Comparison of extracellular vesicle isolation methods for the study of exosome cargo within Toxocara canis and Toxocara cati excretory secretory (TES) products.

Toxocara is a genus of nematodes, which infects a variety of hosts, principally dogs and cats, with potential zoonotic risks to humans. Toxocara spp. larvae are capable of migrating throughout the host tissues, eliciting eosinophilic and granulomatous reactions, while surviving for extended periods of time, unchanged, in the host. It is postulated that larvae are capable of altering the host's immune response through the release of excretory-secretory products, containing both proteins and extracellular vesicles (EVs). The study of EVs has increased exponentially in recent years, largely due to their potential use as a diagnostic tool, and in molecular therapy. To this end, there have been multiple isolation methods described for the study of EVs. Here, we use nanoparticle tracking to compare the yield, size distribution, and % labelling of EV samples acquired through various reported methods, from larval cultures of Toxocara canis and T. cati containing Toxocara excretory-secretory products (TES). The methods tested include ultracentrifugation, polymer precipitation, magnetic immunoprecipitation, size exclusion chromatography, and ultrafiltration. Based on these findings, ultrafiltration produces the best results in terms of yield, expected particle size, and % labelling of sample. Transmission electron microscopy confirmed the presence of EVs with characteristic cup-shaped morphology. These findings can serve as a guide for those investigating EVs, particularly those released from multicellular organisms, such as helminths, for which few comparative analyses have been performed.

Toxocara "omics" and the promises it holds for medicine and veterinary medicine.

Toxocariasis is one of the most neglected worldwide zoonoses that is caused by larval nematode parasites of the genus Toxocara, Toxocara canis, and to a lesser extent, Toxocara cati, whose migration mechanism is still largely unknown. Fortunately, some advanced tools have been employed, such as genomics, transcriptomics, and proteomics, to better understand the molecular biology and regulatory mechanisms of Toxocara. Using genomics and transcriptomics, we can identify a large number of genes that participate in the development of Toxocara and the interaction of parasites and their hosts and can predict the functions of unknown genes by comparing them with other relevant species. Using proteomics, we can identify somatic proteins and excretory and secretory (ES) proteins that perform specific biological functions in tissue degradation, pathogen invasion, immune evasion or modulation. These "omics" techniques also can contribute enormously to the development of new drugs, vaccines and diagnostic tools for toxocariasis. In a word, by utilizing "omics", we can better understand the Toxocara and toxocariasis. In this review, we summarized the representative achievements in Toxocara and the interaction between Toxocara spp. and their hosts based on expressed sequence tags (ESTs), microarray gene expression, next-generation sequencing (NGS) technologies and liquid chromatography-tandem mass spectrometry (LC-MS/MS), hoping to better understand the molecular biology of Toxocara, and contribute to new progress in the application areas of new drugs, vaccines and diagnostic tool for toxocariasis in the future.

Genetic analysis of Toxocara cati (Nematoda: Ascarididae) from Guangdong province, subtropical China.

Toxocara cati (cat roundworm) is a common parasitic nematode that infects humans and other hosts, causing toxocariasis. Although its significance as a pathogen, the epidemiology, genetics and biology of T. cati remain poorly understand in China. In the present study, genetic variation in mitochondrial (mt) cytochrome c oxidase subunit 1 (cox1) gene and internal transcribed spacer (ITS) of rDNA region among T. cati in Guangdong province, subtropical China was examined. A portion of the cox1 (pcox1) and the complete ITS (ITS1 + 5.8S rDNA + ITS2) were amplified separately from individual worms by polymerase chain reaction (PCR) and amplicons were then subjected to sequencing from both directions. The length of the sequences of pcox1, ITS-1, and ITS-2 were 308 bp, 462 bp, and 335 bp, respectively. The intra-specific sequence variations within T. cati were 0-3.6% for pcox1, 0-2.4% for ITS-1, and 0-2.7% for ITS-2. However, the inter-specific sequence differences were significantly higher, being 8.6%, 10.7%, and 11.3% for pcox1, ITS-1, and ITS-2, respectively. Phylogenetic analyses based on the pcox1 sequences indicated that all the isolates in Guangdong province were in genus Toxocara, which confirmed that these parasites represent T. cati. The molecular approach employed provides a powerful tool for elucidating the epidemiology, genetics, and biology of zoonotic T. cati in China and elsewhere.

In vitro translation of mRNA from Toxocara canis larvae.

300 micrograms of total RNA was extracted from 1 ml of packed Toxocara canis larvae by centrifugation through a 5.7 M cesium chloride cushion. 60 micrograms of polyadenylated messenger RNA was separated from 300 micrograms of total RNA in an oligothymidylic acid-cellulose gel column. The in vitro translation of the mRNA, isolated from T. canis larvae, was carried out using the rabbit reticulocyte cell-free translation system. Incorporation of [35S]methionine into trichloroacetic acid precipitable material in the lysate containing mRNA was 4-5 times greater than that of control. Translation products were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. Many polypeptides ranging in molecular weight from 10000 to 100000 were synthesised in the lysate. A T. canis positive human serum was mixed with translation products to form antigen-antibody complexes, which were then absorbed by Staphylococcus aureus Cowan 1 strain and analysed by the autoradiography of SDS-PAGE. Three antigenic polypeptides with molecular weights of 49000, 27000 and 22000 which reacted specifically with IgG antibody in T. canis positive human serum, were demonstrated. The 27000 MW polypeptide reacted particularly strongly with the IgG antibody.

Metabolite mapping of Toxocara canis using one- and two-dimensional proton magnetic resonance spectroscopy.

