Immunization of lambs against Oesophagostomum columbianum, using irradiated third stage larvae

The effect of gamma irradiation at doses of 40- and 50 kR on the development of third stage larvae of Oesophagostomum columbianum and the protection conferred by these irradiated larvae against the nematode, was studied in 6-8 week old male Kashmir Merino lambs. At 40- and 50 kR doses, the third stage larvae failed to develop to the adult stage in the intestine. Though single vaccination with 2000, 50 kR irradiated larvae failed to protect the animals against the infection, vaccination with the same number of 40 kR irradiated larvae conferred a partial protection. The presence of adult worms of O. columbianum in sites outside the intestine of 6-8 week old lambs have been demonstrated for the first time.     

Onchocerca volvulus: immunization of chimpanzees with X-irradiated third-stage (L3) larvae.

To provide a theoretical basis for the potential development of vaccines against Onchocerca volvulus (Ov) a trial has been conducted to assess the protective efficacy of immunization of chimpanzees with X-irradiated L3 larvae. Approximately 1000 larvae were injected at 0, 1, and 7 months. The immunized animals, and unimmunized controls, were then challenged with 250 live L3. In order to provide possibly protective exposure to the immunologically distinct L4 epicuticle, a radiation dose (45 krad) was chosen which preserved about 50% of the molting ability of unirradiated larvae. Despite the presence of a strong immune response to crude adult worm extracts, and to cloned Ov antigens, at the time of challenge little or no significant protection against patent infection was observed: three of four immunized animals developed patent infection as compared to four of four controls. One immunized animal failed to become patent or to manifest the late antibody response to adult worm antigens seen in both subpatent and patent infections in this model, and may have been protected from infection. The implications of these studies for future attempts to immunize against O. volvulus are discussed.     

Dirofilaria immitis: molting process of third-stage larvae

The objective of this study was to determine the molting process of Dirofilaria immitis third-stage larvae (L-3) to fourth-stage larvae (L-4), as it occurred in vitro. After 48 hr in vitro, the L-4 epicuticle was completely formed, and by 72 hr there was a clear separation between the L-3 and L-4 cuticles. The thickness of the newly formed L-4 cuticle was significantly less than that which has been described for larvae recovered from dogs after a similar incubation time period. If culture conditions were lacking in bovine albumin or proper temperature, larvae successfully developed the L-4 epicuticle but did not complete ecdysis. The molting process of D. immitis L-3 was thus shown to be multistepped with different factors required to induce the various developmental phases.     

The response of young Romney lambs to immunization with Trichostrongylus colubriformis larvae

1988. The response of young Romney lambs to immunization with Trichostrongylus colubriformis larvae. International Journal for Parasitology 18: 1035–1038. Groups of weaned Romney ewe lambs were immunized with two doses of 28,000, 35,000 or 42,000 (2000 kg⁻¹) infective larvae of Trichostrongylus colubriformis at 8, 12 or 16 weeks of age, respectively. Each group and helminthfree control lambs of similar age were challenged with T. colubriformis at the same dose rate as used for immunization. Faecal egg counts (FEC) and haematological observations were made during the experiment, and at slaughter, 42 days after challenge, worm burdens were determined and small intestinal histology was examined.

Lambs in each immunized group were identified as ‘responders’ or ‘non-responders’ on the basis of both FEC and worm burdens. A significant (P<0.001) decrease in the worm burdens recovered as a proportion of the challenge infections in both unimmunized and immunized lambs with increasing age was observed.

Globule leukocyte numbers increased with the age of lambs. In addition, within each age group globule leukocyte numbers reflected individual responsiveness to immunization, significantly (P<0.01) greater numbers being present in ‘responders’ than ‘non-responders’ or unimmunized lambs. No difference in haematological responses were found in relation to the lambs' responsiveness to immunization.     

Strategies for the storage of Ancylostoma caninum third-stage larvae

Although cryopreservation protocols for storage of hookworm larvae have been described, the circumstances under which the technique is necessary to ensure larval survival are not well defined. The motility of infective-stage larvae (as judged by observation) and their ability to migrate through canine skin in vitro were measured over a 7-mo period in worms held at room temperature and worms that had been cryopreserved at the start of the experiment. Cryopreserved worms showed motility and migration proportions of 45.6-48.0% and 26.8- 34.0%, respectively, throughout the experiment, compared with percentages of 92.7 and 84.1%, respectively, in the original fresh worms. Larvae held at room temperature showed a gradual decrease in motility and migration ability over the experimental period. Motility and migratory ability of cryopreserved larvae was only significantly higher (P < 0.01) than room temperature-stored larvae from 4 and 5 mo onward, respectively.     

