An improved method to obtain antigen-excreting Toxocara canis larvae

Toxocara canis is a dog helminth which causes visceral larva migrans (VLM) when infecting humans as a larva. The infection is demonstrated by detecting IgG antibodies against excretory-secretory larval antigens (ESLA) in serum by ELISA. The production of ESLA involves the collection of adult worms from dog puppy stools, the separation of eggs from dissected uteri, and the in vitro growing of egg-derived larvae, following the time-consuming and laborious protocol described by De Savigny [De Savigny, D.H., 1975. In vitro maintenance of T. canis larvae and a simple method for the production of Toxocara ES antigen for the uses in serodiagnostic tests for visceral larva migrans. Journal of Parasitology 61, 781-782]. In this work, an improved protocol for obtaining T. canis larvae is described. The modifications proposed improved the efficiency of the original De Savigny method in three ways: (i) increasing the parasite yield up to five fold, (ii) improving the larval purity, and (iii) markedly reducing the execution time of the protocol.     

Toxocara canis: Larval migration dynamics, detection of antibody reactivity to larval excretory-secretory antigens and clinical findings during experimental infection of gerbils (Meriones unguiculatus)

In this study, Mongolian gerbils were used to analyse features of Toxocara infection that included larval migration, humoral immune responses to Toxocara canis excretory-secretory antigens (TES) and aspects of host physiology. At day 10 post-infection (p.i.) most larvae were in the intestine and the lungs while later the total number of larvae was higher in the carcass tissue; the number of larvae per gram of tissue was lower elsewhere other than in the brain. Infected animals showed several neurological abnormalities, an early increase in leukocyte and neutrophil levels, two peaks of peripheral eosinophilia (5 and 40 d.p.i.) and high antibody levels against TES in the circulation and in the vitreous humor. A sequential recognition of eight T.canis larval antigens with MW from 24 to 200 kDa was detected by Western blot. The results obtained in this study further support the use of gerbils as an experimental model for systemic, ocular and cerebral toxocariasis.     

The epidemiology and public health importance of toxocariasis: A zoonosis of global importance

Toxocariasis, caused by infection with larvae of Toxocara canis, and to a lesser extent by Toxocara cati and other ascaridoid species, manifests in humans in a range of clinical syndromes. These include visceral and ocular larva migrans, neurotoxocariasis and covert or common toxocariasis. Toxocara canis is one of the most widespread public health and economically important zoonotic parasitic infections humans share with dogs, cats and wild canids, particularly foxes. This neglected disease has been shown through seroprevalence studies to be especially prevalent among children from socio-economically disadvantaged populations both in the tropics and sub-tropics and in industrialised nations. Human infection occurs by the accidental ingestion of embryonated eggs or larvae from a range of wild and domestic paratenic hosts. Most infections remain asymptomatic. Clinically overt infections may go undiagnosed, as diagnostic tests are expensive and can require serological, molecular and/or imaging tests, which may not be affordable or available. Treatment in humans varies according to symptoms and location of the larvae. Anthelmintics, including albendazole, thiabendazole and mebendazole may be given together with anti-inflammatory corticosteroids. The development of molecular tools should lead to new and improved strategies for the treatment, diagnosis and control of toxocariasis and the role of other ascaridoid species in the epidemiology of Toxocara spp. Molecular technologies may also help to reveal the public health importance of T. canis, providing new evidence to support the implementation of national control initiatives which have yet to be developed for Toxocara spp. A number of countries have implemented reproductive control programs in owned and stray dogs to reduce the number of young dogs in the population. These programs would positively impact upon T. canis transmission since the parasite is most fecund and prevalent in puppies. Other control measures for T. canis include the regular and frequent anthelmintic treatment of dogs and cats, starting at an early age, education and enforcement of laws for the disposal of canine faeces, dog legislation and personal hygiene. The existence of wild definitive and paratenic hosts complicates the control of T. canis. Increasing human and dog populations, population movements and climate change will all serve to increase the importance of this zoonosis. This review examines the transmission, diagnosis and clinical syndromes of toxocariasis, its public health importance, epidemiology, control and current research needs.     