One- and two-dimensional proton (1H) nuclear magnetic resonance (NMR) spectroscopic techniques have yielded detailed in vitro profiles of the metabolites present in the parasitic nematode Toxocara canis. The major intracellular metabolites were found to include trehalose, alanine, succinate, acetate, propionate and alpha-glycerophosphorylcholine.

Surface associated glycoproteins from Toxocara canis larval parasites.

The surface of infective larvae of Toxocara canis, the dog ascarid nematode, reveals relatively few exposed surface proteins which can be recovered in soluble form. The major components identified by surface labelling have molecular weights of 32, 55, 70 and 120 kilodaltons (kDa), and are all significantly glycosylated. All are recognised by the immune response in definitive (canine) and paratenic (murine or human) hosts. Expression of these antigens on the parasite surface begins after the larvae hatch from infective ova in vitro, and presumably in vivo. Each of these molecules may also be found in the set of secreted (ES) glycoconjugates released by larval parasites cultivated in vitro, and currently available biochemical and functional data on the surface/secreted ES glycoproteins are presented. Analysis with monoclonal antibodies (MAbs) confirms the identity of surface and ES molecules, and these MAbs show differing patterns of binding to the epicuticle, the cuticular matrix and to the oral orifice. Alternative mechanisms for antigen synthesis, insertion into the cuticle and export from the parasite are discussed.

Toxocara cati larva migrans in domestic pigs--detected at slaughterhouse control in Norway.

Routine Trichinella meat inspection at the slaughterhouse detected one larva in a pooled batch of 100 pig samples. The larva was sent to the Norwegian Veterinary Institute (NVI) for species identification.Morphological examination revealed that the larva was not Trichinella spp. Molecular analysis was performed. PCR and sequencing of 5S/ITS identified the larva as Toxocara cati. A second round of digests was carried out at the meat inspection laboratory, in smaller batches to try to identify the infected animal. No further larvae were detected and it was not possible to identify which of the 100 animals the larva had come from. This is the first time that Toxocara cati has been reported in slaughterhouse pigs in Norway.Although the infected individual could not be identified, the meat originated from one of six potential farms. A small survey regarding rodent control and cats was sent to each of these farms. Cats had restricted access to food storage areas (two farms reported that cats had access) whilst none of the farms allowed cats into the production housing. Cats were, however, present on all the farms (mostly stray cats of unknown health status). Half of the farms also reported seeing rodents in the pig housing during the previous six months and half reported finding rodents in the feed and straw storage areas. We were unable to narrow down the source of infection - however contamination of food or bedding material, with cat faeces or infected rodents, in addition to the presence of infected rodents in pig housing remain potential routes of infection.

Seroprevalence of toxocariasis in schoolchildren in Northern Iran.

This cross-sectional study was carried out on 1210 randomly selected schoolchildren, attending sixteen primary and secondary schools, during the period between November 2005 and June 2006. Parents who accepted to include their children were requested to be present at sampling time and to fill in a simple questionnaire with personal and epidemiological data. Three milliliter of venous blood were taken by vein puncture under sterile conditions from each subject for detection and titration of antibodies to T. canis and eosinophil counts. Total IgG anti-Toxocara antibodies was evaluated by T. canis IgG ELISA kit. According to the manufacture recommendations, an index positivity >11 U was considered positive. Of the 1210 serum specimens tested, an overall seroprevalence for Toxocara antibodies of 25% was obtained. There was no association between positive seroprevalence and age (p = 0.34). Boys and girls differed significantly with regard to Toxocra titre (p = 0.003). Eosinophilia in peripheral blood (> or = 5%) was detected in 24.5% (297/1210) of the population studied, 97/297 (32.8%) of whom were seropositive for toxocariasis. The findings of this study confirmed that infection with Toxocara is quite high and widespread in children in Northern Iran. Therefore, health promotion efforts must be directed at increasing the awareness of the population about the potential zoonotic hazards associated with the disease and how to minimize them.

Immune-mediated adherence of eosinophils to Toxocara canis infective larvae: the role of excretory-secretory antigens.

The participation of Toxocara canis larval excretory-secretory antigens in immune-mediated adherence was determined in vitro. Adsorption of immune sera with excretory-secretory antigens removed some complement components, removed IgG antibody directed against larval surfaces, and abrogated all adherence observed with untreated immune serum. At least four antigens could be implicated in adherence, by Western blot analysis of adherence mediating sera. Scanning and transmission electron microscopic examination of larval-eosinophil interactions revealed that eosinophils adhered to a membranous sheath-like layer that was frequently detached from the larval epicuticle. The layers appeared to be composed of surface antigens and antibody, and may provide larvae with protection against antibody and eosinophil toxins by preventing their contact with the epicuticle. The release of surface antigens also may be important in allowing larvae to evade the host's immune response by facilitating the removal of antibody and eosinophils from the larval surface.

Persistence of specific antibody response in different experimental infection of mice with toxocara canis larvae

Estudou-se a cinetica de anticorpos anti-Toxocara em camundongos BALB/c infectados experimentalmente segundo tres esquemas: Grupo I (GI): 25 camundongos infectados com dose unica de 200 ovos embrionados de T. canis; grupo II (GII): 25 camundongos infectados com 150 ovos embrionados de T. canis, divididos em tres doses de 50 ovos, administrados no 1§, 5§ e 8§ dias; grupo III (GIII): 25 camundongos infectados com 150 ovos embrionados de T. canis, administrados em tres doses de 50 ovos no 1§, 14§ e 28§ dias...