlabelled worms of the heterogonic strain in families was, however, different for the methods. indicating that subtle variations in pathway can be brought about by the use of injection. If migration involves the Lymphatic system, then the interpretation of immunological experiments in terms of Lymphatic anatomy must take account of such procedural effects. The extent to which these results contribute to theories of migration in Strongyloides ratti is discussed.

Seven geographically varied isolates of Strongyloides ratti were cloned. Each clone was examined for the degree of homogonic development of the free-living generation and by enzyme electrophoresis. Considerable variation was found between the clones in the degree of development that was homogonic. In Contrast, the clones were indistinguishable by enzyme electrophoresis.

Strongyloides ratti has a complex life-cycle with two adult generations, one free-living and dioecious and one parasitic and female only. The parasitic females reproduce by parthenogenesis, but it is unclear whether this is mitotic or meiotic in nature. This question has been addressed genetically by analysing the progeny of parasitic females that were heterozygous at an actin locus for evidence of allelic segregation. Such progeny were similarly heterozygous showing that segregation had not occurred. It was therefore concluded that reproduction in the parasitic female of S. ratti is functionally mitotic.

This study provides the first quantitative assessment of the cutaneous reaction elicited in naive and sensitized rats by invading larvae of Strongyloides ratti. Parasites were identified in skin samples harvested from both naive and sensitized rats between 0·5 and 8 h following percutaneous infection, but were no longer visible at the site of invasion at 24 h. Mast cells, neutrophils, eosinophils and mononuclear cells recruited into the dermis in response to invading larvae were recorded and quantified. Cellular infiltrates were observed as early as 1 h post-invasion in naive/challenged skin, where total cell numbers were up to 3 times greater than in naive control tissue. Peak numbers of all cell types were recorded from 3 to 8 h in both naive/challenged and sensitized/challenged hosts. Interestingly, the reaction in sensitized/challenged rats was neither enhanced nor accelerated, a feature perhaps attributable to lack of recognition of parasite antigens by the host. This possibility was investigated by immunofluorescent labelling, which clearly demonstrated changes in the surface antigen profile of the parasite following penetration of the host skin both in vitro and in vivo. It is proposed that these changes in surface antigenicity constitute an evasive stratagem used by the parasite to deter the host from mounting a potentially lethal inflammatory response.

All mammalian-parasitic stages of a range of nematode species investigated (Brugia pahangi, Acanthocheilonema viteae, Strongyloides ratti, Nippostrongylus brasiliensis, Trichinella spiralis and Ostertagia ostertagi) labelled in a surface-restricted manner with the fluorescent lipid analogues 5-N-(octadecanoyl)aminofluorescein (AF18) or nitrobenzoxadiazole-cholesterol (NBD-chol), but failed to bind other similar probes. In contrast, the surfaces of the ‘pre-parasitic’ infective stages of these species had affinity for neither AF18 nor NBD-chol. This exclusion of lipid analogues changed rapidly upon exposure of the larvae to tissue culture conditions which mimic the mammalian tissue environment (e.g. RPMI 1640/37°C) such that the above probes could then insert into the surface layer of the larvae. The dauer larva of Caenorhabditis elegans also excluded the probes, but became permissive to labelling upon stimulation to emerge from the dauer state. The time taken for the surface transformation to occur ranged from less than 10 min in the vector-borne parasites to approximately 5 h in those which enter by the oral route, with direct skin-penetrators occupying an intermediate position. In all cases, the alteration proceeded too rapidly for it to have been associated with a moult. Fluorescence Recovery After Photobleaching (FRAP) studies of A. viteae larvae showed that approximately 50% of the AF18 probe was free to diffuse within the plane of the surface immediately after transformation. This is only a transitory state because AF18 was found to be highly restricted in its lateral diffusion on the surface of adult parasites. In the larvae of S. ratti, the change in affinity for AF18 was accompanied by the rapid shedding of an otherwise stable surface coat of polyanionic material, here visualized by labelling with fluorescein-conjugated cationized ferritin. Incubation of larvae in lipid-rich host serum during the induction of transformation inhibited subsequent labelling with AF 18. This possibly reflects competition for insertion sites and an in vivo propensity towards the acquisition of host lipid by invading parasites.

The possibility of coproantigen detection in the diagnosis of strongyloidiasis was investigated in rats experimentally infected with Strongyloides ratti. A capture ELISA was developed using affinity-purified polyclonal antibodies raised in rabbits against adult and filariform larval somatic antigens. The first detection of coproantigen coincided with patency and the coproantigen level remained elevated even when the parasite count was low. Following challenge infection, when examination of stool for larvae or ova was negative, coproantigen was detected by anti-adult somatic capture ELISA. Despite the use of antisera raised against crude worm extracts, the test was specific showing only low cross-reactivity with Necator americanus and Syphacia muris.

