Dog walking brings Toxocara eggs to people's homes.

To find out the transmission routes for Toxocara infection, the possibility of transfer of Toxocara eggs from the soil on the paws of animals and on the shoes of people was explored. For this purpose, a study was conducted to find helminth eggs in washings from the paws of dogs after walking, from the shoes of their owners, as well as non-dog owners. Toxocara eggs were detected in 19.4% of the dogs' paws washings and in 11.4% of washings from the shoes of their owners. The number of eggs found on the paws was about twice as high as on the shoes. The mean number of eggs in the sample was 2.9 in washings from the paws and 1.8 from the shoes. In the samples, Toxocara cati eggs prevailed both in occurrence and in abundance. Out of the total number of positive samples, the eggs of T. cati were found in 83%, and T. canis in 42%. 79% of the found eggs belonged to T. cati and 21% to T. canis. In the washings from shoes of people that do not own dogs, the eggs of parasites were not found. This study demonstrates that the helminth eggs can be transferred from contaminated soil to people's homes on the paws and shoe soles. Even animals without a patent infection may take part in propagation of infection causing risks of human toxocariasis. In dogs, in addition, the transferred on paws T. canis eggs can serve as a causative agent of permanent, cumulative subclinical infection with a deferred manifestation in posterity. It is supposed that infestation "through the paws" is one of the probable routes of transmission of toxocariasis in dogs.

Characterization and differential expression analysis of Toxocara canis aquaporin-1 gene.

Toxocara canis is an intestinal nematode of canids with a worldwide distribution, causing an important but neglected parasitic zoonosis in humans. Aquaporins (AQP) are a family of water channel proteins, which function as membrane channels to regulate water homeostasis. In this study, the coding sequence of aquaporin-1 gene of T. canis (Tc-aqp-1) was cloned and characterized. The obtained Tc-aqp-1 coding sequence was 933 bp in length, which predicted to encode 311 amino acids. Two conserved asparagine-proline-alanine (NPA) motifs were identified in the multiple sequence alignments. Phylogenetic analysis revealed the closest relationship between T. canis and Opisthorchis viverrini based on aquaporin-1 amino acid sequence. A structure was predicted with ligand binding sites predicted at H93, N95, N226, L94, I79, and I210 and with active sites predicted at I256 and G207. Gene Ontology (GO) annotations predicted its cellular component term of integral component of plasma membrane (GO: 0005887), molecular function term of channel activity (GO: 0015250), and biological process term of water transport (GO: 0006833). Tissue expression analysis revealed that the Tc-aqp-1 was highly expressed in the intestine of adult male. The findings of the present study provide the basis for further functional studies of T. canis aquaporin-1.

Genetic blueprint of the zoonotic pathogen Toxocara canis.

Toxocara canis is a zoonotic parasite of major socioeconomic importance worldwide. In humans, this nematode causes disease (toxocariasis) mainly in the under-privileged communities in developed and developing countries. Although relatively well studied from clinical and epidemiological perspectives, to date, there has been no global investigation of the molecular biology of this parasite. Here we use next-generation sequencing to produce a draft genome and transcriptome of T. canis to support future biological and biotechnological investigations. This genome is 317 Mb in size, has a repeat content of 13.5% and encodes at least 18,596 protein-coding genes. We study transcription in a larval, as well as adult female and male stages, characterize the parasite's gene-silencing machinery, explore molecules involved in development or host-parasite interactions and predict intervention targets. The draft genome of T. canis should provide a useful resource for future molecular studies of this and other, related parasites.