Seroprevalence and risk factors associated with zoonotic parasitic infections in small ruminants in the Greek temperate environment

A cross-sectional serological study was carried out to screen the sheep and goat population of Thessaly, Greece for evidence of infection with Toxoplasma, Toxocara, Leishmania, and Echinococcus and to determine the risk factors related to herd characteristics, herd management practices, farmer status, and the bioclimatic variables associated with these zoonotic parasitic infections. A total of 540 sheep and goat serum samples were examined. The seroprevalence of infection in all examined animals was 24.5% for Toxoplasma, 32% for Toxocara, 0% for Leishmania and 85.9% for Echinococcus. The final logistic regression model showed that the species of small ruminant, herd size, anthelmintic treatment, class of anthelmintic treatment, grazing with other herds, educational level of farmer, elevation of farm location, and generalized land cover were associated with Toxoplasma gondii infections, while the species of small ruminant, farm type, anthelmintic treatment, class of anthelmintic treatment, rotation of grazing, age of farmer, elevation of farm location, and generalized land cover were associated with Toxocara canis infections. Antibodies to T. gondii were detected in 102 (28.3%) of 360 sheep and in 30 (16.8%) of 179 goats. Animals in small flocks (150–300 animals) had an approximately 0.42-fold lower risk of having positive cases of T. gondii among animals compared with large flocks (> 300 animals). Antibodies to T. canis were found in 155 (42.9%) of 361 sheep and 18 (10.1%) of 179 goats. The later finding constitutes the first report of seropositive goats to Toxocara. The risk of positivity for T. canis was 7.71-fold higher in sheep than in goats. Geographically, animals from plain areas had 2.9 and 2.01-fold higher risk of having positive cases of T. gondii and T. canis respectively. The significant bioclimatic variables (p < 0.05) associated with the occurrence locations of T. gondii infection were related to higher temperature, lower precipitation, and lower elevation compared to the absence locations of T. gondii. The significant bioclimatic variables (p < 0.05) associated with occurrence locations of T. canis infection were related to lower temperature and higher precipitation compared to absence locations of T. canis. These findings are useful to formulate appropriate control strategies for zoonotic parasites of sheep and goats in Greece and other areas with similar climatic conditions.     

Effects of Toxoplasma gondii and Toxocara canis Antigens on WEHI-164 Fibrosarcoma Growth in a Mouse Model

Cancer is the main cause of death in developed countries. However, in underdeveloped countries infections and parasitic diseases are the main causes of death. There are raising scientific evidences indicating that parasitic infections induce antitumor activity against certain types of cancers. In this study, the effects of Toxoplasma gondii and Toxocara canis egg antigens in comparison with Bacillus Calmette Guerin (BCG) (known to have anticancer distinctive) on WEHI-164 fibosarcoma transplanted to BALB/c mice was investigated. Groups of 6 male BALB/c mice injected with T. gondii antigen, BCG, or T. canis egg antigen as case groups and alum alone as control groups. All mice were then challenged with WEHI-164 fibrosarcoma cells. The mice were examined for growth of the solid tumor and the tumor sizes were measured every other day up to 4 wk. The mean tumor area in T. gondii, BCG, or alum alone injected mice in 4 different days of measurements was 25 mm², 23 mm², and 186 mm² respectively. Also the mean tumor area in T. canis injected mice in 4 different days was 25.5 mm² compared to the control group (alum treated) which was 155 mm². T. gondii parasites and T. canis egg antigens induced inhibition of the tumor growth in the fibrosarcoma mouse model. We need further study to clarify the mechanisms of anti-cancer effects.     

