Efficacy of a chlorocresol-based disinfectant product on Toxocara canis eggs.

Toxocara canis is a common parasite of dogs and can cause zoonotic toxocariasis in humans. As a part of control programs for this agent, optimized hygiene including chemical disinfection is considered essential in the prevention and control of zoonotic toxocariasis in humans. However, commonly used disinfectants at present mostly fail to inhibit the embryogenesis and viability of T. canis eggs. To this effect, the present study was designed to evaluate the effect of a chlorocresol-based disinfectant product Neopredisan®135-1 (NP) on embryonic development of T. canis eggs in vitro and to investigate the infectivity of exposed eggs by assessing larval establishment in a mouse model. Under in vitro conditions, NP at a final concentration of 0.25, 0.50, 1, 2, or 4% all exhibited significant killing effect on T. canis embryogenesis compared with the control eggs (P < 0.05), regardless of contact times (30, 60, 90, or 120 min). Such killing activity increased in a concentration- and time-dependent manner, with a maximum killing efficacy of 95.81% at 4% concentration and 120 min exposure time. Comparisons between low and high concentrations and between short and long contact times concluded that a protocol using the 1% concentration of NP with a 90-min contact could be the most suitable for practical application. Additionally, the lower larval recovery in mice inoculated with eggs treated by either 0.25 or 0.5% NP than that from their corresponding controls (P < 0.05) verified once again that NP had an adverse impact on the larval development of T. canis eggs even at a low concentration. To the best of our knowledge, this is the first study to report the effect of the chlorocresol-based disinfectant NP on the embryonation and larval development of T. canis eggs, and the results presented here would contribute to environmental clearance and control of toxocariasis by providing an alternative disinfectant resource. However, it is highlighted that the clearance of the novel and existing sources of infection including larvated eggs in places treated with NP is not guaranteed and therefore continuous monitoring and additional disinfection are still required.

Progesterone in vitro increases growth, motility and progesterone receptor expression in third stage larvae of Toxocara canis.

The in vitro effect of progesterone in T. canis larvae on their enlargement and motility were evaluated, together to the possible presence of progesterone receptors (PRs). T. canis larvae were cultured in RPMI-1640 with different concentrations of progesterone (0, 20, 40, 80, 400 and 800 ng/mL). Enlargement and increases in motility were dependent on the concentration only from 0 to 80 ng/mL (p < 0.05). The mean percentage of PR + cells in newly obtained larvae as measured by flow cytometry was 8.16 ± 0.4. The number of PR + cells increased depending on concentration from 0 to 80 ng/mL (p < 0.001). Cells obtained from larvae stimulated at any of the studied hormone concentrations showed greater mean fluorescence intensity when compared to non-stimulated cells. Additionally, the expression and location of PR + cells were determined in the larvae. The sequence of an amplicon (420-bp) obtained by PCR from T. canis larvae showed 100% homology with a gene fragment that codes for the PR of the dog. PR + cells were immunolocated using confocal microscopy in the intestinal region of the larvae that had been recently obtained. The results of this study show that T. canis larvae can recognize and respond to the presence of progesterone through a molecule possibly able to bind it. Since we previously observed a similar response to prolactin, we suggest that both hormones could participate sequentially in the reactivation of T. canis larvae in pregnant bitches.

Toxocara canis: Larvicidal activity of fatty acid amides.

Considering the therapeutic potential of fatty acid amides, the present study aimed to evaluate their in vitro activity against Toxocara canis larvae and their cytotoxicity for the first time. Linoleylpyrrolidilamide was the most potent, with a minimal larvicidal concentration (MLC) of 0.05 mg/mL and 27% cytotoxicity against murine peritoneal macrophages C57BL/6 mice, as assessed by the MTT assay.

Cross-reactivity of Toxocariasis with Crude Antigen of Toxascaris leonina Larvae by ELISA.

