Carolyn Northern

Infection and immunity in dogs infected with a human strain of Strongyloides stercoralis

The course of infection and immunological responses in dogs infected with a strain of Strongyloides stercoralis of human origin were investigated. The first dog infected developed a chronic infection lasting at least 15 months. Larvae disappeared from the faeces by three months after infection in another four dogs; these animals were resistant to challenge infection. A further dog developed a chronic infection of low intensity which could not be boosted by repeated heavy infections. These differences may be genetically determined. Immune responses in primary infections were measured in four dogs. A blood eosinophilia occurred in infected animals. Anti-Strongyloides antibodies of the IgM class appeared one week after infection, peaked at three weeks then slowly declined in titre while IgG antibodies appeared slightly later and then persisted in high titre. When compared with uninfected control dogs, no significant differences were seen in PHA stimulation of peripheral blood lymphocytes, nor did significant lymphocyte proliferation occur in the presence of Strongyloides antigen. Infected dogs showed marked immediate hypersensitivity to antigen injected intradermally, but Arthus and delayed hypersensitivity reactions were not seen. This model of human strongyloidiasis merits further investigation.

Persistent and disseminated infections with Strongyloides Stercoralis in immunosuppressed dogs

Abstract The effects of immunosuppression on the course of infection in dogs infected with a human strain of S. stercoralis were investigated. Four dogs were infected then 4 weeks later, three animals were begun on prednisolone orally in a dose of 150 mg daily for 6 days each week. Rhabditiform larvae continued to be excreted in the stools. After 5 weeks of immunosuppression, one dog was autopsied; plentiful parasites were found in the upper small bowel but worms were not seen in other tissues. Similar findings were made in a second dog killed after 9 weeks of immunosuppression. In the third dog, the dose of prednisolone was increased to 225 mg daily 15 weeks after infection then azathioprine 100 mg daily was added 6 weeks later. This animal was killed 24 weeks after infection and evidence for multiplication of S. stercoralis was obtained. Large numbers of adult worms, eggs and rhabditiform larvae were recovered from duodenal fluid. Infective larvae were seen in homogenised lung, rhabditiform larvae were noted in homogenised spleen and both rhabditiform larvae and adult worms were found in homogenised kidney. Worms in all stages of development were seen in the urine. Histological examination revealed large numbers of adult worms and enormous numbers of eggs and larvae in the duodenal mucosa. Worms were seen throughout the length of the small intestine as well as in the colonic mucosa. The lungs displayed focal haemorrhages and larvae were seen in the alveolar spaces, bronchioles and bronchi. Evidence of immunosuppression was provided by the gross atrophy of the thymus and the fall in anti-Strongyloides antibody titres. In contrast to these dogs, larvae disappeared from the non-immunosuppressed dog by 13 weeks after infection. This animal was then immunosuppressed for 8 weeks but parasites could not be found in either the stools or at autopsy. Two further dogs were immunosuppressed before infection but they died soon thereafter. It is concluded that immunosuppression permits the persistence of infection with S. stercoralis and if continued for long enough and in sufficient degree, that disseminated infection supervenes. This model may provide a means for assessing the efficacy of various therapeutic regimens in overwhelming strongyloidiasis and for investigations of the host-parasite relationship in this infection.

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 stercoralis infections in the muscles of mice: a model for investigating the systemic phase of strongyloidiasis

&NA; The sequence of events following infection of mice with the intestinal nematode, Strongyloides stercoralis, has been observed. Most infective larvae passed to the muscles where they did not develop further. In mice given primary infections, larvae were found in muscles for the first 9 d or so, then disappeared spontaneously. This was associated with an inflammatory reaction, predominantly eosinophilic and histiocytic in nature, around dying larvae. In mice exposed to the worms previously, both inflammation and worm destruction were hastened indicating the acquisition of resistance. A number of immunological parameters were measured in both primary and challenge infections. Specific antibodies of the IgM and IgG classes appeared, a marked immediate hypersensitivity reaction to injected antigen developed, and a transient blood eosinophilia occurred. No effects on phytohemagglutinin‐induced spleen cell transformation were discerned, nor was transformation induced by specific antigen. It is concluded that this system provides a potentially useful model for investigating the systemic phase of strongyloidiasis, particularly with respect to assessing anthelmintic efficacy and the functions of fractionated antigens.