Toxocara Seroprevalence among Clinically Healthy Individuals,Pregnant Women and Psychiatric Patients and Associated Risk Factors in Shandong Province,Eastern China

Background

Toxocarosis is a widespread zoonosis caused by the ascarid nematodes Toxocara canis and Toxocara cati, which primarily infect dogs and cats, respectively. Most human infections with Toxocara are asymptomatic; however, some infected individuals may develop a serious illness and even death. Nevertheless, epidemiological knowledge regarding the prevalence and risks associated with Toxocara infection is limited in China. Therefore, we performed a cross-sectional pilot study and estimated the seroprevalence of Toxocara infection in humans in Shandong Province, eastern China for the first time, from June 2011 to July 2013, involving clinically healthy individuals, pregnant women and psychiatric patients, aiming to attract public attention to Toxocara infection.

Methodology/Principle Findings

Seroprevalence of Toxocara was determined using an enzyme-linked immunosorbent assay based on a cross-sectional study conducted in Qingdao and Weihai, Shandong Province, eastern China. Factors potentially associated with Toxocara infection were identified by logistic regression analysis. The overall Toxocara seroprevalence among the study population (n = 2866) was 12.25%, and a significantly higher seroprevalence in psychiatric patients (16.40%, 73/445) than that in clinically healthy individuals (13.07%, 187/1431) and pregnant women (9.19%, 91/990) was revealed. Univariate analyses suggested that keeping dogs at home (OR = 0.06, 95% CI 0.05–0.08, P<0.001), contact with cats and dogs (OR = 0.42, 95% CI 0.33–0.53, P<0.001) and exposure with soil (OR = 0.37, 95% CI 0.28–0.49, P<0.001) were risk factors associated with Toxocara infection.

Conclusions/Significance

The present study revealed, for the first time, that human infection with Toxocara is common in eastern China, posing a significant public health concern. Increasing human and dog populations, population movements and climate change all will serve to increase the importance of this zoonosis. Further studies under controlled conditions are necessary to define potential morbidity associated with Toxocara infection.     

Toxocara Seropositivity, Atopy and Wheezing in Children Living in Poor Neighbourhoods in Urban Latin American

Background

Toxocara canis and T. cati are parasites of dogs and cats, respectively, that infect humans and cause human toxocariasis. Infection may cause asthma-like symptoms but is often asymptomatic and is associated with a marked eosinophilia. Previous epidemiological studies indicate that T. canis infection may be associated with the development of atopy and asthma.

Objectives

To investigate possible associations between Toxocara spp. seropositivity and atopy and childhood wheezing in a population of children living in non-affluent areas of a large Latin American city.

Methods

The study was conducted in the city of Salvador, Brazil. Data on wheezing symptoms were collected by questionnaire, and atopy was measured by the presence of aeroallergen-specific IgE (sIgE). Skin prick test (SPT), total IgE and peripheral eosinophilia were measured. Toxocara seropositivity was determined by the presence of anti-Toxocara IgG antibodies, and intestinal helminth infections were determined by stool microscopy.

Findings

Children aged 4 to 11 years were studied, of whom 47% were seropositive for anti-Toxocara IgG; eosinophilia >4% occurred in 74.2% and >10% in 25.4%; 59.6% had elevated levels of total IgE; 36.8% had sIgE≥0.70 kU/L and 30.4% had SPT for at least one aeroallergen; 22.4% had current wheezing symptoms. Anti-Toxocara IgG was positively associated with elevated eosinophils counts, total IgE and the presence of specific IgE to aeroallergens but was inversely associated with skin prick test reactivity.

Conclusion

The prevalence of Toxocara seropositivity was high in the studied population of children living in conditions of poverty in urban Brazil. Toxocara infection, although associated with total IgE, sIgE and eosinophilia, may prevent the development of skin hypersensitivity to aeroallergens, possibly through increased polyclonal IgE and the induction of a modified Th2 immune reaction.     

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.     