Angiostrongylus cantonensis: in vitro cultivation from the first-stage to infective third-stage larvae

The first-stage larvae of Angiostrongylus cantonensis were cultured in various media at 27 degrees C. The most suitable medium for the development was Chernin's balanced salt solution supplemented with 10% L-15, 10% tryptose phosphate broth, 20% fetal calf serum, and 26 mM sodium bicarbonate. Addition of sodium bicarbonate to the medium facilitated early development of the first-stage larvae. When the first-stage larvae were cultured in the medium under 5% CO2 in air, the worms developed gradually to become quiescent and showed the C shape. Thereafter, the larvae developed to the second stage, retaining their first sheath. About 23 days later, the larvae began to develop to the third stage, being enclosed within the sheaths of the first and second molts. Under these conditions, the larvae developed uniformly and 82% of the larvae reached the third stage 50 days later. About 70% of the third-stage larvae discarded their two sheaths, showing almost the same size as those obtained in vivo. When these exsheathed larvae were inoculated into rats, they developed into adult worms and deposited numerous first-stage larvae.     

Strongyloides ratti: thermokinetic behavior of third-stage larvae on a temperature gradient

We examined the thermokinetic behaviors of infective third-stage larvae (L3) of the rodent parasitic nematode Strongyloides ratti on temperature gradients using an in vitro agarose tracking assay method. Observed behaviors included both negative and positive thermokineses, the direction of movement depending both on the gradient temperature at which larvae were initially placed and on prior experience of culture temperature. Larvae isolated from rat feces cultured at 25 degrees C and placed on a gradient at temperatures between 22 degrees and 29 degrees C tended to move toward higher temperatures. At higher placement temperatures, most larvae moved little and showed no directional response, whereas at lower placement temperatures, many migrated toward cooler temperatures. At placement temperatures of 20 degrees C or below, few or no larvae moved toward the zone of higher temperature. Larvae isolated from rat feces cultured at 20 degrees C tended to migrate to a high temperature area regardless of placed temperature. Those cultured at 30 degrees C did not respond to the temperature gradient. L3 cultured at 30 degrees C were significantly less infective to rats than those cultured at 25 degrees or 20 degrees C. Additional experiments were designed to demonstrate thermokinetic behaviors during the period after reaching the L3 stage. Larvae incubated in double distilled water (DDW) for 24 h at 37 degrees C lost their ability to respond to lower temperatures, while in those incubated in DDW at 15 degrees and 25 degrees C, responses were still apparent. The thermokinetic behavior of S. ratti L3 is affected by surrounding environmental temperatures and this may have an important role in host finding.     

Heligmosomoides polygyrus: excretory/secretory antigens released in vitro by exsheathed third-stage larvae

The excretory/secretory antigens released during in vitro culture of infective third-stage Heligmosomoides polygyrus larvae were analyzed by enzyme-linked immunosorbent assay and immunoblotting using sera from repeatedly infected mice. During the first 8-10 hr of culture at 37 C, freshly exsheathed larvae released only one antigen that cosedimented with trypsin (24 kDa) upon ultracentrifugation and was composed of a single 23-kDa polypeptide chain. After 10 hr of culture, the larvae released additional antigens identified by bands equivalent to polypeptides of approximately 18, 25, 26, 32, 58, and 76 kDa on nonreduced Western blots. The release of these molecules was maintained for up to 60 hr. Their staining intensity on blots was in the order 23 much greater than 25 greater than 76 greater than 18 greater than or equal to 58 greater than or equal to 32 greater than or equal to 26 kDa. Velocity sedimentation analysis showed that the 76-kDa component exists as a monomeric 76-kDa "native" antigen. The 32-, 58-, and 76-kDa antigens were specifically adsorbed by concanavalin A (Con A)-Sepharose and the 76-kDa molecule was detected on blots incubated with alkaline phosphatase-conjugated Con A, indicating the presence of mannose-like residues on these molecules. The 18-, 23-, 25-, and 26-kDa antigens did not bind to Con A-Sepharose. Hyperimmune antisera raised against lyophilized larvae had negligible antibody activity against the larval ES antigens, suggesting that the ES antigens are released soon after synthesis rather than being stored in significant quantities within the larvae.     

Angiostrongylus costaricensis: culture of third-stage larvae to young adults in a defined medium

The third-stage larvae of Angiostrongylus costaricensis were successfully cultured to young adults in a chemically defined medium. The most suitable medium for the development was Waymouth's medium among eight defined media examined. Twenty-eight days after cultivation in this medium, 77% of the larvae developed to young adults, although these worms gradually died thereafter. When Waymouth's medium was supplemented with mouse red blood cells, these young adult worms continued their development. The mean body lengths of the worms cultivated in Waymouth's medium supplemented with RBCs were significantly larger than those of the worms in the medium without RBCs on Days 14 and 21 after cultivation. Addition of RBCs was essential for their further development. At 28 days after cultivation, the maximum body length of the worms was 2.1 mm for males and 3.3 mm for females. Additions of serum, yeast extract lactalbumin hydrolysate, and growth factors to Waymouth's medium did not provide any additional benefits for worm development.     

