A family of secreted mucins from the parasitic nematode Toxocara canis bears diverse mucin domains but shares similar flanking six-cysteine repeat motifs

Infective larvae of the parasitic nematode Toxocara canis secrete a family of mucin-like glycoproteins, which are implicated in parasite immune evasion. Analysis of T. canis expressed sequence tags identified a family of four mRNAs encoding distinct apomucins (Tc-muc-1-4), one of which had been previously identified in the TES-120 family of glycoproteins secreted by this parasite. The protein products of all four cDNAs contain signal peptides, a repetitive serine/threonine-rich tract, and varying numbers of 36-amino acid six-cysteine (SXC) domains. SXC domains are found in many nematode proteins and show similarity to cnidarian (sea anemone) toxins. Antibodies to the SXC domains of Tc-MUC-1 and Tc-MUC-3 recognize differently migrating members of TES-120. TES-120 proteins separated by chromatographic methods showed distinct amino acid composition, mass, and sequence information by both Edman degradation and matrix-assisted laser desorption ionization/time of flight mass spectrometry on peptide fragments. Tc-MUC-1, -2, and -3 were shown to be secreted mucins with real masses of 39.7, 47.8, and 45.0 kDa in contrast to their predicted peptide masses of 15.7, 16.2, and 26.0 kDa, respectively. The presence of SXC domains in all mucin products supports the suggestion that the SXC motif is required for mucin assembly or export. Homology modeling indicates that the six-cysteine domains of the T. canis mucins adopt a similar fold to the sea anemone potassium channel-blocking toxin BgK, forming three disulfide bonds within each subunit.     

Toxocara canis: interaction of human blood eosinophils with the infective larvae

This study was carried out to investigate the nature of the immunological responses which took place in a child who had recently recovered from toxocariasis. She had developed a marked eosinophilia and had high titers of toxocara antibodies. Experiments were performed to examine whether Toxocara canis infective larvae could be killed in the presence of her serum and human eosinophils. Eosinophils with human complement, or this patient's serum, adhered to the surface of the larvae within 10 min. By 40 min, using both light and electron microscopy, it was shown that the cells had flattened against the cuticle and degranulated. However, by 3 hr, eosinophils had begun to detach, and the larvae remained alive for at least 1 week afterward. Further addition of serum or of eosinophils, which were shown to be able to immobilize T. spiralis infective larvae, failed to kill the T. canis larvae. It was concluded that, in this patient, the development of an inflammatory response to a T. canis infection was not associated with the appearance of antibodies capable of inducing eosinophil dependent toxicity to the larvae in vitro. Eosinophil dependent killing mechanisms may be less important than other components of the immune response, in immunity to this parasite in humans.     

A novel C-type lectin secreted by a tissue-dwelling parasitic nematode.

Many parasitic nematodes live for surprisingly long periods in the tissues of their hosts, implying sophisticated mechanisms for evading the host immune system. The nematode Toxocara canis survives for years in mammalian tissues, and when cultivated in vitro, secretes antigens such as TES-32. From the peptide sequence, we cloned TES-32 cDNA, which encodes a 219 amino-acid protein that has a domain characteristic of host calcium-dependent (C-type) lectins, a family of proteins associated with immune defence. Homology modelling predicted that TES-32 bears remarkable structural similarity to mammalian immune-system lectins. Native TES-32 acted as a functional lectin in affinity chromatography. Unusually, it bound both mannose- and galactose-type monosaccharides, a pattern precluded in mammalian lectins by a constraining loop adjacent to the carbohydrate-binding site. In TES-32, this loop appeared to be less obtrusive, permitting a broader range of ligand binding. The similarity of TES-32 to host immune cell receptors suggests a hitherto unsuspected strategy for parasite immune evasion.     

Toxocara canis: monoclonal antibodies to carbohydrate epitopes of secreted (TES) antigens localize to different secretion-related structures in infective larvae.

The major secreted glycoproteins of Toxocara canis larvae appear to be derived from two specialized organs within the nematode organism. Using immunogold electron microscopy, we have analyzed the binding patterns of a panel of monoclonal antibodies (Tcn-1 to Tcn-8) reactive with Toxocara excretory-secretory (TES) antigens. We find, first, that the esophageal gland and lumen are strongly reactive with monoclonals Tcn-4, Tcn-5, and Tcn-8, and because the posterior portion of the gut is closed, we hypothesize that products of this gland are released through the oral aperture. Second, a distinct anti-TES antibody (Tcn-2) localizes solely to the midbody secretory column, which opens onto the cuticle at a secretory pore. Thus, the secretory apparatus is probably functional in this stage of parasite as an important source of TES products. Only one monoclonal, Tcn-7, can bind to both esophageal and secretory structures. In addition, another antibody, Tcn-3, binds both to the epicuticle and to a TES antigen, but our data do not directly determine whether antigens located in the cuticle are subsequently released. Thus there are at least two, and possibly three, independent sources of TES antigens within Toxocara larvae.     