Toxocariosis en diferentes poblaciones infantiles vulnerables de la Argentina / Toxocariosis in different vulnerable groups of children in Argentina

La toxocariosis humana es una parasitosis de amplia distribución en el mundo. Se estudiaron 857 muestras de sangre de poblaciones vulnerables de distintas edades. Todas las muestras fueron analizadas mediante el test de ELISA: 346 correspondieron a habitantes de zonas urbanas; 258 eran de niños de entre 0 y 20 años, y 88 eran donantes de sangre; de las 356 muestras estudiadas de poblaciones rurales, 326 correspondieron a niños; los 30 restantes, a adultos de la misma zona; también se analizó una población aborigen de 130 niños y 25 adultos. Entre los niños de zonas urbanas la prevalencia fue de 28.3% y de 46.6% en los adultos. En la zona rural la prevalencia en niños fue de 59.2%, y en los adultos, de 53.3%; en la población aborigen la prevalencia alcanzó el 78.5% en niños y el 80.0% en los adultos. Estos valores son altos y tienen relación con el nivel de educación y con el medio ambiente. El suero de los dadores fue estudiado con dos equipos comerciales, y al compararlos ofrecieron resultados con mayor positividad. Se muestra que la toxocariosis es una infección endémica, que afecta a los niños y tiene alto impacto en la población con déficit educacional. Se discuten los informes internacionales, el diagnóstico y los mecanismos de transmisión. Los resultados justifican que esta parasitosis sea considerada un problema de salud pública...

Molecular characterization and functional analysis of serine/threonine protein phosphatase of Toxocara canis.

Toxocara canis (T. canis) is a widely prevalent zoonotic parasite that infects a wide range of mammalian hosts, including humans. We generated the full-length complementary DNA (cDNA) of the serine/threonine phosphatase gene of T. canis (Tc stp) using 5' rapid amplification of the cDNA ends. The 1192-bp sequence contained a continuous 942-nucleotide open reading frame, encoding a 313-amino-acid polypeptide. The Tc STP polypeptide shares a high level of amino-acid sequence identity with the predicted STPs of Loa loa (89%), Brugia malayi (86%), Oesophagostomum columbianum (76%), and Oesophagostomumdentatum (76%). The Tc STP contains GDXHG, GDXVDRG, GNHE motifs, which are characteristic of members of the phosphoprotein phosphatase family. Our quantitative real-time polymerase chain reaction analysis showed that the Tc STP was expressed in six different tissues in the adult male, with high-level expression in the spermary, vas deferens, and musculature, but was not expressed in the adult female, suggesting that Tc STP might be involved in spermatogenesis and mating behavior. Thus, STP might represent a potential molecular target for controlling T. canis reproduction.

Molecular diagnosis of an ocular toxocariasis patient in Vietnam.

An ocular Toxocara canis infection is reported for the first time in Vietnam. A 34-year-old man residing in a village of Son La Province, North Vietnam, visited the National Eye Hospital (NEH) in August 2011. He felt a bulge-sticking pain in his left eye and loss of vision occurred over 3 months before visiting the hospital. The eye examination in the hospital showed damage of the left eye, red eye, retinal fibrosis, retinal detachment, inflammation of the eye tissues, retinal granulomas, and a parasitic cyst inside. A larva of Toxocara was collected with the cyst by a medical doctor by surgery. Comparison of 264 nucleotides of internal transcribed spacer 2 (ITS2) of ribosomal DNA was done between our Vietnamese Toxocara canis and other Toxocara geographical isolates, including Chinese T. canis, Japanese T. canis, Sri Lankan T. canis, and Iranian T. canis. The nucleotide homology was 97-99%, when our T. canis was compared with geographical isolates. Identification of a T. canis infection in the eye by a molecular method was performed for the first time in Vietnam.

Molecular Diagnosis of an Ocular Toxocariasis Patient in Vietnam

An ocular Toxocara canis infection is reported for the first time in Vietnam. A 34-year-old man residing in a village of Son La Province, North Vietnam, visited the National Eye Hospital (NEH) in August 2011. He felt a bulge-sticking pain in his left eye and loss of vision occurred over 3 months before visiting the hospital. The eye examination in the hospital showed damage of the left eye, red eye, retinal fibrosis, retinal detachment, inflammation of the eye tissues, retinal granulomas, and a parasitic cyst inside. A larva of Toxocara was collected with the cyst by a medical doctor by surgery. Comparison of 264 nucleotides of internal transcribed spacer 2 (ITS2) of ribosomal DNA was done between our Vietnamese Toxocara canis and other Toxocara geographical isolates, including Chinese T. canis, Japanese T. canis, Sri Lankan T. canis, and Iranian T. canis. The nucleotide homology was 97-99%, when our T. canis was compared with geographical isolates. Identification of a T. canis infection in the eye by a molecular method was performed for the first time in Vietnam.

