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.     

Development of a Luminex Bead Based Assay for Diagnosis of Toxocariasis Using Recombinant Antigens Tc-CTL-1 and Tc-TES-26

The clinical spectrum of human disease caused by the roundworms Toxocara canis and Toxocara cati ranges from visceral and ocular larva migrans to covert toxocariasis. The parasite is not typically recovered in affected tissues, so detection of parasite-specific antibodies is usually necessary for establishing a diagnosis. The most reliable immunodiagnostic methods use the Toxocara excretory-secretory antigens (TES-Ag) in ELISA formats to detect Toxocara-specific antibodies. To eliminate the need for native parasite materials, we identified and purified immunodiagnostic antigens using 2D gel electrophoresis followed by electrospray ionization mass spectrometry. Three predominant immunoreactive proteins were found in the TES; all three had been previously described in the literature: Tc-CTL-1, Tc-TES-26, and Tc-MUC-3. We generated Escherichia coli expressed recombinant proteins for evaluation in Luminex based immunoassays. We were unable to produce a functional assay with the Tc-MUC-3 recombinant protein. Tc-CTL-1 and Tc-TES-26 were successfully coupled and tested using defined serum batteries. The use of both proteins together generated better results than if the proteins were used individually. The sensitivity and specificity of the assay for detecting visceral larval migrans using Tc-CTL-1 plus Tc-TES-26 was 99% and 94%, respectively; the sensitivity for detecting ocular larval migrans was 64%. The combined performance of the new assay was superior to the currently available EIA and could potentially be employed to replace current assays that rely on native TES-Ag.     

An improved method to obtain antigen-excreting Toxocara canis larvae

Toxocara canis is a dog helminth which causes visceral larva migrans (VLM) when infecting humans as a larva. The infection is demonstrated by detecting IgG antibodies against excretory-secretory larval antigens (ESLA) in serum by ELISA. The production of ESLA involves the collection of adult worms from dog puppy stools, the separation of eggs from dissected uteri, and the in vitro growing of egg-derived larvae, following the time-consuming and laborious protocol described by De Savigny [De Savigny, D.H., 1975. In vitro maintenance of T. canis larvae and a simple method for the production of Toxocara ES antigen for the uses in serodiagnostic tests for visceral larva migrans. Journal of Parasitology 61, 781-782]. In this work, an improved protocol for obtaining T. canis larvae is described. The modifications proposed improved the efficiency of the original De Savigny method in three ways: (i) increasing the parasite yield up to five fold, (ii) improving the larval purity, and (iii) markedly reducing the execution time of the protocol.     

Highlights of human toxocariasis

Human toxocariasis is a helminthozoonosis due to the migration of Toxocara species larvae through human organism. Humans become infected by ingesting either embryonated eggs from soil (geophagia, pica), dirty hands or raw vegetables, or larvae from undercooked giblets. The diagnosis relies upon sensitive immunological methods (ELISA or western-blot) which use Toxocara excretory-secretory antigens. Seroprevalence is high in developed countries, especially in rural areas, and also in some tropical islands. The clinical spectrum of the disease comprises four syndromes, namely visceral larva migrans, ocular larva migrans, and the more recently recognized "common" (in adults) and "covert" (in children) pictures. Therapy of ocular toxocariasis is primarily based upon corticosteroids use, when visceral larva migrans and few cases of common or covert toxocariasis can be treated by anthelmintics whose the most efficient appeared to be diethylcarbamazine. When diagnosed, all of these syndromes require thorough prevention of recontamination (especially by deworming pets) and sanitary education.     

Anti-inflammatory activity of thiabendazole and its relation to parasitic disease.

In 6 differnet animal assays in the laboratory, thiabendazole had clear anti-inflammatory effect, though it was less potent than aspirin in all assays. These findings add support to clinical suggestions that the drug may have anti-inflammatory properties in man. Such properties may contribute to the clinical response observed following the use of thiabendazole in cases of trichinosis, cutaneous larva migrans, visceral larva migrans, dracunculosis and scabies. In parasitic infections in which corticosteroids are commonly used in clinical management, notably trichinosis, the fact that thiabendazole does not appear to have immunosuppressive activity may confer an added clinical advantage.     

Visceral larva migrans (toxocariasis) in Toronto.