Development of nested multiplex polymerase chain reaction (PCR) assay for the detection of Toxocara canis, Toxocara cati and Ascaris suum contamination in meat and organ meats

Ascarid Larva Migrans Syndrome (ascarid LMS) is a clinical syndrome in humans, caused by the migration of animal roundworm larvae such as Toxocara canis, Toxocara cati and Ascaris suum. Humans may acquire infection by ingesting embryonated eggs, or infective larvae of these parasites in contaminated meat and organ meats. To detect these pathogenic contaminations, a novel nested multiplex PCR system was developed. Our novel nested multiplex PCR assay showed specific amplification of T. canis, T. cati and Ascaris spp. Detection limit of the nested multiplex PCR was tested with serial dilution of T. canis, T. cati or A. suum genomic DNA (gDNA) from 100?pg to 100 ag and found to be 10?fg, 1?fg and 100?fg, respectively. When larvae were spiked into chicken liver tissue, DNA of T. canis and A. suum was detected from the liver spiked with a single larva, while the assay required at least 2 larvae of T. cati. Moreover, the ascarid DNA was detected from the liver of mice infected with 100 and 300 eggs of T. canis, T. cati or A. suum. This nested multiplex PCR assay could be useful for the detection of contamination with ascarid larvae in meat and organ meats.     

Immunopathological Changes in the Brain of Immunosuppressed Mice Experimentally Infected with Toxocara canis

Toxocariasis is a soil-transmitted helminthozoonosis due to infection of humans by larvae of Toxocara canis. The disease could produce cognitive and behavioral disturbances especially in children. Meanwhile, in our modern era, the incidence of immunosuppression has been progressively increasing due to increased incidence of malignancy as well as increased use of immunosuppressive agents. The present study aimed at comparing some of the pathological and immunological alterations in the brain of normal and immunosuppressed mice experimentally infected with T. canis. Therefore, 180 Swiss albino mice were divided into 4 groups including normal (control) group, immunocompetent T. canis-infected group, immunosuppressed group (control), and immunosuppressed infected group. Infected mice were subjected to larval counts in the brain, and the brains from all mice were assessed for histopathological changes, astrogliosis, and IL-5 mRNA expression levels in brain tissues. The results showed that under immunosuppression, there were significant increase in brain larval counts, significant enhancement of reactive gliosis, and significant reduction in IL-5 mRNA expression. All these changes were maximal in the chronic stage of infection. In conclusion, the immunopathological alterations in the brains of infected animals were progressive over time, and were exaggerated under the effect of immunosuppression as did the intensity of cerebral infection.     

A Case of Ocular Angiostrongyliasis with Molecular Identification of the Species in Vietnam

A 23-year-old female residing in a village of Cao Bang Province, North Vietnam, visited the Hospital of Hanoi Medical University in July 2013. She felt dim eyes and a bulge-sticking pain in her left eye for some days before visiting the hospital. In the hospital, a clinical examination, an eye endoscopy, and an operation were carried out. A nematode specimen was collected from the eye of this patient. The body of this worm was thin and long and measured 22.0×0.3 mm. It was morphologically suggested as an immature female worm of Angiostrongylus cantonensis. By a molecular method using 18S rRNA gene, this nematode was confirmed as A. cantonensis. This is the first molecular study for identification of A. cantonensis in Vietnam.     

Parasite-specific antibody profile in the aqueous humor of rabbits with ocular toxocariasis

Human ocular toxocariasis is diagnosed using ophthalmologic and immunologic examinations. Many researchers have suggested that intraocular parasite-specific antibody levels are indicative of ocular toxocariasis, but little is known about the time course of the changes in these levels. We therefore investigated the anti-Toxocara canis antibody profile in the aqueous humor in an animal model of ocular toxocariasis. We intravitreally injected T. canis larvae into the right eye of 4 rabbits; 2 rabbits were orally administered T. canis eggs. We collected serum, aqueous humor, and tear samples weekly and determined the serum and aqueous humor levels of anti-T. canis immunoglobulin (Ig)G, IgA, IgM, and IgE antibodies and the tear IgG antibody level by enzyme-linked immunosorbent assay (ELISA). The severity of vitreous opacity and the aqueous humor IgG levels (measured using optical density [OD]) changed concordantly in the larvae-injected eyes; the OD exceeded 0.1 from 2–4 weeks after infection and remained elevated during active intraocular inflammation. However, the aqueous humor IgG levels were also elevated in 6 out of 8 eyes without intraocular larvae in both groups, and were low in 1 eye with live intravitreal larvae. In contrast, the serum IgG and IgM levels and the tear IgG levels increased in all rabbits, regardless of the presence of intraocular inflammation. Vitreous opacity occurred in all intravitreally infected eyes, but significant histopathological evidence of retinal damage was not detected. Thus, besides the presence of intraocular larvae, some other factors in the host may be required for the development of retinal lesions.     