[Occurrence of Toxocara spp. eggs in household environment of children with diagnosed toxocariasis in Lódz voivodeship]. / Czestosc wystepowania jaj inwazyjnych Toxocara spp. w srodowisku przydomowym dzieci ze zdiagnozowana toksokaroza w woj. lódzkim.

Toxocariasis is a zoonosis due to infection of humans by dog or cat roundworm (Toxocara canis, T. cati). Humans become infected by ingestion of infective eggs either from soil, dirty hands, raw fruits and vegetables or larvae from undercooked meat of paratenic hosts. The aim of the study was to evaluate the level of contamination of soil samples from households of children with diagnosed toxocariasis in rural and urban areas of Lódz voivodeship. In the years 2004-2007 toxocariasis was confirmed in 178 patients of the Polish Memorial Hospital in Lódz. The soil samples were collected from 53 courtyards of patients' domiciles. Toxocara spp. eggs were isolated from the samples using flotation technique (Dada 1979). The examinations revealed the high prevalence of ground contamination with Toxocara eggs in both, rural (30.4%) and urban areas (23.3%). The presence of Toxocara eggs in households enlarges the risk of re-infection for children with diagnosed toxocariasis, especially in rural areas where the high level of contamination was detected.

Utilizing a polymerase chain reaction method for the detection of Toxocara canis and T. cati eggs in soil.

A polymerase chain reaction (PCR) technique has been used for the differentiation of T. canis and T. cati eggs isolated from soil and previously identified from microscopical observations. The method, using specific primers for the identification of the two Toxocara species, was assessed in both the field and laboratory. Successful results were obtained when only a single or large numbers of eggs were recovered from 40 g soil samples. The method is sensitive, allows analysis of material independent of the stage of egg development and can be adapted for the recovery of other species of parasites from soil.

[Molecular techniques applied in species identification of Toxocara]. / Techniki molekularne stosowane w identyfikacji gatunków 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).

Identification of tissue-embedded ascarid larvae by ribosomal DNA sequencing.

Polymerase chain reaction (PCR) was applied to identify tissue-embedded ascarid nematode larvae. Two sequences of the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA), ITS1 and ITS2, of the ascarid parasites were amplified and compared with those of ascarid-nematodes registered in a DNA database (GenBank). The ITS sequences of the PCR products obtained from the ascarid parasite specimen in our laboratory were compatible with those of registered adult Ascaris and Toxocara parasites. PCR amplification of the ITS regions was sensitive enough to detect a single larva of Ascaris suum mixed with porcine liver tissue. Using this method, ascarid larvae embedded in the liver of a naturally infected turkey were identified as Toxocara canis. These results suggest that even a single larva embedded in tissues from patients with larva migrans could be identified by sequencing the ITS regions.

Toxocariasis / Toxocariasia

La entidad clínica como Síndrome de migración larbaria visceral es causada por larvas del parásito Toxocara canis y menos frecuentemente, por Toxocara cati oleolina. Toxocara es un parásito comun en perros y gatos. El hombre es huésped ocasional cuando ingiere huevos del helminto. La toxocariasis adopta dos formas clínicas, ocular y visceral. Presentamos un paciente de 6 años con toxocariasis visceral caracterizada por un síndrome febril prolongado y absceso hepático

Differentiation of Toxocara canis and T. cati eggs by light and scanning electron microscopy.