A 7-year-old child was admitted to Toronto''s Hospital for Sick Children in 1976 with symptoms and laboratory findings compatible with visceral larva migrans, a disease usually caused by Toxocara canis. This prompted a search for other cases seen at the hospital during the period 1952 through 1978. Only 18 cases were discovered that met at least three of six criteria and thus were considered possible or probably cases of the disease. Three possible cases of ocular toxocariasis during the same period were also uncovered. Fever was the commonest presenting symptom. Eosinophilia, leukocytosis and hyperglobulinemia were the most frequent laboratory findings. In view of the small number of cases found in 27 years at this large pediatric hospital with a broad referral base, it is concluded that visceral larva migrans poses little risk to the health of children in the Toronto area.     

Do Toxocara canis larval antigens used in enzyme-linked immunosorbent assay for visceral larva migrans cross-react with AB isohemagglutinins and give false positive results?

A variety of helminth parasites have A and B blood group antigens on their surface. These antigens may cross-react with elevated concentrations of A and B isohemagglutinins in some patients and give false-positive results in the serologic diagnosis of visceral larva migrans caused by T. canis. To clarify this point, serum from patients with visceral larva migrans and elevated T. canis antibody titers as determined by ELISA were absorbed with AB blood cells and retested by ELISA without a demonstrable decline in T. canis antibody titers. Similarly, absorption with T. canis embryonated egg antigens of serum containing elevated levels of anti-A or anti-B isohemagglutinins failed to decrease the isohemagglutinin titer. This indicates that the ELISA using T. canis embryonated egg antigen does not give false positive results with sera containing high concentrations of anti-A or anti-B isohemagglutinins.     

Analysis of Toxocara canis larval excretory-secretory antigens: physicochemical characterization and antibody recognition

Toxocara canis larval excretory-secretory antigens (TEX) were resolved by gradient pore polyacrylamide gel electrophoresis and analyzed using silver, periodic acid-Schiff, and immunoperoxidase stains. At least 15 bands between 29 and 94 kilodaltons (kDa) were detected by silver stain, all of which were recognized by antibodies in serum of a patient with visceral larva migrans. Immunoperoxidase stain detected an additional band at 92 kDa and 4-6 others above 200 kDa. Periodic acid-Schiff stain also detected the high molecular weight components, but did not detect constituents of approximately 53 and 57 kDa. Immunoperoxidase stain using antibody from the vitreous fluid of an ocular larva migrans patient detected 2 TEX components, approximately 76 and 80 kDa. Antigens were compared with respect to batch of larvae and age of larvae in culture. Qualitative differences that correlated with batch were found in the number of constituents above 200 kDa, and in 1 component of 78 kDa. Qualitative differences were noted in many minor components, some of which appeared to correlate with age of larvae in culture. Major TEX constituents were recognized consistently by antibody, regardless of batch or age of larvae. Total protein production per larva was approximately 8 ng/day, and was consistent over time. There was no evidence of neutral proteases in TEX.     

Baylisascaris procyonis (Nematoda: Ascarididae) larva migrans in free-ranging wildlife in Orange County,California

Baylisascaris procyonis is well recognized as a cause of visceral (VLM), ocular, and neural (NLM) larva migrans in birds and mammals, including man. A study of the prevalence of larva migrans in free-ranging wildlife associated with raccoon latrines as well as a retrospective study of wildlife mortalities with neurological disease was conducted in 2000 in Orange County, California. Eighty-seven birds of 18 species and 64 mammals of 8 species were found to have NLM or VLM or both. NLM clinical signs included convulsions, torticollis, opisthotonus, head-tilts, circling, ataxia, paralysis, and visual defects. NLM lesions were characterized by focally disseminated, frequently linear, "tracklike" areas of parenchymal degeneration with varying degrees of astrocytosis in the white matter of the cerebrum, cerebellum, brain stem, and spinal cord. Larvae were rarely found in these lesions but were rather isolated in the brain-spinal cord parenchyma. At least 1 larva was isolated by digestion from each case of NLM and identified as Baylisascaris sp., most likely B. procyonis. VLM lesions consisted of granulomatous reactions surrounding intact or degenerative larvae in the parenchyma of the liver, kidney, diaphragm, and, occasionally, the lymph nodes. This report broadens the range of species of wild birds and mammals that have been found to be susceptible to larva migrans caused by B. procyonis and reaffirms the importance of raccoon latrine sites as contaminative foci for wildlife.     

