Larvicidal activity of coumarin derivatives on Toxocara canis larvae, cytotoxicity analysis, and in silico bioavailability studies.

Human toxocariasis is a neglected anthropozoonosis with global distribution. Treatment is based on the administration of anthelmintics; however, their effectiveness at the tissue level is low to moderate, necessitating the discovery of new drug candidates. Several groups of synthetic compounds, including coumarin derivatives, have demonstrated bioactivity against fungi, bacteria, and even parasites, such as Dactylogyrus intermedius, Leishmania major, and Plasmodium falciparum. The aim of this study was to evaluate the effect of ten coumarin-derived compounds against Toxocara canis larvae using in vitro, cytotoxicity, and in silico tests for selecting new drug candidates for preclinical tests aimed at evaluating the treatment of visceral toxocariasis. The compounds were tested in vitro in duplicate at a concentration of 1 mg/mL, and compounds with larvicidal activity were serially diluted to obtain concentrations of 0.5 mg/mL; 0.25 mg/mL; 0.125 mg/mL; and 0.05 mg/mL. The tests were performed in a microculture plate containing 100 T. canis larvae in RPMI-1640 medium. One compound (COU 9) was selected for cytotoxicity analysis using J774.A1 murine macrophages and it was found to be non-cytotoxic at any concentration tested. The in silico analysis was performed using computational models; the compound presented adequate results of oral bioavailability. To confirm the non-viability of the larvae, the contents of the microplate wells of COU 9 were inoculated intraperitoneally (IP) into female Swiss mice at 7-8 weeks of age. This confirmed the larvicidal activity of this compound. These results show that COU 9 exhibited larvicidal activity against T. canis larvae, which, after exposure to the compound, were non-viable, and that COU 9 inhibited infection in a murine model. In addition, COU 9 did not exhibit cytotoxicity and presented adequate bioavailability in silico, similar to albendazole, an anthelmintic, which is the first choice for treatment of human toxocariasis, supporting the potential for future investigations and preclinical tests on COU 9.

Immunomodulation in the intestinal mucosa of mice supplemented with Lactobacillus rhamnosus (ATCC 7469) and infected with Toxocara canis.

Toxocariasis is an anthropozoonosis caused by the helminth Toxocara canis that shows different clinical manifestations as visceral, ocular, or neurological toxocariasis forms. Probiotics have been studied as alternatives to prevent and treat this parasitosis. Lactobacillus rhamnosus is a prospect that presents immunomodulatory activity that acts to strengthen the intestinal barrier. In this context, the main objective of this study was to evaluate the protective capacity and immunomodulatory action of the probiotic Lactobacillus rhamnosus at the level of the intestinal mucosa in different stages of T. canis infection (acute and chronic). Mice were supplemented by oral gavage with 1 × 107 UFC/mL L. rhamnosus for 15 days before inoculation with 100 embryonated eggs of T. canis. Euthanasia of mice was conducted at three different time points: 2, 15 and 30 days post-inoculation (PI). The brain, lungs and liver were collected to evaluate the intensity of infection. The small intestines were removed, and mucosal cells of the duodenum were collected to perform gene analysis of IFN-γ, IL-10, IL-4 and IL-13 by real-time polymerase chain reaction (qPCR). Jejunum and ileum segments were analysed by histological techniques. A reduction of 51% in infection intensity was observed in the tissue of supplemented animals evaluated 2 days PI; however, analysis of groups 15 and 30 days PI did not show a protective effect. The intestinal mucosa of supplemented animals presented an inflammatory process that initiated at 2 days PI, persisted at 15 days PI and had regressed at 30 days PI. IL-13 transcription was increased in the probiotic group 2 days after supplementation ended; however, the same increase was not observed in the group that was supplemented and infected. Toxocara canis modulated the local immune system, with suppression of IFN-γ at 2 days PI and increased levels of IL-4 and IL-10 at 15 days PI. These results indicate that, under the studied conditions, the protective effect of Lactobacillus rhamnosus against infection caused by T. canis is not related to IL-4, IL-10 or IFN-γ but could be influenced by IL-13 action at 2 days PI. The probiotic stimulated immune cell recruitment to the intestinal mucosa, which can be involved in the diminished capacity of larval penetration in the mucosa, resulting in the reduced infection intensity observed during acute infection.

Protective effect of the probiotic Lactobacillus acidophilus ATCC 4356 in BALB/c mice infected with Toxocara canis.

