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.     

Transmammary transmission of Strongyloides stercoralis in dogs

Vertical transmission of larvae is a major pathway in the life cycle of several species of Strongyloides, but evidence for it occurring in humans or dogs with Strongyloides stercoralis is absent. In an effort to determine if vertical transmission could occur with S. stercoralis, each of 3 female dogs was infected with filariform larvae at a different stage of the reproductive cycle, i.e., preconception, gestation, or postpartum. Results showed that none of 6 pups born to a female infected before conception or any of 6 pups born to another female infected during gestation harbored any stage of S. stercoralis when necropsied at parturition. Conversely, all 5 pups that nursed from the female infected immediately postpartum became infected with adult S. stercoralis in their small intestines (range, 56-129 adult worms). Significantly, live filariform larvae of S. stercoralis were observed on 2 different occasions from milk samples taken from the lactating female. Because arrested development of larvae is not known in S. stercoralis, there is no reservoir of larvae in the parenteral tissues of females to queue for passage to the pups and, thus, it is not surprising that only timely infections, perhaps very late in gestation and during lactation, can be successful. These data support previous work in dogs with S. stercoralis, which concluded that vertical transmission through prenatal pathways does not occur, but they are the first from the dog to indicate that vertical transmission of this parasite through transmammary routes is possible. Whether transmammary transmission of S. stercoralis occurs in humans remains unknown but given its immense pathological potential, it should not be overlooked.     

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.     

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.     

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.     

Life cycle stage-resolved proteomic analysis of the excretome/secretome from Strongyloides ratti--identification of stage-specific proteases

A wide range of biomolecules, including proteins, are excreted and secreted from helminths and contribute to the parasite's successful establishment, survival, and reproduction in an adverse habitat. Excretory and secretory proteins (ESP) are active at the interface between parasite and host and comprise potential targets for intervention. The intestinal nematode Strongyloides spp. exhibits an exceptional developmental plasticity in its life cycle characterized by parasitic and free-living generations. We investigated ESP from infective larvae, parasitic females, and free-living stages of the rat parasite Strongyloides ratti, which is genetically very similar to the human pathogen, Strongyloides stercoralis. Proteomic analysis of ESP revealed 586 proteins, with the largest number of stage-specific ESP found in infective larvae (196), followed by parasitic females (79) and free-living stages (35). One hundred and forty proteins were identified in all studied stages, including anti-oxidative enzymes, heat shock proteins, and carbohydrate-binding proteins. The stage-selective ESP of (1) infective larvae included an astacin metalloproteinase, the L3 Nie antigen, and a fatty acid retinoid-binding protein; (2) parasitic females included a prolyl oligopeptidase (prolyl serine carboxypeptidase), small heat shock proteins, and a secreted acidic protein; (3) free-living stages included a lysozyme family member, a carbohydrate-hydrolyzing enzyme, and saponin-like protein. We verified the differential expression of selected genes encoding ESP by qRT-PCR. ELISA analysis revealed the recognition of ESP by antibodies of S. ratti-infected rats. A prolyl oligopeptidase was identified as abundant parasitic female-specific ESP, and the effect of pyrrolidine-based prolyl oligopeptidase inhibitors showed concentration- and time-dependent inhibitory effects on female motility. The characterization of stage-related ESP from Strongyloides will help to further understand the interaction of this unique intestinal nematode with its host.     

Analysis of polyunsaturated fatty acid composition of Strongyloides ratti in relation to development

The effect of linolenic acid (C18:3 omega 3) 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 (C18:2 omega 6) but its proportion decreased with development. On the contrary, eicosapentaenoic acid (C20:5 omega 3) and C20:4 omega 3 were prominent in the filariform larva (L3). Because C20:5 omega 3 is generally synthesized from C18:3 omega 3 via C20:4 omega 3, the high ratio of C20:5 omega 3/C18:3 omega 3 of L3 in all the free-living generations suggested that polyunsaturated fatty acid metabolism, particularly the omega-3 series, and eicosanoids produced had important roles in the development of S. ratti L1.     

Partial cross-resistance between Strongyloides venezuelensis and Nippostrongylus brasiliensis in rats.