Toxocara canis: T lymphocyte function in murine visceral larva migrans and eosinophilia onset

The second larval stage of Toxocara canis, the major cause of visceral larva migrans syndrome in humans and also the common intestinal roundworm of the dog, elicits an eosinophil-rich granulomatous response in nondefinitive hosts such as man and mice. During murine infection a population of T lymphocytes was identified which responded to a preparation of T. canis larval antigens in a migration inhibition test (MIT). It was further shown that mice infected with T. canis have a substantial eosinophilia which reaches its peak approximately 2 weeks after infection. When mice are depleted of their functional T lymphocytes by use of antilymphocytic antisera, the eosinophil response of infected mice is significantly reduced. These findings are consistent with the findings of others that helminthic parasites can elicit cell-mediated immune responses and the resultant eosinophilia is but one manifestation of this response.     

Cross-reactions with Ascaris suum antigens of sera from mice infected with A. suum, Toxocara canis, and Angiostrongylus cantonensis

Ascaris suum larval excretory-secretory (AsES) antigen and larval (AsLA) as well as adult somatic antigen (AsAA) which were thought to be possibly helpful in the diagnosis of visceral larva migrans (VLM) due to A. suum infection were investigated in the present study. Serum taken from mice orally inoculated with approximately 250 embryonated eggs of A. suum or Toxocara canis, or 40 third-stage larvae of Angiostrongylus cantonensis were assessed by enzyme-linked immunosorbent assay (ELISA) using the AsES antigen, AsLA or AsAA at 1, 2, 3, 4, 6 and 8 weeks post infection (WPI). The titer of serum IgG from mice infected with A. suum increased from 1 WPI and a peak at 4 WPI was observed when it reached approximately three times the level of uninfected control mice. Thereafter, it decreased gradually but remained high as found from 6 to 8 WPI. No cross-reactions of heterologous serum IgG against AsES antigen was observed, whereas heterologous serum IgM exhibited significant cross-reactions to AsES antigen. Cross-reactivities to AsLA and AsAA by heterologous serum IgG as well as IgM antibodies were also observed in the trial. Altogether, the AsES antigen apparently seemed to be superior to the other two somatic antigens when used in the diagnosis of A. suum-induced VLM with serum IgG as tested by ELISA. Moreover, it was the first report to test the possibly antigenic cross-reactivity between A. suum and A. cantonensis.     

Toxocara canis: proteolytic enzymes secreted by the infective larvae in vitro

Second-stage larvae of the dog nematode Toxocara canis are infective to man and cause the syndromes of visceral larva migrans and ocular toxocariasis. Larvae cultured in vitro secrete proteases which degrade components of a model of extracellular matrix and basement membranes. These enzymes have been characterized using a variety of techniques. Multiple enzyme activities were demonstrated by substrate gel electrophoresis, associated with proteins of molecular weights of 120 and 32 kDa. The enzyme activity was inhibited both in substrate gels and in a radiogelatin microplate assay by phenylmethylsulfonyl fluoride. Optimal activity occurred at pH 9, with minor activities apparent at pH 5 and 7; the relationship between these proteolytic activities is currently under investigation.     

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.     

Environmental contamination with Toxocara sp. eggs in public squares and parks from Santiago, Chile, 1999

Most of cases of visceral larva migrans syndrome are caused by Toxocara canis larvae. Man acquires the infection by accidental ingestion of ripe eggs of the helminth. In order to find out the frequency of the presence of Toxocara sp. eggs in dog fecal specimens from public places of Santiago city in 1999 july-september a study was carried out in 84 squares and 12 parks from 32 counties. Three fecal samples were collected in each of these places, making a total of 288. Each sample was processed according to a modified Telemann method and three smears of each were microscopically examined. Toxocara sp. eggs were found in 39 (13.5%) fecal specimens. In 28/84 (33.3%) squares and in 8/12 (66.7%) parks these eggs were detected. These results make necessary the control of dogs and cats populations with the aim of reduce the presence of animals without responsible owners. It should be advisable to prevent dogs and cats defecation in public areas grounds, trying to exclude these animals from recreation areas.     

