Frequency of soil contamination by Toxocara canis eggs in the south region of São Paulo municipality (SP, Brazil) in a 18 month period

Soil contamination by embryonic eggs of Toxocara canis is the main source of human infection by this ascarid larvae resulting, sometimes, in the occurrence of visceral larva migrans syndrome. The objective of the present research is to determine the frequency of T. canis eggs in soil samples monthly collected in nine public places, located at the South Region of São Paulo municipality in a 18-month period, from February 2004 to July 2005. The soil samples collected were treated with a 30% antiformine solution and with a sodium dichromate solution (d = 1.40) and microscopic slides were prepared and examined under light microscopy for searching T. canis eggs. Two peaks of higher frequency had been found, one in February - May 2004 and the other in April - July 2005.

Influence of variables on centrifuge-flotation technique for recovery of Toxocara canis eggs from soil

The purpose of this study was to evaluate the influence of variables in a flotation technique for the recovery of Toxocara canis eggs from soil. The trials were done under standardized conditions on one gram of previously sterilized soil samples contaminated with 200 eggs of T. canis. The following variables were evaluated in serial steps: sieving; type of wash; time of stirring; resuspension of sediment; solution flotation. Centrifuge-flotation in sodium nitrate (d = 1.20 g/cm³) was adopted as an initial technique, using Tween 80 (0.2%) and decinormal sodium hydroxide as solutions for washing the samples. Ten tests were done to compare the variables, using counting in triplicate. The sieving of the material reduced significantly the recovery of eggs (p < 0.001) and the number of eggs recovered was higher when the sediment was resuspended (p < 0.05). After standardization, flotation solutions sodium chloride, zinc sulfate, sodium dichromate, magnesium sulfate, and sodium nitrate (d = 1.20g/cm³) were compared. The best results were obtained by using zinc sulfate solution. In conclusion, the chances of recovering T. canis eggs from samples using flotation solutions can be increased by washing of soil twice using distilled water, and resuspension of sediment. On the other hand, the sieving procedure can drastically reduce the number of eggs.

Toxocara canis (Werner, 1782) eggs in the Pleistocene site of Menez-Dregan, France (300,000-500,000 years before present)

On the archaeological site of Menez-Dregan in Brittany, France, dated 300,000-500,000 years-old, paleoparasitological analysis of cave deposits led to the detection of well-preserved helminth eggs, which morphology and morphometry pointed to the diagnosis of Toxocara canis eggs, a parasite of carnivore mammals. Paleolithic remains suggested a parasitism of the hyena Crocuta spelaea or other canids that inhabited the region.

Persistence of specific antibody response in different experimental infections of mice with Toxocara canis larvae

Anti-Toxocara antibody production and persistence were studied in experimental infections of BALB/c mice, according to three different schedules: Group I (GI) - 25 mice infected with 200 T. canis eggs in a single dose; Group II (GII) 25 mice infected with 150 T. canis eggs given in three occasions, 50 in the 1st, 50 in the 5th and 50 in the 8th days; Group III (GIII) - 25 mice also infected with 150 T. canis eggs, in three 50 eggs portions given in the 1st, 14th and 28th days. A 15 mice control group (GIV) was maintained without infection. In the 30th, 50th, 60th, 75th, 105th and 180th post-infection days three mice of the GI, GII and GIII groups and two mice of the control group had been sacrificed and exsanguinated for sera obtention. In the 360th day the remainder mice of the four groups were, in the same way, killed and processed. The obtained sera were searched for the presence of anti-Toxocara antibodies by an ELISA technique, using T. canis larvae excretion-secretion antigen. In the GI and GII, but not in the GIII, anti-Toxocara antibodies had been found, at least, up to the 180th post-infection day. The GIII only showed anti-Toxocara antibodies, at significant level, in the 30th post-infection day.

