Species-specific differences in heterogonic development of serially transferred free-living generations of Strongyloides planiceps and Strongyloides stercoralis

The direction of free-living development (homogonic vs. heterogonic) in Strongyloides stercoralis and Strongyloides planiceps was examined by successive transplantation of the uterine eggs of free-living females into a test tube culture system containing fresh feces. The eggs from the first-generation free-living females of S. stercoralis did not develop into second-generation free-living adult worms, but all developed into filariform larvae. However, the majority of S. planiceps eggs from the first-generation free-living females developed into second-generation free-living adults. By successive transfer of uterine eggs of each generation, 9 generations developed, and in every cycle more adult worms developed than filariform larvae. However, the number of free-living generations was not infinite; in experiments repeated twice, the number of worms developing from the eggs of eighth or ninth generations was too small to continue further culture. These findings indicate that the pattern of free-living development is different between the 2 species.     

Diagnosis of Strongyloides stercoralis in a peritoneal effusion from an HIV-seropositive man. A case report

BACKGROUND: Strongyloides stercoralis, a nematode parasite in humans with free-living and autoinfective cycles, is often an asymptomatic infection of the upper small intestine. If the host becomes immunocompromised, autoinfection may increase the intestinal worm burden and lead to disseminated strongyloidiasis. The parthenogenetic adult female larvae can remain embedded in the mucosa of the small intestine for years, producing eggs that develop into either rhabditiform, noninfective larvae or filariform, infective larvae. Manifestations of dissemination occur when the filariform larvae penetrate the intestinal wall and migrate into the blood. Pulmonary involvement is common, and the central nervous system may be affected. Blood eosinophilia is typical, and gram-negative sepsis from enteric bacteria may occur. Much less commonly described is invasion of the peritoneal cavity with peritoneal effusion. CASE: A 49-year-old man who came to the United States from Liberia 4 years earlier presented with sudden onset of severe abdominal distention, generalized weakness and marked pedal edema. Diagnostic paracentesis showed numerous filariform larvae of S stercoralis. Stool examination confirmed the presence of both rhabditiform and filariform larvae. Subsequently the patient was found to be HIV seropositive, with a CD4 lymphocyte count of 59. CONCLUSION: Early detection of S stercoralis may alter the often-fatal course of infection. The present case is the second reported one in the English-language literature of the diagnosis of S stercoralis in ascitic fluid.     

A case of fatal hyperinfective strongyloidiasis with discovery of autoinfective filariform larvae in sputum

The autoinfective filariform larva of Strongyloides stercoralis causes hyperinfection in immunosuppressed hosts. Here we report on the case of a male patient who was admitted to the emergency room at Gwangju Veterans Hospital with a complaint of dyspnea, and who was receiving corticosteroid therapy for asthma. Many slender larvae of S. stercoralis with a notched tail were detected in Papanicolaou stained sputum. They measured 269 +/- 21.2 microm in length and 11 +/- 0.6 microm in width. The esophagus extended nearly half of the body length. The larvae were identified putatively as autoinfective third-stage filariform larvae, and their presence was fatal. The autoinfective filariform larva of S. stercoralis has not been previously reported in Korea.     

Strongyloides stercoralis: Hyperinfection in immunosuppressed dogs

Hyperinfective strongyloidiasis involving the threadworm, Strongyloides stercoralis, is well known in humans and primates. Although this nematode also frequently parasitizes dogs, canine hyperinfective strongyloidiasis has not been reported. To determine whether a fulminant pattern of nematode development can occur in dogs, and to test the S. stercoralis/dog system for suitability as a model for human hyperinfective and disseminated strongyloidiasis, five canine infections with a dog-derived strain of S. stercoralis were monitored by the quantitative recovery of larvae from feces. Even 3-month-old pups controlled their initial infections successfully, the number of larvae excreted declining to near zero in 90 days. Immunosuppressive treatment with prednisolone, prednisolone and azathiaprine, or niridazole resulted in a rapid return to former or greater intensities of infection, as judged by larval output. Only first stage (rhabditiform) larvae were passed in the feces, although third stage (filariform) larvae occurred in the intestinal contents of dogs when they were examined at necropsy. In 3 of the 5 dogs, the adult worm recovery exceeded the inoculated dose greatly and, in one of these, adults and rhabditiform larvae were found in distant, extraintestinal sites. In the remaining 2 of the 5 dogs, the adult worm population was less than the inoculated dose, but, in both, the infection was terminated by the host's death before hyperinfection could have developed. The observations demonstrate that autoinfection occurs in dogs infected with S. stercoralis and that, if it is allowed to continue for a sufficiently long time in immunosuppressed hosts, massive hyperinfection, and even disseminated infection, may occur. This spectrum of increasingly invasive parasitism closely resembles strongyloidiasis in humans. Therefore, the S. stercoralis/dog system has excellent potential as a model for human hyperinfective and disseminated strongyloidiasis.     