Toxocara canis: monoclonal antibodies to larval excretory-secretory antigens that bind with genus and species specificity to the cuticular surface of infective larvae

When maintained in culture, the infective-stage larvae of Toxocara canis produce a group of excretory-secretory antigens. Monoclonal antibodies to these antigens have been produced and partially characterized. Hybridomas were made using spleens from mice that had been given 250 embryonated eggs of T. canis followed by immunization with excretory-secretory antigens. Monoclonal antibodies were first screened against excretory-secretory antigens using an indirect enzyme-linked immunosorbent assay. Those antibodies positive in this assay were then screened against the surfaces of formalin-fixed, infective-stage larvae using an indirect fluorescent antibody assay. The two monoclonal antibodies showing fluorescence were also tested against the surfaces of infective-stage larvae of Toxocara cati, Baylisascaris procyonis, Toxascaris leonina, Ascaris suum, a Porrocaecum sp., and Dirofilaria immitis. One of these two antibodies bound to the surface of T. canis and T. cati while the other bound only to the surface of T. canis; neither were reactive with the other ascaridoid larvae or the larvae of D. immitis. Enzyme-linked immunoelectrotransfer blotting techniques were used to demonstrate that the cross-reactive antibody recognized antigens with molecular weights of about 200 kDa while the more specific monoclonal antibody recognized antigens with approximate molecular weights of 80 kDa. The specificity of these two antibodies for T. canis and T. cati should prove helpful in the development of more specific assays for the diagnosis of visceral and ocular larva migrans.     

Toxocara infection in pigs. The use of indirect fluorescent antibody tests and an in vitro larval precipitate test for detecting specific antibodies.

An in vitro larval precipitate test using second-stage Toxocara canis larvae and an indirect fluorescent antibody (IFA) test employing cuticles of T. canis larvae as antigen were evaluated using antisera produced in pigs experimentally infected with T. canis, T. cati, Ascaris suum, Toxascaris leonina and Parascaris equorum. The former test was both specific and sensitive and is suggested as a reliable and simple method of detecting Toxocara antibodies in pigs. The latter test was considered unsuitable because of cross-reactions that occurred when sera from pigs infected with other ascarids were tested. An IFA test for Ascaris antibodies, employing cuticles of A. suum larvae as antigen, is described. The degree of specificity of this test suggests that it may be of value in the detection of antibodies to Ascaris in pigs under natural conditions.     

Demonstration of antibodies during experimental toxocarosis using antigenic extract prepared from the adult parasit]

The detection of antibodies in subjects with visceral larva migrans syndrome caused by Toxocara canis is usually carried out with antigenic larvae, whose preparation presents some difficulties. Results obtained using extracts prepared from adult parasites show that such antigens are quite suitable for the detection of antibodies present in visceral larva migrans syndrome. This phenomenon is easily understood when one considers that there are numerous antigenic points in common between the different stages of the evolution of this parasite.     

Hatching of second-stage larvae of Toxocara canis: a rapid method for processing large numbers of worms.

A method for hatching second-stage larvae of Toxocara canis is described. Whole adult worms are processed by centrifugation in the presence of hypochlorite which allows the grinding step to be eliminated. The technique is more rapid and a high yield is obtained.     

Detection of circulating antigens and antibodies in Toxocara canis infection among children in Chengdu, China

In order to determine the seroprevalence of Toxocara spp. infection in children from Chengdu, we performed an enzyme-linked immunosorbent assay (ELISA) and sandwich ELISA (S-ELISA) with excretory-secretory antigens isolated from second-stage larvae of Toxocara canis (TES-Ag ELISA). The seroprevalences of T. canis antibodies in the children from rural areas, urban districts, and urban districts with recent Ascaris lumbricoides infection were 17.7% (59/333), 2.1% (4/186), and 2.6% (1/38), respectively. Among 63 suspected patients with symptoms of T. canis infection, 31 had positive antibodies. The inhibition assay showed an apparent inhibiting capacity of TES-Ag for the antibody against T. canis larvae. The result of S-ELISA demonstrated that circulating antigens of T. canis larvae could be detected in part of the serum with positive antibodies and that the detection rate for circulating antigens in the sera could be improved by polyethylene glycol-acid treatment. This is the first epidemiological study to confirm the existence of T. canis infection and Toxocara-larvae migrans in Chengdu by the combination of TES-Ag ELISA and S-ELISA.     