Roundworms of Toxocara canis and Toxascaris leonina are common gastrointestinal helminths of canids over the world. Humans are infected with T. canis larvae through ingestion of infective eggs in contaminated environments or larvae by consumption of raw or uncooked meat or livers. Recently, patients of clinically diagnosed toxocariasis are increasing and require correct diagnosis in Korea. The present study investigated serological cross-reactivity between crude antigens of T. canis (TCLA) and T. leonina (TLLA) larvae. We collected serum specimens from 177 toxocariasis patients who were clinically suspected in the Seoul National University Hospital and 115 healthy controls. An ELISA method for toxocariasis was used to evaluate diagnostic efficacy of TLLA for serodiagnosis of human toxocariasis. The IgG ELISA using TLLA gave 14 (14.3%) positives of 98 TCLA positive specimens among 177 suspected toxocariasis patients. Most of them showed high absorbances with TCLA. In conclusion, there is a partial cross reaction between serum specimens of toxocariasis and TLLA.

A comparison of Toxocara canis embryonation under controlled conditions in soil and hair.

Toxocara spp. eggs require a period of time under appropriate environmental conditions to become infective to definitive and paratenic hosts. Temperature and humidity are important factors known to affect the levels of development in soil. We aimed to investigate whether the eggs of T. canis could embryonate in dog hair under controlled conditions of temperature and humidity and, if so, to what degree. No previous work had been carried out on embryonation in hair under controlled conditions. Soil samples exposed to the same conditions as the hair samples were considered a suitable comparison in order to investigate differing levels of development. Development at two temperatures (10°C and 20°C) and the addition of water to samples was investigated over a period of 8 weeks. Importantly, we demonstrated that unembryonated T. canis eggs are capable of development in hair under controlled conditions. The rate of development is lower than that observed in soil, but remains biologically significant in terms of the overall numbers of potentially infective embryonated eggs present. Temperature is responsible for the rate of embryonation while moisture is essential for encouraging development and maintaining egg viability in general. In light of these findings the transmission of Toxocara spp. as a result of direct contact with well-cared-for owned dogs seems unlikely, but should not be ignored.

Temperature and the development and survival of infective Toxocara canis larvae.

The influence of temperature on the development and survival of Toxocara canis larvae was investigated under laboratory conditions, in water at 15, 20, 25, 30 and 35°C and at room temperature 22°C ± 1°C. T. canis eggs were able to develop to the larvated stage at all the tested temperatures. Development rate increased with temperature. Linear regression of development rate against temperature predicted a lower development threshold of 11.8°C. Eggs survived cooling to 1 and -2°C for 6 weeks, and could develop to the infective, larvated stage when transferred to higher temperatures, but their development rates were then retarded compared with non-chilled eggs. Larvated eggs remained viable after 7 weeks of incubation across the tested temperature range, with the highest percentage viability (47%) obtained at 25°C. Development of eggs to the infective larval stage required, on average, 121 degree days between 20°C and 30°C. Results provide a basis for predicting variation in the infectivity of eggs in the environment over time in different climates.

Meningitis by Toxocara canis after ingestion of raw ostrich liver.

Recently reports on toxocariasis are increasing by serodiagnosis in Korea. A previously healthy 17-yr-old boy complained of headache, fever, dyspnea, and anorexia. He showed symptoms and signs of eosinophilic meningitis with involvement of the lungs and liver. Specific IgG antibody to Toxocara canis larval antigen was positive in serum and cerebrospinal fluid by ELISA. He took raw ostrich liver with his parents 4 weeks before the symptom onset. His parents were seropositive for T. canis antigen but had no symptoms or signs suggesting toxocariasis. This is the first report of toxocariasis in a family due to ingestion of raw ostrich liver in Korea.

Imaging of Toxocara canis larvae labelled by CFSE in BALB/c mice.