A case of Strongyloides stercoralis infection.

Strongyloidiasis has been recognized as one of the life-threatening parasitic infections in the immunocompromised patients. We report an intestinal infection case of Strongyloides stercoralis in a 61-year-old man. Rhabditiform larvae were detected in the stool examination and developed to filariform larvae having a notched tail through the Harada-Mori filter paper culture. The patient received five courses of albendazole therapy but not cured of strongyloidiasis.     

A case of strongyloidiasis accompanied by duodenal ulcer]

A 58-year-old chronic alcoholism patient, who complained of epigastric discomfort, nausea, and frequent loose stool was diagnosed as strongyloidiasis accompanied by duodenal ulcer. The symptoms first appeared two years ago and aggravated during the recent 3 months, and he lost 4 kg of his body weight. Stool examination revealed rhabditoid nematode larvae, which were confirmed as those of Strongyloides stercoralis after cultivation of them to filariform larvae. At duodenoscopy, duodenal ulcer was found. The patient was treated with albendazole (200 mg, bid, for 14 days) for strongyloidiasis and with colloidal bismuth sulfate (240 mg, bid, for 6 weeks) for duodenal ulcer. After the medication, the symptoms of loose stool and epigastric discomfort were much improved and he was discharged with no clinical problems. This is an interesting case which suggests that S. stercoralis infection could be related with ulceration of the duodenal mucosa.     

Radiolabeling of infective third-stage larvae of Strongyloides stercoralis by feeding [75 Se]selenomethionine-labeled Escherichia coli to first- and second-stage larvae

A technique is described for radiolabeling Strongyloides stercoralis larvae with [75Se]selenomethionine. Cultures of an auxotrophic methionine-dependent stain of Escherichia coli were grown in a medium containing Dulbecco's modified Eagle's medium supplemented with 5% nutrient broth, amino acids, and [75Se]selenomethionine. When the 75Se-labeled bacterial populations were in the stationary phase of growth, cultures were harvested and the bacteria dispersed on agar plates to serve as food for S. stercoralis larvae. Use of nondividing bacteria is important for successful labeling because the isotope is not diluted by cell division and death of larvae attributable to overgrowth by bacteria is prevented. First-stage S. stercoralis larvae were recovered from feces of infected dogs and reared in humid air at 30 C on agar plates seeded with bacteria. After 7 days, infective third-stage larvae were harvested. The mean specific activity of 6 different batches of larvae ranged from 75 to 330 counts per min/larva with 91.8 +/- 9.5% of the population labeled sufficiently to produce an autoradiographic focus during a practicable, 6-wk period of exposure. Labeled infective larvae penetrated the skin of 10-day-old puppies and migrated to the small intestine, where the developed to adulthood.     

Strongyloides stercoralis excretory/secretory protein strongylastacin specifically recognized by IgE antibodies in infected human sera

The infective, microscopic Strongyloides stercoralis larvae in contaminated soil can penetrate human skin with the help of excretory/secretory proteases. These proteases play a critical role in infection and transmigration of the parasite to the intestines. Strongylastacin is similar to astacin (from the digestive gland of the crayfish Astacus astacus), a multi-domain protein with a signal peptide, a pro-enzyme, a catalytic domain containing the zinc binding consensus astacin family signature sequence HEXXHXXGFXHEXXRXDR, and a second conserved zinc binding motif SIMHY at N- terminal region. An EGF-1 like domain and a CUB domain are located at the COOH- terminal. In this study, the excretory/secretory Strongylastacin gene from S. stercoralis infective larval stage was cloned and expressed as a 45 kDa in Escherichia coli. Immunoblot analysis showed the presence of natural IgG antibodies against strongylastacin in six infected and six non-endemic normal sera. These findings were confirmed in an ELISA of 32 S. stercoralis infected and 32 presumed normal human sera; all contained natural anti-strongylastacin IgG antibodies. By contrast, IgE antibodies specific to strongylastacin were present in sera from individuals infected with S. stercoralis but not in uninfected control sera. Moreover, recombinant strongylastacin did not cross-react with IgE antibodies either from patients infected with filaria or patients with tropical pulmonary eosinophilic (TPE) who had increased IgE antibodies. The present authors conclude that strongylastacin, an excretory/secretory antigen, elicits specific IgE antibodies in S. stercoralis infected humans. Non-specific IgG antibodies to strongylastacin are present in both infected and normal humans. Further investigation is needed to understand the role of the host protective response against strongylastacin.     

