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.     

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.     

The application of mass spectrometry to identify immunogenic components of excretory/secretory products from adult Dictyocaulus viviparus

Proteomics has come to the forefront in the post-genomic era. The ability to compare and identify proteins expressed in a particular cell type under specific physiological or pathological states requires a range of technologies, including separation of complex protein or peptide mixtures, densitometry-based or isotope-coded methods for comparison of multiple proteomes, and mass spectrometric methods for identification of individual low abundance proteins. Although an emergent technology, thus far, proteomics has provided new perspectives on many problems in biomedical science. In parasitology, proteomics has been used to answer specific biological questions relating to survival and development, and also to identify candidates for vaccines. Here, we describe an ongoing research programme in which proteomics is being used to identify potential vaccine candidates for the bovine lungworm, Dictyocaulus viviparus. This work is focusing on antibody responses to the adult parasite excretory/secretory (ES) products, with selection of candidate antigens based on differential screening with serum from immune versus non-immune animals to simplify the proteome and the ensuing analytical challenges. Thus far, we have identified seven candidate proteins using this strategy. Of these, one protein showed significant identity to a previously cloned gene from D. viviparus, whilst the other six proteins have shown no significant identities. Isolation of further peptide sequences is now warranted to facilitate cloning of the genes encoding these antigens.     

Trichinella spiralis and Strongyloides ratti: immune interaction in adult rats.

The consequences of prior and concurrent infection with two species of nematodes were studied in rats. Primary infection with Strongyloides ratti adversely affected the development of a secondary Trichinella spiralis infection. Both immediate and delayed challenge with T. spiralis, following the expulsion of the previous S. ratti infection, reduced the percentage of worm recovery of the former as well as their fecundities and lengths. It is suggested that nonspecific inflammation produced by one species, during the peak period of worm expulsion, was not responsible for the accelerated rate of expulsion of the other; instead a direct, specific cross-immunity was probably operative affecting the survival of the challenge species. The response elicited by previous experience of the intestinal phase was reciprocal, but there was evidence of an enhancing effect by the muscle larval stages of T. spiralis on S. ratti. Rats concurrently infected with both species expelled S. ratti more rapidly than T. spiralis. Possible mechanisms underlying the interaction between the two species are suggested and discussed.     

Strongyloides ratti: the role of enteral antigenic stimuli by adult worms in the generation of protective immunity in rats

Immunogenicity of adult Strongyloides ratti was studied in rats. Immunization of rats by intraduodenal implantation of adult worms could completely inhibit the egg production and hasten the expulsion of challenged worms which were developed from subcutaneously inoculated L3 or were implanted intraduodenally as adults. Enteral immunization by intraduodenal implantation of adult worms was, however, not able to affect the esophageal larval output of the challenge infection with L3. In contrast to enteral immunization with adult worms, immunization by full sequence of a primary infection or by a combination of drug-abbreviated infection and adult worm implantation could suppress the esophageal larval output of the challenge infection. The relationship between the host defense mechanism and the life cycle of S. ratti is discussed.     

Biochemical analyses of secretory and excretory products of adult Dipetalonema viteae in culture

Radioisotopically labeled glucose and pyruvate were employed to elucidate biochemical mechanisms utilized by the filariid Dipetalonema viteae during cultivation. Adults isolated from amicrofilaremic hamsters were incubated at 37 C in a mixture of NCTC135:IMDM (NI), with either D-[14C-(U)]glucose or [1-14C]pyruvate, under a gas phase of 5% CO2/N2 for 3 days. Labeled organic acids were separated and quantified by ion exchange chromatography. High performance liquid chromatography (HPLC) was used for separation and quantification of the 23 free amino acids in the NI medium. Ion exchange chromatography revealed that lactate was the major glycolytic end product, accounting for 90-97% of the original carbon utilized. Small amounts of radioactivity were recovered in succinate and variably in acetate fractions. HPLC analysis demonstrated that some amino acids increased, some decreased, and some remained at the initial concentration. Alanine exhibited the greatest change, consistently increasing from 2 to 4 times the original concentration. Analyses of purified amino acid peaks revealed radioactivity only in the alanine peak, accounting for 2-4% of the original carbon utilized.     

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.     

Fasciola hepatica: attempts to induce protection against infection in rats and mice by injection of excretory/secretory products of immature worms

In vitro excretory/secretory products of 4-week (immature) and 8-week-old (mature) Fasciola hepatica parasites, derived from rats, were injected together with adjuvant into naive rats and mice. Resistance to infection was assessed in rats by counting adults in the bile ducts at 9 weeks, or in mice by recording deaths after oral challenge with a high dose of viable metacercariae. Exposure of rats to excretory/secretory products of immature F. hepatica conferred a significant degree of resistance which was comparable to the level of resistance induced following oral administration of a low number of metacercariae. No protection against infection was seen in rats injected with excretory/secretory products from mature, bile duct-derived worms. In mice, no obvious mouse strain variation in susceptibility to first infection existed and hypothymic nude mice were as susceptible to infection as intact mice. As determined by protection against death, vaccination with excretory/secretory products derived from immature F. hepatica was without effect in mice. It is concluded that "host protective antigens", at least for rats, were present in the excretory/secretory products of immature F. hepatica larvae.     