Shared carbohydrate epitopes on distinct surface and secreted antigens of the parasitic nematode Toxocara canis

The nematode parasite Toxocara canis is found in all dog populations and poses a poorly defined health hazard to humans. We have studied excretory-secretory antigen (ES) and surface antigens of the infective larval stage which is tissue-invasive in mammalian hosts. Antigens were probed with a panel of eight monoclonal antibodies raised in mice to whole ES. Six of eight antibodies reacted with periodate-sensitive carbohydrate epitopes on ES molecules, and the remaining two (Tcn-3 and Tcn-6) recognized either peptide or periodate-resistant sugar determinants. By immunoprecipitation and immunoblotting, the anti-carbohydrate monoclonals each reacted with several distinct ES molecules, known from previously published work to possess contrasting biochemical properties. Tcn-3 and -6 were directed predominantly against 32,000 and 120,000 m.w. molecules, respectively. Iodinated surface antigens of similar m.w. were precipitated by each antibody after detergent solubilization, but only two clones (Tcn-2 and -8) were able to bind exposed sites on the epicuticle of intact Toxocara larvae. Significantly, these antibodies do not bind to newly hatched larvae, and their target antigens are poorly expressed until the second day of in vitro cultivation. The specificities of the monoclonals were further studied by cold antibody inhibition of radiolabeled monoclonal binding, and by a matrix of two-site binding assays. These data show that Tcn-2, -4, -5, and -8 recognize a related group of repetitive carbohydrate epitopes, whereas Tcn-1, -6, and -7 bind discrete determinants on the same molecules. These studies are being continued to define further the structure of antigenic Toxocara carbohydrates and to compare the diagnostic utility of carbohydrate and peptide antigens.     

The in vitro adherence of murine eosinophils, neutrophils and non-induced and induced macrophages to infective larvae of Toxocara canis (Nematoda, Ascarididae)

Infective larvae of the parasite nematode Toxocara canis were incubated in vitro with murine eosinophils, neutrophils and non-induced and induced macrophages. The interactions between the different types of cells and the worms were observed in the presence or absence of immune mouse serum and/or complement. Cells showed considerable differences in the manner, duration and outcome of this interaction. Despite the adhesion of cells to the larvae of T. canis, there was no evidence of damage to the worms. Scanning and transmission electron microscopic observations suggest that the cells adhere to the cuticular surface via an electron-dense material. This material might play a protective role against the helmintotoxic capacity of the inflammatory cells.     

Visceral larva migrans: examinations by means of enzyme-linked immunosorbent assay of human sera for antibodies to excretory-secretory antigens of the second-stage larvae of Toxocara canis

An enzyme-linked immunosorbent assay is described for the detection of serum antibodies to visceral larva migrans (Toxocariasis). Excretory-secretory antigens of the second-stage larvae of Toxocara canis were used as antigen to coat the polystyrene plates. With sera from patients high antibody titers were observed in both ocular and visceral disorders. Cross-reactions due to other parasitic infections could be excluded, including other migrating larval infections such as ascariasis, trichinellosis, strongyloidiasis, filariasis, and anisakiasis. In a small seroepidemiologic survey of healthy primary schoolchildren, a remarkably high percentage (7.1) reacted positively to this method. These children showed eosinophilia as compared to the seronegative group. The data were compared with those observed in other countries and the results prompt reconsideration of the significance of T. canis for public health.     

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.     

Direct evidence that the cuticle surface of Trichinella spiralis muscle larvae shares antigenicity with stichocyte alpha-granules and the esophagus-occupying substance.

Antibodies against the cuticle surface of Trichinella spiralis muscle larvae were purified by means of immunoaffinity chromatography and incubated with ultrathin sections of muscle larvae. Major constituents of the parasite reactive with the purified antibodies included the cuticle surface, stichocyte alpha-granules, and the esophagus occupying substance of the muscle larvae. Thus the present data suggest that the cuticle surface is an antigenically different entity from the cuticle inner layers and its origin is likely stichocyte alpha-granules.     