Mice are used most often as a model for human toxocariasis caused by Toxocara canis larvae. Variety of symptoms developing during the infection reflects behaviour of the larvae, which are able to escape from the intestine and further invade and damage various host organs. In order to find an approach enabling observation on parasite behaviour in mouse in vivo, we used an epifluorescence method and a small animal imaging system (SAIS). Larvae of T. canis were labelled by carboxyfluorescein succinimidyl ester (CFSE) which incorporated on the parasite gastrointestinal tract. Following infection of BALB/c mice by CFSE-labelled larvae it has been observed that staining had no influence on viability and further migratory activity of the parasites through the host organs (the intestine, liver, lungs and brain) where they were detected by SAIS until day 17 p.i. In addition, the dye did not affect larval antigenic activity as well as the development of related immune response. Imaging of parasites labelled by CFSE, therefore, may represent a promising way to study behaviour of T. canis larvae in a paratenic host.

Toxocara canis: potential activity of natural products against second-stage larvae in vitro and in vivo.

The anthelmintic activity of extracts from Chenopodiumambrosioides, Pycnanthusangolensis and Nutridesintox was in vitro and in vivo investigated, against Toxocaracanis larvae. The in vitro assays results showed that the aqueous extract of Nutridesintox was the most effective, followed by C. ambrosioides extracts, hexane, dichloromethane and the infusion. P. angolensis extracts showed a lower anthelmintic activity compared to the other natural products. For the in vivo assays, Nutridesintox, the hexane extract and the infusion of C. ambrosioides were administered orally to T. canis-infected mice, in single doses, during three consecutive days. The efficacy was evaluated on the 17th day post-infection, not only by counting T. canis larvae in the tissues but also by ELISA detection of IgM and IgG antibodies and histological analysis of liver and lungs. The different treatments did not reduce the larvae burden and had no influence on the antibodies dynamic. Interestingly, a reduction on the inflammatory infiltrates was observed in the liver and lung sections of the group treated with the hexane extract of C. ambrosioides. In conclusion, the hexane extract of C. ambrosioides is of further research interest, as it showed an anthelmintic activity in vitro and a reduction on the inflammatory reaction produced by the infection of T. canis larvae in vivo.

Quantification of Toxocara canis DNA by qPCR in mice inoculated with different infective doses.

The nematode Toxocara canis is of public health importance and is the main causative agent of toxocariasis in humans. This disease is difficult to diagnose due to several factors, including the possibility of cross-reactions with other nematodes in the ELISA. To overcome this problem, molecular tests have been recommended as an alternative to identify the parasite. The quantitative real-time polymerase chain reaction (qPCR) technique was used in this study to identify and quantify the parasite load of T. canis in the mouse brain. To this end, 24 mice were divided into six groups, five of which were challenged with different infective doses of T. canis larvae (L3) (1000, 500, 250, 100 and 50 larvae), while the sixth group, uninfected, acted as negative control. Forty-five days after infection, the animals were euthanized to collect the brain, from which two portions of 20 mg of tissue were taken for DNA extraction, while the rest of the brain tissue was digested to quantify the number of larvae by microscopy. The number of DNA copies was calculated from the standard DNA quantification curve, showing values of E = 93.4%, R2 = 0.9655 and Y = -3.415. A strong positive correlation (R = 0, 81; p < .001) was found between the number of copies and the recovery of larvae from brain. However, the parasite's DNA was also identified even in animals from whose brain no larvae were recovered after tissue digestion. The results of this study therefore confirm that the qPCR technique can be a valuable tool for the detection and quantification of T. canis DNA in murine hosts, even in animals whose with tissues contain very few parasites.

Serum metabolomic alterations in Beagle dogs experimentally infected with Toxocara canis.