Rats were immunized through an initial infection with 1,000 filariform larvae (L3) of Nippostrongylus brasiliensis and after complete expulsion of worms they were challenged with 1,000 L3 of Strongyloides venezuelensis to investigate whether cross-resistance developed against a heterologous parasite. Nippostrongylus brasiliensis-immunized rats developed a partial cross-resistance against S. venezuelensis migrating larvae (MSL3) in the lungs and adult worms in the small intestine. The population of MSL3 in the lungs were significantly lower (P < 0.05) in immunized rats (22.0 +/- 7.4) compared with controls (105.0 +/- 27.6). The populations of adult worms, egg output and fecundity were initially decreased but from day 14 post-challenge they did not show any significant difference between immunized and control rats. However, the length of worm in immunized rat was revealed as retardation. Peripheral blood eosinophilia was significantly decreased (P < 0.05) on day 7 post-challenge and then gradually increased, which peaked on day 42 post-challenge when most of the worms were expelled. These results suggest that peripheral blood eosinophilia is strongly involved in the worm establishment and expulsion mechanisms.     

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.     

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.     

A case of gastric strongyloidiasis in a Korean patient

A 69-year-old Korean man was admitted to emergency room with complaints of abdominal pain, vomiting, and diarrhea. Laboratory tests revealed eosinophilia, anemia, hypoproteinemia, and hyponatremia. The gastric mucosa showed whitish mottled and slightly elevated lesions on the body angle of antrum. Microscopically, chronic gastritis with incomplete intestinal metaplasia was observed. Many adult worms, larvae, and eggs in cross sections were located in the crypts. Furthermore, the filariform larvae of Strongyloides stercoralis with a notched tail were detected through the culture.     

Strongyloides ratti: Transfer of immunity in rats by lymph node cells or serum

Immune mesenteric lymph node cells (IMLNC) and hyperimmune serum, alone or in combination, were transferred to recipient, syngeneic Lewis rats. On the day of cell transfer, the recipients, along with appropriate controls, were infected with 1,000Strongyloides ratti larvae. Twelve days later, the number of adult worms in the anterior small intestine was counted and the number ofS. ratti eggs in the terminal uterus of representative female worms was determined. Using these methods, we showed that worm fecundity was reduced in the IMLNC recipients, the hyperimmune serum recipients, and the recipients of both cells and serum as compared with regular controls (averages of 2.45, 2.93, and 2.98 eggs, respectively, vs. 4.73 eggs). The number of adult worms recovered from the IMLNC recipients was not reduced (474.6 worms). However, significantly fewer worms were recovered from the recipients of IMLNC-hyperimmune serum and the recipients of hyperimmune serum only as compared with regular controls (184.8 and 171.5 worms, respectively, vs. 567.2 worms). A working hypothesis, involving an interaction of serum and cellular components, is postulated as the mechanism for this protective response.     

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.     

The free-living generation of the nematode Strongyloides papillosus undergoes sexual reproduction

The nematode genus Strongyloides consists of parasites that live as parthenogenetic females in the small intestines of their hosts. They can also form a facultative free-living generation with males and females. Recently, research on Strongyloides cellular and molecular biology has concentrated on Strongyloides ratti and Strongyloides stercoralis. We propose that the related nematode Strongyloides papillosus, a common parasite of ruminants, is well suited for comparative and evolutionary studies and we show that it is phylogentically basal to S. ratti and S. stercoralis. Based on cytological observations several reports have proposed that Strongyloides males do not contribute genetically to the next generation, leaving open the question of why males still exist. In contrast, the only study employing molecular markers showed that S. ratti males do pass on genetic material. Here, we demonstrate that in S. papillosus males also contribute molecular genetic markers to the next generation. This is interesting for two reasons. First, it shows that S. papillosus is amenable to genetic analysis and second, it indicates that sexual reproduction is more common in Strongyloides than previously assumed.     

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 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.     

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.     

How effective is the agar plate method for Strongyloides stercoralis?

The sensitivity of the agar plate method for the diagnosis of Strongyloides stercoralis was studied experimentally. Results demonstrated that this method was sensitive enough to detect S. stercoralis even when only a few worms were present.     

<Emphasis Type="Italic">Strongyloides stercoralis</Emphasis> infection in a Finnish kennel

Background  

Intestinal threadworm Strongyloides stercoralis is a parasite of dog, cat and primates that occurs worldwide being most prevalent in tropical and subtropical countries. The adult parasitic worm is about 2 mm long and slender. It possesses both parasitic and free-living lifecycles. The parasitic worms are females. Strongyloides stercoralis infects the host via percutaneous, peroral or transmammary transmission in addition to autoinfection. Clinical disease varies from inapparent to severe enteritis and pneumonia. The diagnosis is based on demonstration of larvae in fresh faeces, which is best made by Baermann technique.