Cell viability analysis of Toxocara cati larvae with LIVE / DEAD® Viability/Cytotoxicity kit.

Toxocara spp. are responsible for causing toxocariasis, a zoonotic disease of global significance. In some countries of South America, toxocariasis is considered the most prevalent human helminthic infection. The objective of this study was to evaluate LIVE/DEAD® Viability/Cytotoxicity kit as an alternative method to analyze the viability of Toxacara cati larvae. Two control groups were used to confirm the usage of this methodology: 100 untreated T. cati larvae as a negative control (G1) and 100 T. cati larvae killed by thermal shock as a positive control (G2). Subsequently, the viability of T. cati larvae was assessed by the exclusion of the trypan blue dye and by LIVE/DEAD® Viability/Cytotoxicity kit, as well as observation of motility and morphology. In order to confirm the larvicidal effect, T. cati larvae G1 and G2 were inoculated in mice to evaluate their progression in vivo. As expected, G1 showed negative staining by Trypan blue and was stained green by LIVE/DEAD® Viability/Cytotoxicity kit in all the exposure periods. Moreover, G1 presented 100% of relative motility (RM) (score of 5). G2 group was stained blue by Trypan blue and red by LIVE/DEAD® Viability/Cytotoxicity kit, and had 0% RM (score zero) in 24 h of incubation period. In mice, G2 was not viable and, therefore, was not able to infect the animals. In mice inoculated with G1, however, larvae were recovered from all the evaluated organs, except eyes. These results demonstrate that the viability of T. cati larvae was accurately obtained by the LIVE/DEAD® Viability/Cytotoxicity kit, making it an alternative method for viability evaluation.

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.

Morphological and Molecular Characterization of Toxocara tanuki (Nematoda: Ascaridae) from Korean Raccoon Dog, Nyctereutes procyonoides koreensis.

Present study was performed to describe the morphological and molecular characterization of Toxocara tanuki (Nematoda: Ascaridae) from Korean raccoon dog, Nyctereutes procyonoides koreensis, naturally infected in the Republic of Korea (Korea). Juvenile and adult worms of T. tanuki were recovered in 5 out of 10 raccoon dogs examined and the larval worms were detected in 15 out of 20 muscle samples (75%). Small lateral alae were observed on the cranial end of the body in male and female adults and 2 long spicules (3.0-3.5 mm) were characteristically observed in the posterior end of males. In SEM observation, 18 pairs of proximal precloacal, a precloacal median, a postcloacal median and 5 pairs of postcloacal papillae were uniquely revealed in the posterior portion of males, but the proximal papillae were not shown in the lateral ends of females. Molecular analysis on the 18S rRNA partial DNA sequences was revealed the same finding in both samples, adult worms and muscle larvae, which are closely related to T. tanuki. In conclusion, it was confirmed for the first time that T. tanuki is indigenously distributed, the Korean raccoon dog is acted as the natural definitive host of this nematode in Korea and the morphological characteristics of T. tanuki were shown in specific structure for single postcloacal median papilla in male.

Efficacy of methanolic extract of Balanites aegyptiaca fruits on Toxocara vitulorum.

In this study, the effect of the methanolic extract of Balanites aegyptiaca fruits (BAE) on adult Toxocara vitulorum was evaluated after incubating the parasites in Ringer solution containing 10, 30, 60, 120 and 240 µg/ml of the methanolic extract, for 24h using light and scanning electron microscopic observations. Differences in response to BAE action were concentration dependent. These changes occurred in definite sequences in response to BAE concentration and were consisted of slightly swelling which became pronounced and so severe, with lips showed wrinkled cuticular surface and deformed sensory papillae on increasing the BAE concentration. The strongest effects were reached with the highest BAE concentration, where disorganization of the cuticle and body musculature was observed. Additionally, the ovicidal effect of BAE, at the previous concentrations, on the development of T. vitulorum eggs was examined after 12h exposure. The inhibitory activity of BAE on egg development was concentration dependent and the highest value reached to 100% with the concentration of 240 µg/ml. These results were compared with those observed in the worm cuticle and eggs following incubation in albendazole, as it was a broad-spectrum nematodicidal compound with well-known ovicidal activity.

Cryopreservation and long-term in vitro maintenance of second-stage larvae of Toxocara canis.

Second stage larvae of Toxocara canis were isolated from developed eggs, frozen in Eagle's Minimal Essential Medium with 5% dimethyl sulfoxide or 10% glycerol as cryoprotectants according to two cooling schedules and maintained in liquid nitrogen for 1 week. After thawing, the previously frozen larvae (FL) and unfrozen controls (CL) were maintained in a chemically defined medium in vitro for 35 weeks. While CL had motility rates around 95% to 97% throughout the experiment, previously frozen larvae (FL) exhibited rates of 48%-58% at the beginning and of 19%-39% at the end of the 35 week in vitro maintenance period. The surviving FL and CL larvae proved to be infective for mice. Excretory/secretory (ES) antigens isolated from several batches of culture medium in which FL and CL had been maintained reacted in the ELISA with human sera containing antibodies against Toxocara. Antigens from FL and CL separated by SDS-PAGE and silver-stained showed some differences in polypeptide patterns. Western-blot analysis revealed that these differences were not related to antigenic polypeptides but were most likely caused by substances without antigenic properties originating from dead and/or degenerating larvae. It can be concluded that ES antigens produced by previously frozen larvae are essentially the same as those derived from unfrozen controls. The value of cryopreservation of T. canis larvae for routine production of ES antigens will be further evaluated.