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Identification of toxocara canis antigens by Western blot in experimentally infected rabbits.

Toxocariasis is a frequent helminthiasis that can cause visceral and ocular damage in humans specially in children. The identification of specific antigens of Toxocara canis is important in order to develop better diagnostic techniques. Ten rabbits were infected orally with a dose of 5000 Toxocara canis embryonated eggs. Rabbits were bled periodically and an ELISA assay was performed to determine levels of specific Toxocara IgG antibodies. ELISA detected antibodies at day 15 after infection. Western blot (WB) assay was performed using excretory/secretory antigens (E/S) of T. canis second stage larvae. Different antigen concentrations were evaluated: 150, 200, 250 and 300 micro g/mL. The concentration of 250 micro g/mL was retained for analysis. Rabbit sera were diluted 1:100. Secondary antibody was used at a dilution of 1:1000. Results of WB indicated that in the first month after infection specific antibodies against the 200 KDa, 116 KDa, 92 KDa and 35 KDa antigens were detected; antibodies against the 92 KDa, 80 KDa, 66 KDa, 45 KDa, 31 KDa and 28 KDa antigens appeared later. All positive sera in the ELISA test were also positive in WB. Two antigen bands, 92 KDa and 35 KDa, were identified since the beginning and throughout the course of infection. These antigens merit further evaluation as candidates for use in diagnosis.

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Adult Toxocara cati infections in U.S. children: report of four cases.

We report four cases of passage of subadult or adult Toxocara cati worms by young children ages 20 months to seven years. Worms were expelled rectally in two cases and in two cases they were vomited. A single worm was passed in two cases, three worms in one case, and 15 worms in the fourth case. All worms that were available for study were identified as T. cati by morphologic criteria, including the arrow-shaped cervical alae and the digitiform shape of the male tail. None of the four children exhibited clinical signs of ocular or visceral larva migrans, and in two cases where serum samples were available, neither child had a titer to Toxocara. These results further the argument that these children acquired the worms through the ingestion of immature worms passed by infected cats, not through the ingestion of infective eggs. Although the children were generally not ill as a result of these unusual infections, it does serve to reinforce the public health issue that potential serious consequences can occur where children have exposure to an environment that has been contaminated with cat feces, or, more specifically, infective eggs, and could become infected with larval forms of Toxocara.

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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.

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Prevalence of Toxocara spp. eggs in the soil of public parks in Ankara, Turkey.

One hundred and seventy soil samples from forty-six public parks in Ankara area were examined to determine the level of contamination with Toxocara spp. eggs. 30.6% of the 170 soil samples were contaminated with Toxocara spp. eggs. The number of eggs in the positive soil samples varied from 1 to 10. A high proportion of the eggs was fully embryonated. The findings demonstrate the common occurrence of these eggs in locations likely to be important in the transmission of visceral larva migrans. Of 19 fecal collected, 5 (26.3%) contained Toxocara spp. eggs. Eggs of Ancylostomidae, Toxascaris leonina, Trichuris spp., Taenia spp. and Enterobius vermicularis were also recovered from 17.6, 4.1, 2.4, 1.8 and 1.2% of soil samples, respectively.

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[Soil contamination with Toxocara sp. eggs in squares and public places from the city of La Plata. Buenos Aires, Argentina].

This study consisted of a stratified sampling, randomly taken, of the soil from the squares and parks of the city of La Plata, Province of Buenos Aires, in order to establish the prevalence of contamination caused by Toxocara sp. A total 242 soil samples was examined. From each sample a 10 grams aliquot was taken, washed in a 0.2% Tween 80 solution, and processed using the technique of concentration by flotation with sugar solution. There was a prevalence of 13.2%. In each positive sample, the quantity of eggs varied from 1 to 4. Toxocara sp. eggs were observed in 15 out of 22 squares and parks investigated. The sampling design and the processing method employed were satisfactory for the recovering and identification of Toxocara sp. eggs.

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[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.

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[Incidence of antibody detection against Toxocara canis and clinical symptoms in inhabitants of North-Eastern Poland].