Strongyloides ratti is a parasitic nematode of rats. The host immune response against S. ratti affects the development of its free-living generation, favouring the development of free-living adult males and females at the expense of directly developing, infective 3rd-stage larvae. However, how the host immune response brings about these developmental effects is not clear. To begin to investigate this, we have determined the effect of non-immune stresses on the development of S. ratti. These non-immune stresses were subcurative doses of the anthelmintic drugs Ivermectin, Dithiazanine iodide and Thiabendazole, and infection of a non-natural host, the mouse. These treatments produced the opposite developmental outcome to that of the host immune response. Thus, in infections treated with subcurative doses of Ivermectin, Dithiazanine iodide and in infections of a non-natural host, the sex ratio of developing larvae became more female-biased and the proportion of female larvae that developed into free-living adult females decreased. This suggests that the mechanism by which the host immune response and these non-immune stresses affect S. ratti development differs.

The host immune response has profound effects on parasitic nematode infections. Here we have investigated how a range of infection parameters are affected by host immune responses and by their suppression and enhancement. The infection parameters considered were the number of parasitic females, their size, per capita fecundity and intestinal position. We found that in immunosuppressive treatments worms persist in the gut, sometimes with a greater per capita fecundity, maintain their size and have a more anterior gut position, compared with worms from control animals. In immunization treatments there are fewer worms in the gut, sometimes with a lower per capita fecundity and they are shorter and have a more posterior gut position, compared with worms from control animals. Worms from animals immunosuppressed by corticosteroid treatment reverse their changes in size and gut position. This description of these phenomena pave the way for a molecular biological analysis of how these changes in infection parameters are brought about by the host immune response.

In theory, the age at which maturation occurs in parasitic nematodes is inversely related to pre-maturational mortality rate, and cross-species data on mammalian nematodes are consistent with this prediction. Immunity is a major source of parasite mortality and parasites stand to gain sizeable fitness benefits through short-term adjustments of maturation time in response to variation in immune-mediated mortality. The effects of thymus-dependent immune responses on maturation in the nematode parasites Strongyloides ratti and Nippostrongylus brasiliensis were investigated using congenitally thymus-deficient (nude) rats. As compared with worms in normal rats, reproductive maturity of parasites (presence of eggs in utero) in nude rats occurred later in S. ratti but earlier in N. brasiliensis. Immune-mediated differences in maturation time were not associated with differences in worm length. Thymus-dependent immunity had no effect on pre-maturational mortality. Results are discussed in relation to theoretical expectations and possible explanations for the observed patterns in parasite maturation.

Factors constraining the evolution of host-specificity were investigated using a gastrointestinal parasitic nematode, Strongyloides ratti. S. ratti is a natural parasite of rats which can also reproduce, with decreased success, in laboratory mice. Observed host-specificity arose from lower establishment, reduced per capita fecundity and more rapid expulsion of parasites from mice relative to rats. Variation in the efficacy of thymus-dependent immunity between host species (rats and mice) was insufficient to explain the majority of the observed differences in parasite establishment and reproductive success. The role of natural selection in determining host-specificity was addressed using experimental selection followed by reciprocal fitness assays in both host species. Experimental selection failed to modify the host-specificity of S. ratti to any measurable degree, suggesting either a lack of genetic variation for this trait or the involvement of as yet unidentified factors underlying the differences in S. ratti fitness in rats and mice respectively. These results are discussed in relation to competing theoretical models of ecological specialization, host immunology and previous attempts to experimentally alter the host-specificity of parasitic nematodes

In Strongyloides ratti-infected rats, 2 peaks of egg excretion were observed; a large one with maximum egg production on days 7–8 of infection and a small more inconspicuous one around day 25. The second peak, which had been ignored in most studies, was produced by adults in the caecum and the colon. The adults were larger in length and had more embryonated eggs in the uterus compared with adults in the small intestine at day 25 post-infection. It is suggested that parasitic adults once expelled from the small intestine resettle and recover in the large intestine. Filter paper faecal culture carried out for 9 days at different days post-infection revealed that the total number of infective larvae that developed during the second peak was twice the number that developed during the first peak, despite the fact that total egg output during the second peak was less than one twentieth of the first peak. The results suggest that the small second peak was as important as the first one in the transmission of S. ratti.

The distribution of helminth parasites within their host population is usually overdispersed and can be described by the negative binomial distribution. The causes of this overdispersion are poorly understood, but heterogeneity in the distribution of infective stages within the environment has been implicated as a possible factor. Here we describe the distribution of infective stages of the rat intestinal nematode parasite Strongyloides ratti among the faecal pellets of its host. The distribution of infective stages between faecal pellets is overdispersed and well described by the negative binomial distribution. This overdispersion increases during the course of infection and occurs over a range of infection intensities. Overdispersion of nematode infective stages among faecal pellets may result in increased spatial heterogeneity of the infective stages in the environment and thus may contribute to the generation of overdispersion of adult parasitic stages. In addition, these findings raise important issues regarding the accurate quantification of helminth egg counts from faecal samples.