The effect of oxygen and carbon dioxide on the development of the free-living stages of Strongyloides ratti in axenic culture

The effect of oxygen and carbon dioxide concentration on the development of the free-living stages of Strongyloides ratti in axenic culture was examined. Hatching of S. ratti eggs was inhibited at very low oxygen levels. Development of all organisms was inhibited in medium gassed with less than 4% O2 in 0.03% CO2 with the remainder N2. Between 8 and 21% O2 there was no significant difference in the percentage developing directly to filariform larvae, free-living females, or free-living males. Cultures gassed with CO2 concentrations greater than 1% in 21% O2 with the remainder N2 manifested inhibited development to filariform larvae, whereas concentrations between 5 and 7.5% enhanced development to free-living females. There was a positive effect on increasing the CO2 concentration from 0.03% to 5% on development to filariform larvae and male worms at levels of 1% and 2% oxygen in nitrogen, respectively. Direction of development, either directly to the filariform larva or to the free-living female, was influenced by CO2 and O2 concentrations encountered by the egg or the newly hatched larva of the parasitic female in axenic culture. The axenic culture system permits an important experimental approach to the study of factors modulating the differentiation of the free-living stages of Strongyloides.     

Strongyloides stercoralis: Amphidial neuron pair ASJ triggers significant resumption of development by infective larvae under host-mimicking in vitro conditions

Resumption of development by infective larvae (L3i) of parasitic nematodes upon entering a host is a critical first step in establishing a parasitic relationship with a definitive host. It is also considered equivalent to exit from the dauer stage by the free-living nematode Caenorhabditis elegans. Initiation of feeding, an early event in this process, is induced in vitro in L3i of Strongyloides stercoralis, a parasite of humans, other primates and dogs, by culturing the larvae in DMEM with 10% canine serum and 5mM glutathione at 37 degrees C with 5% CO(2). Based on the developmental neurobiology of C. elegans, resumption of development by S. stercoralis L3i should be mediated, in part at least, by neurons homologous to the ASJ pair of C. elegans. To test this hypothesis, the ASJ neurons in S. stercoralis first-stage larvae (L1) were ablated with a laser microbeam. This resulted in a statistically significant (33%) reduction in the number of L3i that resumed feeding in culture. In a second expanded investigation, the thermosensitive ALD neurons, along with the ASJ neurons, were ablated, but there was no further decrease in the initiation of feeding by these worms compared to those in which only the ASJ pair was ablated.     

Soluble extract from the nematode Strongyloides stercoralis induces CXCR2 dependent/IL-17 independent neutrophil recruitment

Neutrophil recruitment via CXCR2 is required for innate and adaptive protective immunity to the larvae of Strongyloides stercoralis in mice. The goal of the present study was to determine the mechanism of CXCR2-mediated neutrophil recruitment to S. stercoralis. Mice deficient in the receptor for IL-17A and IL-17F, upstream mediators of CXCR2 ligand production, were infected with S. stercoralis larvae; there was no difference in larval survival, neutrophil recruitment, or production of CXCR2 ligands compared with wild type mice. In vivo and in vitro stimulation of neutrophils with S. stercoralis soluble extract resulted in significant neutrophil recruitment. In vitro assays demonstrated that the recruitment functioned through both chemokinesis and chemotaxis, was specific for CXCR2, and was a G protein-coupled response involving tyrosine kinase and PI3K. Finally, neutrophil stimulation with S. stercoralis soluble extract induced release of the CXCR2 ligands MIP-2 and KC from neutrophils, thereby potentially enhancing neutrophil recruitment.     

Cryopreservation of first-stage and infective third-stage larvae of Strongyloides stercoralis

Infective third-stage larvae of Strongyloides stercoralis were frozen over liquid nitrogen and remained infective to dogs when thawed. Successful cryopreservation depended on a 30-60-min incubation in a cryoprotectant (10% DMSO and 10% dextran) before freezing and thawing the frozen larvae into RPMI. First-stage larvae could also be frozen by this method. Thawed first-stage larvae remained viable and continued their development to third-stage larvae, which were shown to be infective to dogs.     