Ovigerous-hair stripping substance (OHSS) in an estuarine crab: purification, preliminary characterization, and appearance of the activity in the developing embryos

Ovigerous-hair stripping substance (OHSS) is an active factor in crab hatch water (i.e., filtered medium into which zoea larvae have been released). This factor participates in stripping off the egg attachment structures (i.e., egg case, funiculus, and the coat investing ovigerous hairs) that remain attached to the female's ovigerous hairs after larval release. Thus this activity prepares the hairs for the next clutch of embryos. OHSS activity of an estuarine crab, Sesarma haematocheir, eluted as a single peak on molecular-sieve chromatography, but this peak still showed two protein bands at 32 kDa and 30 kDa on SDS-PAGE. The two protein bands stained with a polyclonal antiserum raised to the active fractions from molecular-sieve chromatography. Moreover, antibodies purified from this polyclonal OHSS antiserum also recognized both the 32-kDa and 30-kDa bands. OHSS immunoreactivity and biological activity were associated with the attachment structures that remained connected to the ovigerous hairs after hatching. In developing embryos, both protein bands could be stained immunochemically at least 10 days before hatching. But OHSS biological activity appeared only 3 days before hatching. The immunoreactive protein bands were not observed in the zoea, but OHSS bioreactivity was present, though greatly reduced. The 32-kDa protein, at least, is probably an active OHSS, and the 30-kDa protein band may also be OHSS-related. The OHSS appears to be produced and stored by the developing embryo. Upon hatching, most of the material may be trapped by the remnant structures, and the remainder is released into the ambient water.     

Hemoimmuno-adherence test (HIAT) for the detection of antibodies anti-Toxocara canis L2 larvae

The use of a hemoimmuno-adherence test (HIAT) for detecting antibodies anti-L2 larvae of Toxocara canis is presented. The HIAT allows to reveal the presence of antibodies in the sera of both naturally and experimentally infected animals. The sensitivity of HIAT appears to be comparable to that of methods such as immunoenzymatic ones.     

Nematophagous fungi of Toxocara canis eggs in a public park of La Plata, Argentina

Fungi have showed a great potential for the biological control of nematodes. However, they have not been evaluated for the control of animal and/or human parasites transmitted by egg contaminated soils. Environmental contamination with Toxocara spp. eggs is a public health problem. Accidental swallowing of Toxocara canis eggs (a nematode of dogs) usually results on a zoonotic infection (toxocarosis). The objectives of this research were: 1) To test the presence of antagonistic fungi against T. canis in the soil in public places of La Plata city, Argentina, infected with eggs of this parasite, 2) To determine the possible association between biotic and abiotic factors of the soil with the presence of fungal parasites of egg nematodes. Soil samples were tested for: textural type, organic matter (%), pH, presence of egg-parasite fungi, of larvae and of nematode eggs, in particular of Toxocara spp. The studied area showed the following characteristics: pH: 6.6-8.0, organic matter: 1.2-70%, with a predominantly loam texture. The following antagonistic fungal genera were identified: Acremonium, Aspergillus, Chrysosporium, Fusarium, Humicola, Mortierella, Paecilomyces and Penicillium. A prevalence of 70% was detected for nematode eggs, of 33% for Toxocara spp. eggs and of 90% for larvae. No association between the presence of egg-parasite fungi and the considered factors was found. More studies are necessary to know the natural antagonism factors to T. canis eggs for its in situ biological control.     

Evaluation of chemotherapy in experimental toxocarosis by determination of specific immune complexes

Parasitism by the larval phase of Toxocara canis is a chronic process in which the larvae survive in the tissues, resulting in the constant stimulation of the immune system. As a result, the detection of specific antibodies may not reflect the active state of the parasite. We have studied the dynamics of the production of specific immune complexes by ELISA with the monoclonal antibody TC-1 in rabbits inoculated with single and multiple doses of T. canis eggs. We also compared this with the production of specific antibodies and their possible modification after treatment with mebendazole. The specific antibodies against excretory-secretory antigen were detected with peaks at 10 and 12 weeks depending on the dose and remained positive during the entire experiment (62 weeks). Treatment caused an increase in the level of detectable antibodies dropping to similar levels to the controls. Specific immune complexes were detected only in multiple doses, and were then positive during the entire experiment. From the beginning of treatment the values of immune complexes fell quickly, remaining at undetectable levels during the rest of the experiment. For this reason the detection of specific immune complexes is a valid technique for monitoring the efficiency of treatment.     

Initial stage of development and migratory behavior of Toxocara canis larvae in BALB/c mouse experimental model

In the present study, the initial developmental stage of Toxocara canis eggs and larvae, and number of recovered larvae from BALB/c mouse-infected organs are described. In vitro culture of T. canis detects the frequencies of interphasic, mitotic and embryonated eggs only within a 7-day period. Analysis by egg counting was carried out for 32 days. The results showed that at 7 days after cultivation, the frequency of larvae was 50.4% and that this frequency reached 52.8% in 32 days. In the experimental infection of BALB/c mice with T. canis, the number of recovered larvae statistically increased in the brain and liver, with doses of approximately 200 and 1000 eggs. After 7 days of infection, a larger number of larvae were obtained in the lung and liver, although a maximum amount was found in the brain after a 15- or 30-day post-infection period.