Nematodes transmitted to man by fish and aquatic mammals

Zoonotic nematodes may cause disease in man through migrating larva (larva migrans), through direct infection or possibly through allergic responses. The parasitic genera Ancylostoma, Uncinaria, Bunostomum and Toxocara can cause larva migrans. The cod worm (Phocanema decipiens) a parasite found in fish and seals, can infect man, as can Anisakis, Dioctophyme renale and Gnathostoma hispidium larvae obtained from eating raw fish.     

First outbreak of Baylisascaris procyonis larva migrans in rabbits in Japan

A syndrome of progressive neurological signs was noticed in rabbits (Oryctolagus cuniculus) kept in a small wildlife park in mid-July 2000. Three out of 12 common raccoons (Procyon lotor) kept in this park were infected with Baylisascaris procyonis, and the larvae were found from affected rabbits. This outbreak is the first proven B. procyonis larva migrans in Japan, and the potential risk of serious zoonosis by this ascarid species should be considered by pet owners, veterinarians, physicians and public health authorities in this country as in North America where raccoons are endemic.     

Two imported cases of cutaneous larva migrans

Cutaneous larva migrans (CLM) is a rare serpiginous cutaneous eruption caused by accidental penetration and migration in the skin with infective larvae of nematode that normally do not have the human as their host. Although CLM has a worldwide distribution, the infection is most frequent in warmer climates. More recently, they have been increasingly imported from the tropics or subtropics by travelers. We experienced two patients who had pruritic serpiginous linear eruption in their skin for a few weeks after traveling to the endemic areas (Brazil and Thailand, respectively). After the treatment with albendazole, the skin lesions resolved with post-inflammatory hyperpigmentation. We report herein two cases of cutaneous larva migrans successfully treated with albendazole.     

Fine binding specificities to Ascaris suum and Ascaris lumbricoides antigens of the sera from patients of probable visceral larva migrans due to Ascaris suum

Fine binding specificities to Ascaris suum and A. lumbricoides antigens of the sera from patients with probable visceral larva migrans (VLM) due to A. suum infection were examined. Although multiple-dot enzyme-linked immunosorbent assay (ELISA) was found to be useful for the primary screening of patients, identification of the responsible species was sometimes difficult due to extensive cross reactions with other ascarid parasite antigens. Fine resolution to determine the causative pathogen was obtained by a rather classical Ouchterlony's double immunodiffusion test. The difference in the binding of the patients' sera to A. suum and A. lumbricoides antigens was also demonstrated by an inhibition ELISA. The patients' antibodies bound with higher avidity to the A. suum antigen than to the A. lumbricoides and Toxocara canis antigens. Combination of at least two different immunological assay methods is recommended for the diagnosis of VLM due to ascarid parasites.     

Immunocytolocalization study of the external covering of Trichinella spiralis muscle larva

The antibody-binding sites of the muscle larva of Trichinella spiralis were investigated by immunogold staining on the ultrathin sections of LR white resin. The antibodies, which were produced in the course of T. spiralis infection in rats, specifically bound to the inner layers of the body cuticle and the cuticle of the hindgut, but not to the cuticle of the esophagus. This is the first report that reveals the antigenic nature of the inner layers of the external coverings of T. spiralis larva.     

Baylisascaris procyonis (Nematoda: Ascaridoidea) eggs in raccoon (Procyon lotol) latrine scats in Orange County,California

Baylisascaris procyonis is the common intestinal nematode of the raccoon and is well-recognized as a cause of visceral, ocular, and neural larva migrans in many species of wild and domestic birds and mammals, including humans. To develop data on the prevalence of B. procyonis in Orange County, California, 800 distinct raccoon latrine sites were sampled in 4 spatial zones from 15 January to 31 December 2000. Counts of fecal eggs per gram (EPG) were determined and evaluated with reference to spatial zone and season of collection. No significant differences in EPG were noted among the spatial zones. However, EPG exhibited a significant rise (37,730 +/- 1,865) in the fall and a significant decline (26,204 +/- 1,446) in the winter (ANOVA, P = 0.045). The overall egg prevalence was 100%, and the overall mean EPG was 30,265 +/- 867.