Toxocara canis: T lymphocyte function in murine visceral larva migrans and eosinophilia onset

The second larval stage of Toxocara canis, the major cause of visceral larva migrans syndrome in humans and also the common intestinal roundworm of the dog, elicits an eosinophil-rich granulomatous response in nondefinitive hosts such as man and mice. During murine infection a population of T lymphocytes was identified which responded to a preparation of T. canis larval antigens in a migration inhibition test (MIT). It was further shown that mice infected with T. canis have a substantial eosinophilia which reaches its peak approximately 2 weeks after infection. When mice are depleted of their functional T lymphocytes by use of antilymphocytic antisera, the eosinophil response of infected mice is significantly reduced. These findings are consistent with the findings of others that helminthic parasites can elicit cell-mediated immune responses and the resultant eosinophilia is but one manifestation of this response.     

Serodiagnosis of Toxocariasis by ELISA Using Crude Antigen of Toxocara canis Larvae

Toxocariasis is a worldwide zoonosis caused by larvae of ascarid nematodes of dogs or cats, Toxocara canis or T. cati. Diagnosis of human toxocariasis currently relies on serology that uses T. canis excretory-secretory antigen to detect specific IgG antibodies by ELISA. We investigated the serodiagnostic efficacy of ELISA using crude antigen of T. canis larvae (TCLA). Serum specimens of 64 clinically confirmed toxocariasis, 115 healthy controls, and 119 other tissue-invading helminthiases were screened by ELISA using TCLA. The ELISA using TCLA showed 92.2% (59/64 patient samples) sensitivity and 86.6% (103/119) specificity. Its positive diagnostic predictivity was 78.7% and negative predictivity was 97.8%. No serum of healthy controls reacted but that of anisakiasis (45.5%), gnathostomiasis (19.2%), clonorchiasis (15.8%), sparganosis (11.1%), and cysticercosis (6.3%) cross-reacted. Immunoblot analysis on TCLA recognized antigenic proteins of 28- and 30-kDa bands in their dominant protein quantity and strong blotting reactivity. The present results indicate that the ELISA using our TCLA antigen is acceptable by the sensitivity and specificity for serodiagnosis of human toxocariasis. ELISA with TCLA is recommended to make differential diagnosis for patients with any sign of organ infiltration and eosinophilia.     

Toxocariasis and epilepsy: systematic review and meta-analysis

Objective

Human toxocariasis is a zoonotic infection caused by the larval stages of Toxocara canis (T. canis) and less frequently Toxocara cati (T. cati). A relationship between toxocariasis and epilepsy has been hypothesized. We conducted a systematic review and a meta-analysis of available data to evaluate the strength of association between epilepsy and Toxocara spp. seropositivity and to propose some guidelines for future surveys.

Data Sources

Electronic databases, the database from the Institute of Neuroepidemiology and Tropical Neurology of the University of Limoges (http://www-ient.unilim.fr/) and the reference lists of all relevant papers and books were screened up to October 2011.

Methods

We performed a systematic review of literature on toxocariasis (the exposure) and epilepsy (the outcome). Two authors independently assessed eligibility and study quality and extracted data. A common odds ratio (OR) was estimated using a random-effects meta-analysis model of aggregated published data.

Results

Seven case-control studies met the inclusion criteria, for a total of 1867 participants (850 cases and 1017 controls). The percentage of seropositivity (presence of anti-Toxocara spp. antibodies) was higher among people with epilepsy (PWE) in all the included studies even if the association between epilepsy and Toxocara spp. seropositivity was statistically significant in only 4 studies, with crude ORs ranging 2.04–2.85. Another study bordered statistical significance, while in 2 of the included studies no significant association was found. A significant (p<0.001) common OR of 1.92 [95% confidence interval (CI) 1.50–2.44] was estimated. Similar results were found when meta-analysis was restricted to the studies considering an exclusively juvenile population and to surveys using Western Blot as confirmatory or diagnostic serological assay.