Coinfection by Strongyloides stercoralis in blood donors infected with human T-cell leukemia/lymphoma virus type 1 in São Paulo City, Brazil

The frequency of coinfection with Strongyloides stercoralis and human T-cell leukemia/lymphoma virus type 1 (HTML-1) was determined in 91 blood donors examined at the blood bank of a large hospital in S?o Paulo city, Brazil. As control group 61 individuals, not infected by HTLV-1, were submitted to the same techniques for the diagnosis of S. stercoralis infection. In HTLV-1 infected patients the frequency of S. stercoralis infection was 12.1%; on the other hand, the control group showed a frequency significantly lower of S. stercoralis infection (1.6%), suggesting that HTLV-1 patients should be considered as a high risk group for strongyloidiasis in S?o Paulo city.     

Ancylostoma caninum: Adult worm removal,corticosteroid treatment,and resumed development of arrested larvae in dogs

Beagles, 2 months old and helminth naive, were infected with chilled arrest-prone larvae of Ancylostoma caninum. Eighteen days after infection, the pups were treated with an adulticidal anthelmintic (disophenol or dichlorvos) to remove adult worms while allowing dormant larvae to survive. Examinations of treated pups at necropsy demonstrated that the remaining hypobiotic larvae could develop and mature in the same dogs within which their development was arrested. Removal of adult worms was not a stimulus for resumption of larval development. Indeed, larvae resumed development in untreated control dogs harboring substantial populations of adult worms. Prednisolone treatment of dogs, although apparently producing some degree of immunodepression as judged by lymphocyte transformation assays, did not release larvae from dormancy. In fact, the dogs treated with the corticosteroid harbored significantly greater populations of hypobiotic larvae at 100 days after infection than did their untreated controls. Some hypobiotic larvae appeared to resume development spontaneously and idiosyncratically during the 2 to 3 month duration of these experiments. Whether a synchronous resumption of development would occur given other stimuli or spontaneously after a longer period of dormancy remains to be determined.     

Successful transgenesis of the parasitic nematode Strongyloides stercoralis requires endogenous non-coding control elements

Critical investigations into the cellular and molecular biology of parasitic nematodes have been hindered by a lack of modern molecular genetic techniques for these organisms. One such technique is transgenesis. To our knowledge, the findings reported here demonstrate the first heritable DNA transformation and transgene expression in the intestinal parasite Strongyloides stercoralis. When microinjected into the syncitial gonads of free-living S. stercoralis females, a construct fusing the S. stercoralis era-1 promoter, the coding region for green fluorescent protein (gfp) and the S. stercoralis era-1 3' untranslated region was expressed in intestinal cells of normally developing F1 transgenic larvae. The frequency of transformation and GFP expression among F1 larvae was 5.3%. By contrast, expression of several promoter::gfp fusions incorporating only Caenorhabditis elegans regulatory elements was restricted to abortively developing F1 embryos of S. stercoralis. Despite its lack of regulated expression, PCR revealed that one of these C. elegans-based vector constructs, the sur-5::gfp fusion, is incorporated into F1 larval progeny of microinjected female worms and then transmitted to the F2 through F5 generations during two host passages conducted without selection and punctuated by free-living generations reared in culture. Heritable DNA transformation and regulated transgene expression, as demonstrated here for S. stercoralis, constitute the essential components of a practical system for transgenesis in this parasite. This system has the potential to significantly advance the molecular and cellular biological study of S. stercoralis and of parasitic nematodes generally.     

Comparative effect of Neospora caninum infection in BALB/c mice at three different gestation periods

Neospora caninum has been recognized as a major cause of infectious bovine abortion worldwide. In the present study, the effect of N. caninum infection in mice at the 3 gestation periods (first, second, and third period) was investigated. In dams, tissue distribution of N. caninum was evaluated by nested polymerase chain reaction. In the progeny, fetal mortality, stillbirth, litter size, neonatal mortality/morbidity, vertical transmission, and parasite burden in neonatal tissues were evaluated. Pregnant BALB/c mice were infected subcutaneously with 2 x 10(6) NC-1 tachyzoites on days 0, 7, or 14 of gestation. Dams from each group were sequentially killed during gestation and postpartum (PP). Pups were killed on days 1 and 7 PP. Infection on day 0 of gestation produced a high vertical transmission rate, although no changes in fetal mortality, stillbirth, and littermate size were observed. The highest level of vertical transmission, together with an increase in fetal mortality and stillbirth and a decrease in litter size, were observed when infection was done on day 7 of gestation. Finally, infection on day 14 of gestation produced the lowest vertical transmission. Furthermore, infection at any time during gestation compromised the postnatal development of pups, because neonates from infected dams showed less body weight and a delay in the hair development.     

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.     

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.