A highly purified allergen from excretory and secretory products of Dirofilaria immitis

Preceding data revealed that the allergen concentrated mainly in excretory and secretory (ES) products exhausted by adult Dirofilaria immitis. The present paper reported that a highly purified allergen was obtainable from ES products more easily and effectively. An allergen in ES products was purified by a combination of DEAE-Sephadex A-50 chromatography, Sephadex G-200 and Sephadex G-100 gel filtration. The purified preparation was proved to be one protein by sodium dodecyl sulfate (SDS) gel electrophoresis and to be exact the same allergen with the one obtained from the crude extract of adult Dirofilaria worms. The molecular weight of the purified allergen was estimated to be 15,000, and the allergen was inclined to aggregate in the buffered solution.     

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.     

Characterization of endonuclease activity from excretory/secretory products of a parasitic nematode, Trichinella spiralis.

Double-stranded endonuclease activity was demonstrated for the first time in the excretory/secretory (ES) products of a parasitic nematode, Trichinella spiralis, which can reorganize host muscle cells. The endonuclease introduced double-stranded breaks to the native DNA. The ES double-stranded endonuclease(s) was sequence nonspecific, with a pH optimum below 6, and required divalent cations as a cofactor. Its activity was inhibited by the Zn2+ ion. It was detected mainly in the ES products of the infective-stage larvae of T. spiralis collected at 37 degrees C and was present in much smaller amounts in samples collected at 43 degrees C and in the products of T. pseudospiralis, a nonencapsulated species. The activity of endonuclease was blocked by antibodies against ES products. Zymographic analysis showed that the endonuclease activity was associated with at least three molecular forms, designated approximately 25, 30 and 58 kDa, respectively.     

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.     

On the biological and biochemical nature of cloned populations of Strongyloides ratti.

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.     

Demonstration of collagenase activity in adult Strongyloides ratti (Nematoda:Strongyloididae) and its absence in the infective larvae

Larvae and adults of Strongyloides ratti were examined for collagenolytic activity on 14C proline-labelled, native, guinea-pig skin collagen substrate. The activity was measured by determining either the amount of hydroxyproline released or the amount of radioactivity in the solubilized fraction of the collagen substrate. Bacterial collagenase was used for enzyme control and trypsin served as substrate control. No collagenolytic activity was found in living larvae, their extracts or metabolites. The collagenolytic activity of the metabolites of adult worms appeared weak, whereas that of the extracts of the adults was pronounced. It is suggested that collagenase is active in the adult females at the time of migration in the intestinal mucosa during oviposition.     

Strongyloides stercoralis: oral transfer of parasitic adult worms produces infection in mice and infection with subsequent autoinfection in gerbils.

Oral transfer of parasitic adult Strongyloides stercoralis produced patent infections in gerbils, C57BL/6J and SCID mice. In gerbils receiving adult worms, 7.3% of the transferred worms established and autoinfective L3 were found beginning on day 5 post-transfer, with peak numbers seen on days 6 and 7 post-transfer and few seen by 9 days post-transfer. These results suggest that development of autoinfective L3 in the gerbil is limited by the immune response of the host. When given orally to mice, between 7.2% (C57BL/6J) and 19.5% (SCID) of the adult worms established. These levels are higher than those previously obtained by the subcutaneous infection of SCID mice with infective larvae. No autoinfective larvae were found in infected mice and the ratio of L1/adult worms was small compared with that seen in gerbils. Thus, mice infected orally can be used as a model to study the interaction between the adult worm and the host, and since autoinfection has not been seen in the murine model, as developed to date, orally infected mice may be useful as a model to study mechanisms preventing autoinfection.     

Identification and partial characterization of excretory/secretory products with proteolytic activity in Giardia intestinalis

This report examines the presence of proteolytic activity detected in media collected from in vitro cultures of Giardia intestinalis, and the partial characterization by gelatin-substrate polyacrylamide gel electrophoresis and inhibition studies. Gelatin-substrate polyacrylamide gel electrophoresis revealed 6 bands with proteolytic activity, with estimated molecular weights of 36, 59, 63, 72, 103, and 175 kDa. These bands were not present in the control medium. On the other hand, G. intestinalis trophozoite lysates showed proteolytic bands at 16, 20, 66, 82, 108, and 120 kDa, thus indicating that intracellular proteases could be different from the excretory/secretory (E/S) products. Based on inhibition studies, 2 bands of 59 and 63 kDa were inhibited by iodoacetic acid, indicating the presence of cysteine proteases. Partial inhibition of a band of 36 kDa was found with EDTA, a metal-chelating agent, suggesting the possible presence of metalloproteases. The presence of aspartic and serine proteases were not detected under the assay conditions used. As G. intestinalis E/S may be involved in differentiation mechanisms of the parasite and also be responsible for the mucosal alterations that occur in giardiasis, the characterization of these proteases may facilitate their evaluation as targets in the therapy of the disease.