In this study, we investigated the morphological identification of Toxocara canis and T. cati eggs on the basis of light and scanning electron microscopic (SEM) observations. T. canis and T. cati eggs used in this study were recovered from the uteri of respective gravid female worms. Measurement of egg size was not helpful in the differentiation of these species, because approximately 90% of eggs measured were of similar size. Using SEM, we were able to differentiate T. canis eggs from T. cati eggs based on their respective characteristic surface structures. Both species have subspherical eggs with markedly pitted surfaces like those of a golf ball, but the surface pitting in T. canis is more coarse than that in T. cati. In this study, however, these differences were not absolute, as 16% of T. canis and 29% of T. cati eggs showed surface pitting that was uncharacteristic of their species. Of the 16% of T. canis eggs that could not be differentiated by surface structure, 3% had pitting resembling T. cati, and the remaining 13% showed intermediate type surface pitting. Similarly, 5% of T. cati eggs resembled T. canis, and 25% of these were of intermediate type. Light microscopic observation yielded results similar to those of SEM, indicating that light microscopy is also a useful tool for the identification of Toxocara eggs.

Characterisation of Tc-cpl-1, a cathepsin L-like cysteine protease from Toxocara canis infective larvae.

Cysteine proteases play vital biological roles in both intracellular and extracellular environments. A cysteine protease migrating at 30 kDa was identified in somatic extracts of Toxocara canis larvae (TEX), by its binding to the biotinylated inhibitor Phe-Ala-CH2F. TEX proteases readily cleaved the cathepsin L- and B-specific peptide substrate Z-Phe-Arg-AMC and to a lesser extent, the cathepsin B-specific peptide Z-Arg-Arg-AMC. Excretory/secretory (TES) products of T. canis larvae did not cleave either substrate. Partial sequence encoding the 5' end of a cysteine protease cDNA from infective T. canis larvae was then obtained from an expressed sequence tag (EST) project. The entire cDNA (termed Tc-cpl-1) was subsequently sequenced and found to encode a preproenzyme similar to cathepsin L-like proteases (identities between 36 and 69%), the closest homologues being two predicted proteins from Caenorhabditis elegans cosmids, a cathepsin L-like enzyme from Brugia pahangi and a range of parasite and plant papain-like proteases. Sequence alignment with homologues of known secondary structure indicated several charged residues in the S1 and S2 subsites involved in determining substrate specificity. Some of these are shared with human cathepsin B, including Glu 205 (papain numbering), known to permit cleavage of Arg-Arg peptide bonds. The recombinant protease (rTc-CPL-1) was expressed in bacteria for immunisation of mice and the subsequent antiserum shown to specifically react with the 30 kDa native protease in TEX. Sera from mice infected with the parasite also contained antibodies to rTc-CPL-1 as did sera from nine patients with proven toxocariasis; control sera did not. Larger scale studies are underway to investigate the efficacy of rTc-CPL-1 as a diagnostic antigen for human toxocariasis, the current test for which relies on whole excretory/secretory antigens of cultured parasites.

Detection of sequence variation in parasite ribosomal DNA by electrophoresis in agarose gels supplemented with a DNA-intercalating agent.

This study evaluated the use of a commercially available DNA intercalating agent (Resolver Gold) in agarose gels for the direct detection of sequence variation in ribosomal DNA (rDNA). This agent binds preferentially to AT sequence motifs in DNA. Regions of nuclear rDNA, known to provide genetic markers for the identification of species of parasitic ascarid nematodes (order Ascaridida), were amplified by polymerase chain reaction (PCR) and subjected to electrophoresis in standard agarose gels versus gels supplemented with Resolver Gold. Individual taxa examined could not be distinguished reliably based on the size of their amplicons in standard agarose gels, whereas they could be readily delineated based on mobility using Resolver Gold-supplemented gels. The latter was achieved because of differences (approximately 0.1-8.2%) in the AT content of the fragments among different taxa, which were associated with significant interspecific differences (approximately 11-39%) in the rDNA sequences employed. There was a tendency for fragments with higher AT content to migrate slower in supplemented agarose gels compared with those of lower AT content. The results indicate the usefulness of this electrophoretic approach to rapidly screen for sequence variability within or among PCR-amplified rDNA fragments of similar sizes but differing AT contents. Although evaluated on rDNA of parasites, the approach has potential to be applied to a range of genes of different groups of infectious organisms.