Cross-Reactions between Toxocara canis and Ascaris suum in the diagnosis of visceral larva migrans by western blotting technique

Visceral larva migrans (VLM) is a clinical syndrome caused by infection of man by Toxocara spp, the common roundworm of dogs and cats. Tissue migration of larval stages causes illness specially in children. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. After the introduction of the enzyme-linked immunosorbent assay (ELISA) using the larval excretory-secretory antigen of T. canis (TES), the diagnosis specificity was greatly improved although cross-reactivity with other helminths are still being reported. In Brazil, diagnosis is routinely made after absorption of serum samples with Ascaris suum antigens, a nematode antigenicaly related with Ascaris lumbricoides which is a common intestinal nematode of children. In order to identify T. canis antigens that cross react to A. suum antigens we analyzed TES antigen by SDS-PAGE and Western blotting techniques. When we used serum samples from patients suspected of VLM and positive result by ELISA as well as a reference serum sample numerous bands were seen (molecular weight of 210-200 kDa, 116-97 kDa, 55-50 kDa and 35-29 kDa). Among these there is at least one band with molecular weight around 55-66 kDa that seem to be responsible for the cross-reactivity between T. canis e A. suum once it disappears when previous absorption of serum samples with A. suum antigens is performed

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.

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.

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.

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.

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.

IgG Antibody responses in mice coinfected with Toxocara canis and other helminths or protozoan parasites

The immune response expressed by IgG antibodies in BALB/c mice experimentally infected with Toxocara canis, was studied with the aim of verifying the possible in vivo cross-reactivity between antigens of T. canis and other parasites (Ascaris suum, Taenia crassiceps, Schistosoma mansoni, Strongyloides venezuelensis and Toxoplasma gondii). Experiments included three groups of mice: one infected only by T. canis, another with one of the other species of parasites and a third concomitantly infected with T. canis and the other species in question. Animals were bled by orbital plexus at 23, 38 and 70 days post infection (p.i.). Sera were analyzed for anti-Toxocara antibodies by ELISA and Immunoblotting, using excretion-secretion antigens (ES), obtained from culture of third-stage larvae of T. canis. For all experiments a control group comprised by ten non-infected mice was used. Only in the case of A. suum infection, in these experimental conditions, the occurrence of cross-reactivity with T. canis was observed. However, in the case of co-infection of T. canis - S. mansoni, T. canis - S. venezuelensis and T. canis - T. crassiceps the production of anti-Toxocara antibodies was found at levels significantly lower than those found in mice infected with T. canis only. Co-infection with S. mansoni or S. venezuelensis showed lower mortality rates compared to what occurred in the animals with single infections. Results obtained in mice infected with T. canis and T. gondii showed significant differences between the mean levels of the optical densities of animals infected with T. canis and concomitantly infected with the protozoan only in the 23rd day p.i.

Behavioral changes in Rattus norvegicus coinfected by Toxocara canis and Toxoplasma gondii

Using an elevated plus maze apparatus and an activity cage, behavioral changes in Rattus norvegicus concomitantly infected by Toxocara canis and Toxoplasma gondii were studied, during a period of 120 days. Rats infected by Toxocara canis or Toxoplasma gondii showed significant behavioral changes; however, in the group coinfected by both parasites a behavioral pattern similar to that found in the group not infected was observed thirty days after infection, suggesting the occurrence of modulation in the behavioral response.

PROTECTIVE EFFECT OF THE PROBIOTIC Saccharomyces boulardii IN Toxocara canis INFECTION IS NOT DUE TO DIRECT ACTION ON THE LARVAE

SUMMARY In a previous study our group found that the probiotic Saccharomyces boulardii was capable of reducing the intensity of infection in mice with toxocariasis. In order to assess whether the mechanism involved would be a direct action of the probiotic on Toxocara canis larvae, this study was designed. Both probiotics were singly cultivated in plates containing RPMI 1640 medium and T. canis larvae. S. boulardii and B. cereus var. toyoi cultures presented 97.6% and 95.7% of larvae with positive motility, respectively, and absence of color by the dye trypan blue, not representing significant difference to the control group (p > 0.05). We conclude that none of the probiotics showed in vitro effects on T. canis larvae and that the interaction with the intestinal mucosa is necessary for the development of the protective effect of S. boulardii.

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.