The course of infection in rats given small primary doses of Strongyloides ratti and S. venezuelensis

Development of exact doses (less than 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 (less than 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.     

Studies of the effect of contralateral versus ipselateral challenge of rats primed on one side with Strongyloides ratti

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 10,000 [corrected] 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 infection. Challenge infections of less than 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.     

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.     

The acquirement of growth ability for third-stage larvae of Strongyloides ratti during the head-passage in the rat

The role of larval passage through the head in the course of the migration of Strongyloides ratti in rats was investigated. Third-stage larvae (L3) recovered from various portions of donor rats were re-injected into the skin, cranial cavity and small intestine of recipient rats to check their ability for further growth. Cultured L3 (L3c) and the L3 recovered from the skin of donor rats (L3s) did not survive in the small intestine after intestinal inoculation. However, intestinal inoculation of L3 recovered from the head of donor rats (L3h) revealed growth to the adult stage. Cultured L3 injected into the cranial cavity of rats also became adult worms in the small intestine. L3 incubated in the cranial cavity for more than 24 h could grow in the small intestine of the recipient rats. These experiments suggest that S. ratti L3 acquire their ability to mature in the small intestine during their migration through the head of rats.     

Strongyloides ratti: formation of protection in rats by excretory/secretory products of adult worms

Formation of a marked protective immunity against the challenge infection was found in the rats immunized with excretory/secretory (ES) products of Strongyloides ratti adult worms. Immunization by intraduodenal injection of ES products reduced both the fecal egg counts and the adult worm burden by subcutaneous inoculation of infective larvae and by an intraduodenal implantation. The duration of parasitism in the immunized rats, however, was not shortened compared with that of control rats. The normal migration of subcutaneously challenged larvae was not affected by ES product immunization. Intestinal mastocytosis occurred according to the appearance of adult worms in the small intestine of the immunized rats earlier than it did in controls. This result suggests that mastocytosis is involved in the induction of protection by ES products of S. ratti adult worms.     

The effects of single and repeated inoculations of various larval doses on Strongyloides ratti burden and distribution in rats

The changes in worm burden, distribution, length, and fecundity after and during single and repeated inoculations of 10, 50, or 500 larvae of Strongyloides ratti were examined in rats. Worm burden after a single inoculation of a higher larval dose reduced rapidly. Repeated inoculations of lower larval doses at weekly intervals led to a delayed peak and slower reduction of worm burden; the repeated inoculations of 10 larvae did not induce worm expulsion for at least 7 wk. In repeated inoculations at 3-wk intervals, a primary inoculation of 500 larvae induced strong resistance to reinfection at week 3, whereas no resistance was induced until week 6 in rats receiving repeated inoculations of 10 or 50 larvae. Similar dose-dependent reductions in worm length and fecundity were observed in single and repeated inoculations, and the reductions began earlier than worm expulsion. Intestinal migration of worms from the upper small intestine to the large intestine was observed during the course of single and repeated inoculations. Earlier and clearer migration was observed in rats receiving higher doses. These findings indicate that in S. ratti infection, the changes of worm burden, distribution, length, and fecundity are dependent on the inoculated larval dose.     

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.     

Strongyloides ratti in virgin female rats: studies of oestrous cycle effects and general variability

There were no differences in mean intestinal worm burdens 8 days after subcutaneous injection of 4000 infective larvae of Strongyloides ratti into rats in dioestrus, pro-oestrus, oestrus and metoestrus. Thus, changes in the hormonal environment of the migrating larvae dependent on the oestrous cycle did not alter the worms' destination or affect their potential for development. In particular, the results are prima facie evidence that prolactin is not, on its own, responsible for the re-orientation of larvae in the tissues of nursing mothers. Other sources of variability in experimental S. ratti infections are analysed and the 'exact dose' technique offered as a corrective for some procedural errors.     

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 infection in the large intestine of wild rats, Rattus norvegicus

The large intestine of a rat has been neglected almost completely as a site of Strongyloides sp. infection. We reported that adult Strongyloides ratti remained in the large intestine for more than 80 days, producing more number of infective larvae than small intestine adults, and therefore hypothesized that parasitism in this site could be a survival strategy. In wild rats, however, no study has focused on large intestine infections of Strongyloides. The present study revealed that 32.4% of 68 wild rats, Rattus norvegicus, had the infection of S. ratti in the large intestine, with an average of 4.7 worms. These worms harbored normal eggs in the uterus. In a laboratory experiment with S. ratti and Wister rats, daily output of infective larvae by 4.7 females in the large intestine was estimated to be 4,638.4, suggesting that a few parasites could play a role in the parasite transmission. Five species of nematode found in the wild rats showed seasonality in infection intensity, with highest intensities in March-May. The number of S. ratti in the large intestine was also highest in these months.     