Western blot analysis of reactivity to larval and adult Strongyloides ratti antigens in mice

Summary Mice were infected once, twice or many times with Strongyloides ratti infective larvae, and the parasite was allowed to complete its development. Other mice were infected many times with either infective larvae only, by aborting the infection with cambendazole. or with adult worms transferred by intraoesophageal intubation. Sera from these animals were analysed by immunoblotting against SDS‐PAGE separations of larval and adult worm water‐soluble, deoxycholate‐soluble, sodium dodecyl sulphate‐soluble and excretory/secretory antigens. Minimal antibody responses were observed after primary and secondary infections. Mice exposed to multiple complete infections reacted strongly to both larval and adult antigens but greater responses were observed against the larval preparations. Stage‐specific effects were noted in mice infected with larvae only or adult worms only. Mice exposed only to larvae reacted with larval antigens and to a minor degree to somatic adult worm antigens while those mice which were exposed only to adult worms failed to react with any of the antigen preparations. Some cross‐reactions were found, however, as mice infected only with larvae displayed strong reactions against both larva] and adult excretory/secretory products. These data demonstrate differences in sero‐reactivity to infective larvae and adult worms and suggest that humoral immunity is induced by larvae migrating through the tissues and not by adult worms in the gut.

Antigenic analysis of Strongyloides ratti infective larvae and adult worms

The protein composition of Strongyloides ratti infective larvae and adult worms extracted sequentially in water, sodium deoxycholate and sodium dodecyl sulphate (SDS) and their excretory/secretory products were analysed by both one‐ and two‐dimensional SDS‐polyacrylamide gel electrophoresis. While many bands common to all preparations and both stages of the worm were seen, a number of bands unique to each stage and preparation were identified. Western blot analysis of these larval and adult preparations for reaction with IgG and IgM antibodies in hyperimmune mouse sera revealed a large number of antigens. These data provide a framework for analysis of protective and diagnostically useful antigens.

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

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.

The effects of thiabendazole, mebendazole and cambendazole in normal and immunosuppressed dogs infected with a human strain of Strongyloides stercoralis

The effects of three benzimidazole anthelmintics in dogs infected with a human strain of Strongyloides stercoralis were investigated. Cambendazole, but not thiabendazole or mebendazole, abrogated the subsequent development of a patent infection when administered at the same time as infection to immunocompetent dogs. None of the drugs eradicated infection when given after the onset of patency in immunosuppressed animals, although worm burdens were greatly reduced in dogs treated with cambendazole. The implications of these findings for the treatment of patients with strongyloidiasis, particularly those with disseminated infections, are discussed.

Light and electron microscopical studies of the location of strongyloides stercoralis in the jejunum of the immunosuppressed dog

Abstract This study was undertaken to define the precise anatomical location of S. stercoralis in the intestinal mucosa of the dog. In order to facilitate finding worms, hyperinfection was induced by immunosuppression of the host with prednisolone. Light microscopy showed adult worms in vacuoles in close relationship with the columnar epithelium although portions of worms were sometimes seen in the intestinal lumen. Electron microscopy demonstrated that adult worms were situated between the enterocytes. The enterocytes were compressed and distorted and appeared to form a tunnel through which the worm probably moved. Adult worms were never observed to penetrate the basement lamina and enter the lamina propria. Rhabditiform and filariform larvae were never seen in the mucosa. These data suggest that autoinfection does not occur by rhabditiform larvae being seeded directly into the tissues, but supports the hypothesis that they are deposited in the small bowel lumen, moult, then penetrate the mucosa of the lower gastrointestinal tract. Since adult worms dwell in the epithelial layer of the mucosa, they are susceptible to attack by cellular elements of the hosts defences, although this was not observed in the present study because the host was immunosuppressed.