Identification of toxocara canis antigens by Western blot in experimentally infected rabbits

Toxocariasis is a frequent helminthiasis that can cause visceral and ocular damage in humans specially in children. The identification of specific antigens of Toxocara canis is important in order to develop better diagnostic techniques. Ten rabbits were infected orally with a dose of 5000 Toxocara canis embryonated eggs. Rabbits were bled periodically and an ELISA assay was performed to determine levels of specific Toxocara IgG antibodies. ELISA detected antibodies at day 15 after infection. Western blot (WB) assay was performed using excretory/secretory antigens (E/S) of T. canis second stage larvae. Different antigen concentrations were evaluated: 150, 200, 250 and 300 µg/mL. The concentration of 250 µg/mL was retained for analysis. Rabbit sera were diluted 1:100. Secondary antibody was used at a dilution of 1:1000. Results of WB indicated that in the first month after infection specific antibodies against the 200 KDa, 116 KDa, 92 KDa and 35 KDa antigens were detected; antibodies against the 92 KDa, 80 KDa, 66 KDa, 45 KDa, 31 KDa and 28 KDa antigens appeared later. All positive sera in the ELISA test were also positive in WB. Two antigen bands, 92 KDa and 35 KDa, were identified since the beginning and throughout the course of infection. These antigens merit further evaluation as candidates for use in diagnosis.

Environmental contamination by Toxocara sp. eggs in Ribeirão Preto, São Paulo State, Brazil

Toxocariasis is a zoonosis mainly caused by Toxocara canis, an intestinal nematode of dogs. Man acquires the infection through accidental ingestion of viable eggs, and the toxocariasis clinical manifestations may vary from an asymptomatic infection up to the Visceral Larva Migrans syndrome. Seventy eight public squares of Ribeirão Preto, São Paulo, Brazil, including Bonfim Paulista district were visited aiming to evaluate the soil contamination by Toxocara eggs. The squares were divided in five different areas corresponding to the Sanitary Districts of the city. From May to December 2003, soil samples weighting about 250 g each were collected from five distinct sites of each public square. The laboratorial analysis was done by centrifugal-flotation techniques in magnesium sulphate solutions with 5% of potassium iodide (d = 1.33) and zinc sulphate (d = 1.20), and by the sedimentation- flotation in conic chalices with zinc sulphate (d = 1.20). Toxocara sp. eggs were found on 16 (20.5%) squares, with the lowest prevalence (12%) at the central area. From these results, it is expected that the legal authority will adopt protection measures for the city public areas, reducing thus the contamination risk by Toxocara sp. eggs.

Muscular strength decrease in Rattus norvegicus experimentally infected by Toxocara canis

The muscular strength of experimental infected Rattus norvegicus with 3rd. stage Toxocara canis larvae was investigated. Fifty Wistar rats, divided in three groups (G1 - 20 rats infected by 300 eggs of T. canis; G2 - 20 rats infected by 2,000 eggs of T. canis and G3 - 10 rats without infection) had been used. Ten and 30 days after infection the muscular strength in the fore-feet of the rats was checked; at the same time, the body weight was determined. No significative differences in the body weight were noted among the infected and control rats in both occasions. Otherwise, an impairment on the muscular strength was observed in rats infected with T. canis 30 days after inoculation.

Behavioral changes in Rattus norvegicus experimentally infected by Toxocara canis larvae

Toxocara canis is a common canine nematode parasite and one of its possible transmission mechanisms is the predation of infected rodents by canids. Fifty Rattus norvegicus were used to study behavioral alterations in rodents infected by T. canis larvae. The rats were divided into three groups: G1, 20 rats infected with 300 T. canis eggs; G2, 20 rats infected with 2,000 T. canis eggs; and G3, 10 non-infected rats. Thirty and 60 days post-infection, rats from all the groups were submitted to an open-field apparatus for five min and subsequently, to an elevated plus-maze apparatus, again for five min. The data obtained indicated improvement in mobility (total locomotion time and rearing frequency) and exploratory behavior in infected rats, principally in G2, which provides some support for the hypothesis that behavioral alterations in rodents infected by Toxocara canis larvae enhance the transmission rate of this ascarid to dogs.