The purpose of this work was to evaluate the the level of exposition of humans on Toxocara eggs in North Eastern Poland on a base of serological tests and epidemiological and clinical data. 1025 inhabitants of North Eastern region of Poland were examine to detect antibodies against Toxocara antigens. 214 (20.7%) showed presence of antibodies against Toxocara. They were mostly males (71%) and country inhabitants (58.9%). No abnormalities in laboratory tests were found among these people. Some of them had complaints like joint pains (19.6%), temporary skin rash 2.3%).

Adult↗

Prevalence of species of Toxocara in dogs, cats and red foxes from the Poznan region, Poland.

The prevalence of toxocariasis was evaluated for 445 dogs, 105 cats and 92 foxes from the Poznan region during 1997-1998. Forty one cats were infected (39%), 140 dogs (32%) and 15 red foxes (16%). Toxocara canis was found most frequently in puppies up to 3 months old (58%) and T. cati in kittens 4-6 months old (64%). Toxocariasis was much more prevalent amongst adult foxes (14%) than adult dogs (3%). In contrast to cats, female dogs and foxes were less infected than males. The present study suggests that cats may constitute an underestimated risk of transmission of Toxocara spp. to humans and the progressive synatropization of red foxes may also increase the sources of environmental contamination with Toxocara eggs.

Age Factors↗

Serologic differentiation of adult Toxocara and Ascaris.

The antigenic analysis of Toxocara canis a causative agent of visceral larva migrans and Ascaris suum has been carried out. Agar gel double diffusion techniques and its modifications were employed to determine whether or not crude antigens, obtained from the adult parasites: (a) contained components unique for each, (b) could be used to differentiate the parasites serologically. Adult Toxocara canis and Ascanis suum antigens used in this study were found to contain several unique antigenic components. It was shown that antisera produced in rabbits artificially immunized against adult Ascaris and Toxocara could be specifically differentiated from each other by using intragel absorption test.

Agar↗

Serologic differentiation of larval Toxocara and Ascaris.

Antigenic extracts of adult and larval T. canis and A. suum have been used in microgel double diffusion technique to differentiate antibodies produced in animals, experimentally infected with these parasites. Antigenic extracts from adult Ascaris reacted with the sera from animals infected with A. suum but the extracts from adult Toxocara failed to react or reacted feebly with the sera from animals infected with T. canis. Interestingly the antigenic extract from adult Toxocara reacted with the sera from animals infected with Ascaris. The antigens extracted from embryonated eggs of both the worms reacted with anti-Toxocara larval serum but could not absorb all the specific antibodies in the interagel absorption test.

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[Frequency of seropositivity to Toxocara canis in children of different socioeconomic strata].

Frequency of seropositivity for Toxocara in children from different socioeconomic strata in the city of Brasilia (Brazil) was measured. Six hundred and two children of both sexes, aged one to 12 years were distributed in two socioeconomically distinct groups. The samples of sera of the first group were obtained from blood drawn for routine tests in the laboratory of a public hospital attending children from low-income families. Samples from the second group were obtained from private laboratories attending children from middle-class families. Anti-toxocara antibodies were detected by ELISA, using Toxocara canis excretory-secretory antigens previously absorbed with Ascaris suum extract. The prevalence of seropositivity was 21.8% (66/302) in the first group and 3% (9/300) in the second (p< 0.0001). No differences in frequency according to age or sex could be detected. Our results suggest a high prevalence of childhood toxocariasis in Brasilia, with children from lower income brackets being the most affected.

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[Prevalence of infection by Toxocara in schoolchildren in the community of El Moján, Zulia state, Venezuela].

The systemic Toxocariasis or migrant larva is a helminto-zoonosis caused by the migration of nematode larvae through human tissues mainly, of the canine helminth Toxocara canis, which can infect humans, especially children. The aim of this study was to determine the prevalence of anti-T. canis antibodies in an infantile population. Serum samples of 73 children of either sex with ages between 4 and 6 years were examined. The determination of anti-Toxocara antibodies was carried out through an ELISA test (using excretory/secretory antigen from the Toxocara larvae). The statistical analysis was carried out through the Chi square test, the Fisher exact test and the correlation of Spearman. The overall prevalence of infection was 9.72%, the highest percentage (50%) was observed in 4 year-old children (3/6), there were significant differences among the ages. No differences were observed with regard to sex. An inverse correlation was observed between the age and ELISA positivity. These results suggest that the toxocariasis is frequent in children from this community.