Conclusion

Our results support the existence of a positive association between Toxocara spp. seropositivity and epilepsy. Further studies, possibly including incident cases, should be performed to better investigate the relationship between toxocariasis and epilepsy.     

The First Human Case of Thelazia callipaeda Infection in Vietnam

A 26-year-old man residing in a village of Thai Nguyen Province, North Vietnam, visited the Thai Nguyen Provincial Hospital in July 2008. He felt a bulge-sticking pain in his left eye and extracted 5 small nematode worms by himself half a day before visiting the hospital. Two more worms were extracted from his left eye by a medical doctor, and they were morphologically observed and genetically analyzed on the mitochondrial cytochrome c oxidase 1 gene. The worms were 1 male and 1 female, and genetically identical with those of Thelazia callipaeda. By the present study, the presence of human T. callipaeda infection is first reported in Vietnam.     

Immunodiagnosis of parasitic zoonoses: purification of Toxocara canis antigens by affinity chromatography

A method of affinity chromatography developed for the purification of species-specific antigens from Toxocara canis adult worms is described. Immunochemical analyses by polyacrylamide gel electrophoresis, immunoelectrophoresis and immunodiffusion showed that ‘pure’ antigen contained fewer but more specific proteins than ‘crude’ antigen. Purified antigens and parasite sections from four parasite species (Toxocara canis, Dirofilaria immitis, Angiostrongylus cantonensis and Ascaris lumbricoides) were used in immunofluorescence tests to measure serum antibody levels in animals with natural or experimental T. canis infections and people with zoonotic toxocariasis. ‘Pure’ antigen showed higher specificity and sensitivity than ‘crude’ antigen in serological testing.     

Establishment of a serodiagnosis system for the detection of Toxocara spp. and Ascaris suum infection in chickens

Chickens are considered to act as paratenic hosts for agents, Toxocara canis, T. cati and Ascaris suum; which cause ascarid larva migrans syndrome (ascarid LMS) in humans. In addition, they are the definitive host for Ascaridia galli, considered not to be infective for humans. All ascarid parasites can have a high homology of antigenicity, leading to cross-reactivity in serodiagnostic assays. This study was conducted to establish a procedure for the serological detection of those roundworm infections in chickens.Twenty-five male Julia chickens were divided into five groups (n = 5); T. canis-, T. cati-, Ascaris suum- and Ascaridia galli-infected, and an uninfected control group. In Ascaris suum-soluble worm antigen preparation (As-SWAP) ELISA, all infected groups showed an elevation of anti-ascarid antibodies, indicating the usefulness of As-SWAP as a screening antigen for the detection of ascarid infections. For infecting species identification, T. canis-excretory/secretory (Tc-ES) and Ascaris suum-ES (As-ES) antigen ELISA were conducted by serial dilution sera. Toxocara spp.-infected sera showed stronger binding to Tc-ES than As-ES, while Ascaris suum and Ascaridia galli-infected sera bound to As-ES more strongly than Tc-ES. To discriminate between Ascaris suum and Ascaridia galli infection, sera were pre-incubated with Ascaridia galli-SWAP antigen and applied to Tc-ES and As-ES ELISAs. In this pre-adsorbed ES antigen ELISAs, only the Ascaris suum infected group showed positive binding to As-ES, resulting from the adsorption of cross-reactive antibodies in Ascaridia galli-infected sera. Finally, anti-Toxocara specific antibodies were confirmed by Tc-ES western blot (WB). Toxocara spp.-infected sera showed toxocariasis-specific band pattern in Tc-ES WB, while no specific band appeared on any strip incubated with Ascaris suum, Ascaridia galli-infected and uninfected sera.In conclusion, the serodiagnostic assays evaluated in this study are useful for the detection of ascarid infections in chickens.