Resumption of feeding in vitro by hookworm third-stage larvae: a comparative study.

Third-stage infective larvae of the canine hookworm Ancylostoma caninum resume feeding in vitro in response to several stimuli. Experiments were conducted to characterize the in vitro feeding behavior of several hookworm species. Reduced glutathione and, to a lesser extent, canine and human serum stimulated third-stage larvae of Ancylostoma duodenale to resume feeding. Glutathione-induced feeding reached a maximum by 16 hr and was concentration-dependent between 0- and 15-mM glutathione. Oxidized glutathione and the reducing agents dithiothreitol and L-cysteine failed to induce feeding, suggesting that reducing conditions alone were not stimulatory. Serum incubated with glutathione was the most efficient stimulus for Ancylostoma ceylanicum, Ancylostoma braziliense, and Ancylostoma tubaeforme larvae, whereas Uncinaria stenocephala larvae responded best to canine serum alone. Necator americanus larvae did not resume feeding in response to glutathione, serum, glutathione plus serum, or linoleic acid (0.1-10 mM). These differences in feeding behavior suggest that generalizations concerning hookworm biology must be interpreted cautiously.     

Strongyloides ratti: chemokinesis of glycolytic enzyme- and lectin-treated third-stage infective larvae in vitro

The infective third-stage larvae (L3s) of Strongyloides ratti, a parasitic nematode in rodents, showed two types of chemokinesis on a gradient of sodium chloride (NaCl) in an in vitro agarose tracking assay. The types were a consistent directional avoidance behavior under unfavorable environmental conditions and a reduced avoidance behavior under favorable conditions. We examined the effects of treatments with glycolytic enzymes and lectins by analyzing the avoidance behavior. L-Fucose dehydrogenase, hyaluronidase, beta-glucosidase, alpha-mannosidase, beta-galactosidase, concanavalin A, wheat germ agglutinin and soybean agglutinin exhibited inhibitory or enhancive effects on chemokinesis. We also confirmed the sites of the amphids of L3s aside from the mouth at the anterior end by scanning electron microscopy, and that concanavalin A-binding sites existed in the vicinity of the amphids using lectin-histochemistry. The carbohydrate moieties in the amphids of S. ratti L3s may play an important role as chemosensors in perceiving environmental cues.     

Lagochilascaris minor third-stage larvae secrete metalloproteases with specificity for fibrinogen and native collagen

Dissemination of parasitic infections depends on migration through tissues and evasion from both hemostatic processes and immune responses from hosts. Metalloproteases play major roles in these mechanisms of pathogen-host interactions and, thus, are considered drug targets. In this study, we characterized metalloprotease activities in excretory/secretory (ES) products from third stage larvae (L3) of the ascarid Lagochilascaris minor, the causative agent of lagochilascariosis, which demonstrates an impressive migrating capacity across host tissues, including bone. Gel enzymography showed that ES products of L3 display two major gelatinolytic activities. Optimal proteolytic activity was found to occur at neutral/alkaline pH and was associated with two L. minor-secreted metalloproteases of 59 (SM59(Lm)) and 114kDa (SM114(Lm)). We next showed that ES products of L3 were able to hydrolyze fibrinogen and collagen I at neutral pH, but not BSA, in an extensive manner. Furthermore, we demonstrated that ES products of L3 mediate hydrolysis of the triple helical structure of collagen I fibers in mouse mesentery. These results suggest that ES proteases of L3 might facilitate both L. minor migration through host tissues by hydrolyzing collagens of the extracellular matrix and evasion from host hemostatic mechanisms by degrading fibrinogen.     

Gnathostoma binucleatum: excretion-secretion antigen analysis obtained from advanced third-stage larvae in in vitro culture

The paper describes an introductory characterization of antigenic stimulation of excretion-secretion products (ESP) of Gnathostoma binucleatum advanced third-stage larvae cultured in vitro and proteinases present in this products. Excretory and secretory proteins were obtained after 10 larvae were maintained in 5% CO(2) RPMI medium. The supernatant was collected each week for two months. The proteins were dialyzed, concentrated, and separated in 10% SDS-PAGE gels under reducing conditions and transferred to nitrocellulose paper for immunoblot analyses. G. binucleatum immunized mice serum was used to determine protein antigenicity. Four proteins of 40, 80, 120, and 208 kDa persisted for two months and three proteins, 80, 120, and 208 kDa were recognized for antibodies of mice. In SDS-PAGE gelatin substrate gels ESP resolved as two proteins with molecular weight of 80 and 208 kDa that were sensitive to a metalloproteinase inhibitor, and thus it may be inferred that they might be used for diagnosis of gnathostomiasis.