Tissue migration capability of larval and adult Brugia pahangi

Infection with mosquito-born filarial nematodes occurs when hosts are bitten by a vector carrying the infective third stage larvae (L3) of the parasites. These larvae, deposited on the skin by the feeding mosquito, are presumed to enter the skin via the vector-induced puncture wound. Larvae of Brugia spp. must then migrate from the entry site, penetrate various skin layers, and locate a lymphatic vessel that leads to their lymphatic predilection site. We have recently established an intradermal (ID) infection model using B. pahangi and the Mongolian gerbil, allowing us to investigate the migratory capability ofB. pahangi. Larval and adult parasites recovered from the peritoneal cavities of gerbils were capable of establishing an infection following ID (larvae) or subcutaneous (adult) injection. Third and fourth stage larvae both migrated away from the injection site within hours, although data suggest they localize to different lymphatic tissues at 3 days postinfection (DPI). Immature adult (28 day) B. pahangi also migrated away from their SC inoculation site within 7 DPI. Mature (45 day) adult B. pahangi displayed little migration away from the SC infection site, suggesting tissue migration may be limited to developing stages of the parasite.     

Strongyloides spp.: demonstration and partial characterization of acidic collagenolytic activity from infective larvae

Infective larvae of Strongyloides spp. have been shown to contain azocollytic enzymes which may aid in host skin penetration. Attempts to demonstrate classical, neutral pH-active collagenase activity in Strongyloides ratti were unsuccessful. In the current study, we investigated the presence of acidic collagenolytic activity in the infective larvae of Strongyloides ransomi, S. ratti, and S. stercoralis. All three species demonstrated collagenolytic activity in acidic homogenates as well as in neutral freeze-thaw fractions. Biochemical characterization of this collagenolytic activity from S. ratti and S. ransomi indicated that it was active over an acidic pH range, although it was stable at a neutral pH. This, along with molecular weight estimates and inhibitor susceptibilities, suggested that the collagenolytic activity was similar to vertebrate acidic cysteinyl proteinases. These studies also indicated that this activity is similar to the acidic cysteinyl proteinases in extracts of S. ransomi.     

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.     

The occurrence of Strongyloides ratti in the tissues of mice after percutaneous infection

The migration of infective larvae of Strongyloides ratti has been examined in C57Bl/6 mice after percutaneous infection of the anterior abdominal wall. Lateral migration of larvae through the skin and subcutaneous tissues was not seen. Large numbers of larvae were recovered from the muscles between 2 and 24 hours after infection and larvae were seen in the cerebrospinal fluid 24 and 48 hours after infection. Insignificant numbers of larvae were seen in the blood, serosal cavities, liver, spleen, kidneys, brain or nasopharynx. Larvae arrived in the lungs between 24 and 72 hours after infection and worms were first noted in the small intestines at 48 hours. It is concluded that larvae migrate preferentially to the muscles and CSF before passing to the lungs, but the exact mode of travel is uncertain.     

Skin penetration of infective hookworm larvae. I. The path of migration of infective larvae of Ancylostoma braziliense in canine skin

Migratory behaviour of Ancylostoma braziliense was studied in relation to the structure of the skin in dogs after primary infections. Data were obtained studying serial sections of lateral skin areas 6 mm in diameter, which had been exposed to larvae. The sections were stained either with Harris' haematoxylin and eosin or with P.A.S. or as outlined by Crossmon. Most of the larvae managed to penetrate the skin within 1/2 hr after the application. Hairs did not seem to constitute sites of entry. The larvae moved into the horny layer where edges of keratinized cells provide uneven spots. They migrated approximately parallel to the surface from the horny layer into the living epidermis and continued into an external root sheath of a hair follicle. They could only leave this site via sebaceous glands for the dermis or via apocrine sweat glands for the hypodermis. Tunnels from the epidermis into the dermis, however, suggested that a direct trans-epidermal migration had occurred. The vessels invaded by larvae were hypodermal lymphatic vessels. The first ones were found in these structures 1/2 h after the onset of the exposure.     

Strongyloides ratti: Dissociation of the rat's protective immunity into systemic and intestinal components

Analysis of the early stages of a challenge infection with Strongyloides ratti has shown that protection is expressed against the developing third-stage larval worms (L₃) and prevents the maturation to adulthood of most larvae. Challenge after an immunizing infection that was restricted to the parenteral L₃ migratory phase showed that some 10–40% of overall protection could be ascribed to systemic antilarval immunity. Some larvae were trapped in the skin at the site of injection whereas others failed to migrate to the head and lung of immune rats. Larvae arriving in the intestine at Days 3, 4, and 5 did not persist beyond Day 7 and 8. Studies using [⁷⁵Se]methionine-labeled L₃ showed a significant increase in fecal label in rats immunized by a complete infection. This loss did not occur to the same extent in rats immunized only with parenteral larvae. Significant rejection of worms transplanted to the intestine also indicated intestinal protection. The possible existence of large numbers of worms in a state of “arrested development” was excluded by their failure to appear after cortisone treatment and the absence of worm accumulation in radiolabeling studies. It is concluded that at least two responses operate against larval S. ratti, one is systemic and the other operates in the intestine against larvae in a manner that resembles the “rapid expulsion” rejection of Trichinella spiralis in immune rats.     

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.     

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.     

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.