BACKGROUND: Toxocara canis, a globally distributed roundworm, can cause debilitating disease in dogs and humans; however, little is known about the metabolomic response of the hosts to T. canis infection. There is an increasing need to understand the metabolic mechanisms underlying the pathogenesis of T. canis infection in dogs. Here, we examined the metabolomic changes in Beagle dogs' serum following T. canis infection using LC-MS/MS. RESULTS: The metabolic profiles of Beagle dogs' serum were determined at 12 h, 24 h, 10 d and 36 d after oral infection with 300 infectious T. canis eggs by LC-MS/MS. We tested whether the T. canis-associated differentially abundant metabolites could distinguish the serum of infected dogs from controls, as measured by the area under the receiver operating characteristic (ROC) curve (AUC). The differentially expressed metabolites were further evaluated by principal components analysis and pathway enrichment analysis. A total of 5756 and 5299 ions were detected in ESI+ and ESI- mode, respectively. ROC curve analysis revealed nine and five metabolite markers, at 12 hpi and 24 hpi to 36 dpi, respectively, with potential diagnostic value for toxocariasis. The levels of taurocholate, estradiol, prostaglandins and leukotriene were significantly changed. Primary bile acid biosynthesis pathway, steroid hormone biosynthesis pathway and biosynthesis of unsaturated fatty acids pathway were significantly altered by T. canis infection. CONCLUSIONS: These findings show that T. canis infection can induce several changes in the dog serum metabolome and that the metabolic signature associated with T. canis infection in dogs has potential for toxocariasis diagnosis.

Prolactin evokes lactational transmission of larvae in mice infected with Toxocara canis.

We investigated the trans-lactational maternal-neonatal transmission of Toxocara canis larvae in mice, with particular interest in the role of prolactin in their migration to the mammary gland. Two female mice were infected with 300 T. canis eggs soon after delivery of 27 offspring. After 1 week of breast-feeding, seven larvae were recovered from 4 of 13 offspring. After 2 weeks of lactation, 101 larvae were recovered from all the remaining offspring. Daily prolactin administration (5 microg) was performed 2 weeks before T. canis infection and continued until 2 weeks after infection in six non-pregnant female mice, which resulted in larval accumulation in the mammary gland. Furthermore, prolactin administration in female mice that had been infected with T. canis 4 weeks prior to prolactin treatment induced migration of larvae into the mammary gland. These findings suggest that prolactin is a promoting factor contributing to lactational transmission of T. canis larvae in mice.

Initial stage of development and migratory behavior of Toxocara canis larvae in BALB/c mouse experimental model.

In the present study, the initial developmental stage of Toxocara canis eggs and larvae, and number of recovered larvae from BALB/c mouse-infected organs are described. In vitro culture of T. canis detects the frequencies of interphasic, mitotic and embryonated eggs only within a 7-day period. Analysis by egg counting was carried out for 32 days. The results showed that at 7 days after cultivation, the frequency of larvae was 50.4% and that this frequency reached 52.8% in 32 days. In the experimental infection of BALB/c mice with T. canis, the number of recovered larvae statistically increased in the brain and liver, with doses of approximately 200 and 1000 eggs. After 7 days of infection, a larger number of larvae were obtained in the lung and liver, although a maximum amount was found in the brain after a 15- or 30-day post-infection period.

[Developmental stages and viability of Toxocara canis eggs outside the host].

INTRODUCTION: Toxocariasis is a soil-transmitted zoonotic disease caused mainly by ingestion of larvated eggs of Toxocara canis. OBJECTIVES: To study the morphology of the intraovular developmental stages of Toxocara canis in culture, characterize non-viable eggs and the sequences of larval molting and compare the viability of eggs at the early stages of division and at reaching full maturation. MATERIAL AND METHODS: Observation of developing embryos and characterization of non-viable eggs were done using light microscope. The proportions of viable eggs during embryonation were compared to the proportions of viable mature eggs. RESULTS: Cell division commenced after 24 hours of cultivation. Early stages were found to be present over a period of 3-5 days. The developmental stages identified were eggs with: One cell, two cells, three cells, four cells, early morula, late morula, blastula, gastrula, tadpole, pre-larva, first, second and third stage larva. Two larval molts occurred. Non-viable eggs had degenerated cytoplasm, thin or collapsed shell and the larvae did not move after exposure to light. No significant differences were found between the proportions of viable eggs from day five to day 21 as compared to viability of fully mature eggs (30 days). CONCLUSION: Developing embryos in the environment may be considered as a potential threat to the public health. The precise identification of developmental stages and the clear differentiation of viable and non-viable eggs can help in determining an accurate baseline rate of development that could be used in studies of ovicidal compounds.