BEHAVIORAL AND MEMORY CHANGES IN Mus musculus COINFECTED BY Toxocara canis AND Toxoplasma gondii

Several researchers have stated that parasites can alter the behavior of their hosts, in order to increase the transmission rate, principally when prey-predator relationships are a reliable way of infection transmission. The aim of this study was to verify the occurrence of changes in anxiety and short-term memory patterns in experimentally infected Mus musculus by Toxocara canis and/or Toxoplasma gondii. Forty male Mus musculus (Balb/c) eight-week-old were divided into four groups of 10 mice each. One group was infected with 300 eggs of Toxocara canis; a second group was submitted to infection with 10 cysts of Toxoplasma gondii; a third group was concomitantly infected with both parasites with the same inoculums and the last group was maintained without infection. The anxiety levels were evaluated using an elevated plus maze and an actometer; the short-term memory was determined by a two-way active avoidance equipment. The determination of anxiety levels were conducted 40 and 70 days after infection and the short-term memory was evaluated 140 days after infection. Mice chronically infected by Toxoplasma gondii showed impaired learning and short-term memory, but no significant differences were found in mice infected by Toxocara canis or concomitantly infected by Toxocara canis and Toxoplasma gondii when compared to non infected mice.

ELEVATED TRANS-MAMMARY TRANSMISSION OF Toxocara canis LARVAE IN BALB/c MICE

Toxocariasis is a widespread zoonosis and is considered an important worldwide public health problem. The aim of this study was to investigate the frequency of trans-mammary Toxocara canis infection in newborn BALB/c mice nursed by females experimentally infected with 1,200 eggs after delivery. After 50 days of age, the presence of larvae in different organs of the offspring was investigated. Trans-mammary infection was confirmed in 73.9% of the mice that had been nursed by infected females. These data show a high trans-mammary transmission of T. canis and confirm the significance of this transmission route in paratenic hosts.

EFFICACY OF NITAZOXANIDE AGAINST Toxocara canis: LARVAL RECOVERY AND HUMORAL IMMUNE RESPONSE IN EXPERIMENTALLY INFECTED MICE

SUMMARY The efficacy of nitazoxanide (NTZ) against toxocariasis was investigated in an experimental murine model and results were compared to those obtained using mebendazole. Sixty male BALB/c mice, aged six to eight weeks-old, were divided into groups of 10 each; fifty were orally infected with 300 larvaed eggs of T. canisand grouped as follows, G I: infected untreated mice; G II: infected mice treated with MBZ (15 mg/kg/day) 10 days postinfection (dpi); G III: infected mice treated with NTZ (20 mg/kg/day) 10 dpi; G IV: infected mice treated with MBZ 60 dpi; G V: infected mice treated with NTZ 60 dpi; GVI: control group comprising uninfected mice. Mice were bled via retro-orbital plexus on four occasions between 30 and 120 dpi. Sera were processed using the ELISA technique to detect IgG anti- Toxocaraantibodies. At 120 dpi, mice were sacrificed for larval recovery in the CNS, liver, lungs, kidneys, eyes and carcass. Results showed similar levels of anti- ToxocaraIgG antibodies among mice infected but not submitted to treatment and groups treated with MBZ or NTZ, 10 and 60 dpi. Larval recovery showed similar values in groups treated with NTZ and MBZ 10 dpi. MBZ showed better efficacy 60 dpi, with a 72.6% reduction in the parasite load compared with NTZ, which showed only 46.5% reduction. We conclude that administration of these anthelmintics did not modify the humoral response in experimental infection by T. canis. No parasitological cure was observed with either drug; however, a greater reduction in parasite load was achieved following treatment with MBZ.

Ocular lesions in gerbils (Meriones unguiculatus) infected with low larval burden of Toxocara canis: observations using indirect binocular ophthalmoscopy

To study the frequency of ocular lesions in 30 gerbils infected with 100 embryonated eggs of Toxocara canis, indirect binocular ophthalmoscopy was performed 3, 10, 17, 24, 31 and 38 days after infection. All the animals presented larvae in the tissues and 80% presented ocular lesions. Hemorrhagic foci in the choroid and retina were present in 92% of the animals with ocular lesions. Retinal exudative lesions, vitreous lesions, vasculitis and retinal detachment were less frequent. Mobile larvae or larval tracks were observed in four (13.3%) animals. Histological examination confirmed the ophthalmoscopic observations, showing that the lesions were focal and sparse. In one animal, there was a larva in the retina, without inflammatory reaction around it. The results demonstrated that gerbils presented frequent ocular lesions after infection with Toxocara canis, even when infected with a small number of embryonated eggs. The lesions observed were focal, consisting mainly of hemorrhages with signs of reabsorption or inflammation in different segments of eye, and differing from the granulomatous lesions described in ocular larva migrans in humans.