Age Factors↗

[Soil contamination with Toxocara spp. eggs in the Kraków area and two nearby villages].

The distribution of Toxocara spp. eggs was studied in the Kraków city and nearby villages Grodkowice and Lazkowice. In Kraków out of 80 samples surveyed 30% were positive and the mean egg density was 3.7 eggs/100g soil. Court-yards and squares in the centre of the city were the most heavily contaminated areas (58% samples positive). In two nearby villages Toxocara spp. eggs were present in 16% samples examined and the mean egg density was 0.8 eggs/100g soil. Almost 90% of Toxocara spp. eggs recovered were infective. At least 80% of the eggs were classified as T. cati by egg's morfology.

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[Soil contamination with Toxocara spp. eggs in the Elblag area].

The distribution of Toxocara spp. eggs in Elbl4g was studied. Out of 72 soil samples collected in public places of the city 13.9% were positive and the mean egg density was 3.75/100g soil. The city backyards were much more contaminated with Toxocara spp. eggs (18.0%) than the playgrounds (4.5%). In sandpits examined the eggs were not found. Almost 80% of Toxocara spp. eggs recovered were infective. T. cati eggs were more frequent than T. canis eggs. Additionally in examined samples two eggs of Ancylostoma caninum and one egg of Ascaris lumbricoides were recognized.

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Intestinal nematode infections in Turkish military dogs with special reference to Toxocara canis.

The prevalence and potential zoonotic risk factors of intestinal nematodes of military working dogs, which are used for different military purposes, were assessed. Faecal samples from 352 defined-breed Turkish military dogs were investigated and 107 (30.4%) dogs were found to be infected with one or two nematode species. The following nematodes, with their respective prevalences, were diagnosed in the faecal samples: Toxascaris leonina (21.8%), Toxocara canis (13.3%), Trichuris vulpis (2.9%) and Uncinaria stenocephala (1.2%). Toxocara canis infections were more frequently seen in puppies (0-6 months old). The prevalence of T. canis was significantly higher in male than in female dogs and also higher in dogs which were exercised daily than in those without exercise. The highest prevalence was found in Belgian malinois breed dogs. Toxocara canis infections were not influenced by the floor type of the kennels (i.e. concrete or soil floor). There was no difference in the occurrence of T. canis infection when the last anthelmintic treatment was carried out less or more than 3 months prior to sampling. It is suggested that T. canis infected military dogs would be a threat not only for dog trainers but also for military personnel, notably during national and international operations.

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[Molecular techniques applied in species identification of Toxocara].

Toxocarosis is still an important and actual problem in human medicine. It can manifest as visceral (VLM), ocular (OLM) or covert (CT) larva migrans syndroms. Complicated life cycle of Toxocara, lack of easy and practical methods of species differentiation of the adult nematode and embarrassing in recognition of the infection in definitive hosts create difficulties in fighting with the infection. Although studies on human toxocarosis have been continued for over 50 years there is no conclusive answer, which of species--T. canis or T. cati constitutes a greater risk of transmission of the nematode to man. Neither blood serological examinations nor microscopic observations of the morphological features of the nematode give the satisfied answer on the question. Since the 90-ths molecular methods were developed for species identification and became useful tools being widely applied in parasitological diagnosis. This paper cover the survey of methods of DNA analyses used for identification of Toxocara species. The review may be helpful for researchers focused on Toxocara and toxocarosis as well as on detection of new species. The following techniques are described: PCR (Polymerase Chain Reaction), RFLP (Restriction Fragment Length Polymorphism), RAPD (Random Amplified Polymorphic DNA) and SSCP (Single Strand Conformation Polymorphism).

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[Transplanted infections with Toxocara canis as a model for the effectiveness testing of anthelmintics].

Patent infections of adult dogs with Toxocara canis induced by transplantation of immature, intestinal stages were examined for their suitability for testing of anthelmintics. Each of 5 dogs were infected four times by transplantation of 80 immature, intestinal stages of Toxocara canis. The dogs were treated with various anthelmintics of well established efficacy (pyrantel, nitroscanate, mebendazole, piperazine) 20 dpi. All anthelmintics tested showed the same efficacy as had been assessed earlier by treatment of dogs infected prenatally with Toxocara canis.

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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.

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