Comparative transcriptomic analyses of male and female adult Toxocara canis.

Toxocariasis is an important, neglected zoonosis caused mainly by Toxocara canis. Although our knowledge of helminth molecular biology is improving through completed draft genome projects, there is limited detailed information on the molecular biology of Toxocara species. Here, transcriptomic sequencing of male and female adult T. canis and comparative analyses were conducted. For each sex, two-thirds (66-67%) of quality-filtered reads mapped to the gene set of T. canis, and at least five reads mapped to each of 16,196 (87.1%) of all 18,596 genes, and 321 genes were specifically transcribed in female and 1467 in male T. canis. Genes differentially transcribed between the two sexes were identified, enriched biological processes and pathways linked to these genes established, and molecules associated with reproduction and development predicted. In addition, small RNA pathways involved in reproduction were characterized, but there was no evidence for piwi RNA pathways in adult T. canis. The results of this transcriptomic study should provide a useful basis to support investigations of the reproductive biology of T. canis and related nematodes.2.

Flotation of Toxocara canis Eggs in Commercial Bleach and Effects of Bleach Treatment Times on Larval Development in These Eggs.

Toxocara canis is a common intestinal nematode of young dogs. Puppies contaminate the environment with large numbers of eggs that can embryonate and become infective in less than a month. Embryonated eggs are infectious for humans and other paratenic hosts. Most T. canis infections in humans are asymptomatic; however, migration of T. canis larvae in the eye and in the central nervous system can result in vision loss, blindness, and even death. The eggs of T. canis are highly resistant to harsh environmental conditions and routinely used chemical disinfectants. The objective of this study was to evaluate the effects of full-strength commercial bleach (5.25% sodium hypochlorite solution) treatment on development of T. canis eggs and to report our serendipitous finding that T. canis eggs in dog feces can float in passive fecal flotation tests using bleach. We also demonstrated that T. canis eggs could be identified using the McMaster's fecal eggs counting test using 100% bleach. Toxocara canis eggs collected from the feces of naturally infected 4-8 wk old puppies were treated with full-strength bleach (5.25% sodium hypochlorite solution) for 15 min, 30 min, 60 min, and 120 min; washed free of bleach smell by centrifugation; and resuspended in 0.1 N sulfuric acid solution to undergo larval development at room temperature for 18 days after exposure to bleach. Motile larvae were observed in T. canis eggs in all groups treated for 15-120 min and eggs continuously exposed to bleach for 18 days. Our results indicate that bleach may not be an appropriate disinfectant for dog kennels, cages, or laboratory utensils and work surfaces. Toxocara canis eggs are resistant to bleach treatment and continue to pose a risk for canine and human infections. Further study is needed to find the most appropriate methods for disinfection and removal of eggs to reduce the risk of transmission of this parasite.

Population dynamics and epidemiology of Toxocara canis in Danish red foxes.

Toxocara canis, an intestinal helminth of canids with zoonotic potential, was found in 618 (59%) of 1,040 red foxes (Vulpes vulpes) collected from all Danish provinces (1997-2002). The prevalence and average worm burden were significantly higher for cubs than older foxes and in males than in females. A multiple logistic regression demonstrated that the prevalence was influenced significantly by sex and age of foxes in addition to location, season, and year of collection. The highest prevalence and worm burden were found in rural areas. The size and number of female worms was positively correlated to the fecal egg excretion. The length and fecundity of the worms was significantly higher in male foxes, and a general intensity dependence was suggested from a negative correlation between worm numbers and worm lengths. As compared to intestinal recovery of worms, somatic larvae were recovered from 20% of muscle samples and fecal eggs in 41% of fecal samples. The consistent finding of T. canis larvae in somatic tissues of naturally infected foxes is new.

The in vitro effect of prolactin on the growth, motility and expression of prolactin receptors in larvae of Toxocara canis.

The in vitro effect of prolactin (PRL) on the growth and motility of Toxocara canis larvae was assessed. Additionally, the expression and location of prolactin receptors (PRL-Rs) were determined in the larvae. Larvae of T. canis were incubated with different concentrations of PRL for different periods of time. The stimulated larvae accelerated their enlargement and increased their motility. The mean percentage of PRL-R+ cells in non-stimulated larvae, measured by flow cytometry was 7.3±0.3%. Compared with non-stimulated larvae, the mean fluorescence intensity (p<0.05) increased in larvae incubated with 40ng/mL of PRL for 10 days. A 465-bp length fragment was amplified from larvae gDNA by PCR. The sequence of this fragment showed 99% similarity with the gene fragment that codes for the PRL-R of the domestic dog. A high concentration of PRL-Rs was immune-located in the posterior region of the larval intestine; therefore, the intestinal cells in this region were most likely the targets for this hormone. Based on these results, PRL-Rs were identified in T. canis larvae, and the in vitro stimulation with PRL increased the number of these receptors, accelerated the growth and modified the activity of larvae. All of the above suggest that T. canis larvae are evolutionarily adapted to recognize the PRL of their definitive host and furthermore might explain the reactivation of tissue-arrested larvae during the gestation of bitches, which does not occur in gestating females of other species.

The effect of free and polyethylene glycol-liposome-entrapped albendazole on larval mobility and number in Toxocara canis infected mice.

As part of our exploratory drug research on the larva migrans that causes roundworm in dogs and cats, this study was carried out to clarify the effect of free and liposome-entrapped (LE) albendazole in Toxocara canis infected mice. In infected mice, evaluation of mobility and number of larva were examined in detail in the brain, skeletal muscle and liver. Larva mobility was evaluated by using the relative mobility (RM) value. Albendazole was LE as one of the drug delivery systems (DDSs). Polyethylene glycol (PEG) was added to the liposome in order to avoid evoking a response by the reticuloendothelial system (RES). By using the albendazole PEG-LE delivery system, it was possible to target the larvae in the mouse brain and liver resulting in a decrease in the number of larvae. In the skeletal muscle of the infected mice, the intraperitoneal dosages of PEG-LE albendazole did not cause a complete decrease in the number of larvae, even though free albendazole did cause the number to decrease. Therefore, it is necessary to take into consideration the migrating stage of the larvae before the initiation of any drug administration.

Giardia intestinalis and other zoonotic parasites: prevalence in adult dogs from the southern part of Mexico City.

The protozoan Giardia intestinalis is a mammalian-infecting parasite. It produces diarrhoea and malabsorption in its hosts. There is growing evidence that dogs could be reservoirs and play an important role in transmission. In Mexico, there are few data on the frequency of G. intestinalis. Therefore, we studied the small intestine of stray dogs, euthanazed at the "Culhuacan" Control Canine Centre, towards the end of 1997 and during the summer of 1998. We microscopically analysed intestinal contents and mucus samples taken every 3cm. During the cold season (winter), parasites were not found in 38/100 dogs, in contrast to 8/100 through the warm season. We found that 42/100 in winter and 51/100 in summer harboured G. intestinalis. To our knowledge, these G. intestinalis frequencies are the highest found in adult dogs worldwide. The results showed a rise in Ancylostoma spp. from 23/100 to 67/100 during the cold and warm seasons. Toxocara canis frequencies varied between 12/100 and 18/100, respectively. The data suggest that the probability of infection is higher during the hottest months compared to the coldest months of the year. Both puppies and adult dogs are highly infected. Dogs are reservoirs for zoonotic parasites; for this reason, it is imperative for humans to avoid fecal contamination in streets, public gardens and parks.