Human toxocariasis consists of chronic tissue parasitosis that is difficult to treat and control. This study aimed to evaluate the action of the probiotic Lactobacillus acidophilus ATCC 4356 on larvae of Toxocara canis and the effect of IFN-γ cytokine on parasite-host in vivo (1.109 CFU) and in vitro (1.106, 1.107, 1.108, 1.109 CFU) interactions. Four groups of six BALB/c mice were formed: G1 - L. acidophilus supplementation and T. canis infection; G2 - T. canis infection; G3 - L. acidophilus supplementation; and G4 - PBS administration. Mice were intragastrically suplemented with probiotics for 15 days before inoculation and 48 h after inoculation with 100 T. canis eggs. The inoculation of T. canis was also perfomed intragastrically. The recovery of larvae took place through digestion of liver and lung tissues; the evaluation of IFN-γ gene transcription in leukocytes was performed by qPCR. The in vitro test consisted of incubating the probiotic with T. canis larvae. The supplementation of probiotics produced a reduction of 57.7% (p = 0.025) in the intensity of infection of T. canis larvae in mice, whereas in the in vitro test, there was no larvicidal effect. In addition, a decrease in the IFN-γ gene transcription was observed in both, T. canis-infected and uninfected mice, regardless of whether or not they received supplementation. The probiotic L. acidophilus ATCC 4356 reduced T. canis infection intensity in mice, however, the probiotic did not have a direct effect on larvae, demonstrating the need of interaction with the host for the beneficial effect of the probiotic to occur. Yet, the proinflammatory cytokine IFN-γ did not apparently contributed to the observed beneficial effect of probiotics.

Saccharomyces boulardii reduces the mean intensity of infection in mice caused by the consumption of liver contaminated by Toxocara canis.

Probiotics have shown promising results as a potential method to control toxocariasis in mice inoculated with embryonated eggs of Toxocara canis. This study aimed to evaluate the protective effect of Saccharomyces boulardii in mice fed in natura chicken livers infected with T. canis. Twenty 15-day-old male Sussex chickens were inoculated with 300 T. canis embryonated eggs via intragastric catheter (GI). After 72 h of infection, each liver was collected and individually offered to a group of 20 mice. Mice that received supplemented ration with S. boulardii (1.107 colony forming units) and consumed in natura chicken liver showed reduction in infection intensity of 67.1%. This study demonstrated that administration of S. boulardii has potential as a probiotic to assist in controlling visceral toxocariasis caused by the consumption of viscera from paratenic hosts containing infective parasite larvae.

Lactobacillus rhamnosus reduces parasite load on Toxocara canis experimental infection in mice, but has no effect on the parasite in vitro.

Human toxocariasis is a neglected global parasitic zoonosis. The efficacy of drug treatment for this disease has been hindered by the biological complexity of the main etiological agent, the nematode Toxocara canis. Experimental studies have shown the potential of probiotics to promote a reduction in the parasite load of T. canis larvae. This study aimed to evaluate the effect of probiotic Lactobacillus rhamnosus ATCC 7469 on the parasite load of BALB/c mice with acute toxocariasis and evaluate the direct effect of this probiotic on T. canis larvae in vitro. In vivo administration of probiotics reduced the parasite load of T. canis larvae by 53.3% (p = 0.0018) during the early stage of infection in mice. However, when analyzed in vitro, it was observed that the probiotic did not present a deleterious effect on the larvae, as approximately 90% of these remained viable. These results demonstrate the potential of the probiotic L. rhamnosus in the reduction of T. canis larvae in BALB/c mice and suggest it could be used as an alternative means for the controlling of visceral toxocariasis. However, further studies are required to elucidate the mechanisms of action promoted by this probiotic.

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.

Elevated trans-mammary transmission of Toxocara canis larvae in balb/c mice / Elevada transmissão transmamária de larvas de Toxocara canis em camundongos BALB/c

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.

A toxocaríase é zoonose amplamente difundida e considerada importante problema de saúde pública. O objetivo deste estudo foi avaliar a frequência da transmissão transmamária de Toxocara canis em camundongos BALB/c neonatos amamentados por fêmeas experimentalmente infectadas com 1.200 ovos logo após o parto. Após 50 dias de idade, foi avaliada a presença de larvas em diferentes órgãos dos neonatos. A infecção por via transmamária foi confirmada em 73,9% dos camundongos amamentados por fêmeas infectadas. Estes dados demonstram elevada transmissão transmamária de T. canis e confirmam a importância desta via de transmissão em hospedeiros paratênicos.

Transmammary infection in BALB/c mice with chronic toxocariasis.

Human toxocariasis is a neglected public health problem. Infection of humans generally results from the accidental ingestion of embryonated Toxocara canis eggs, but it is important to broaden knowledge about other forms of transmission. This study aimed to demonstrate the prevalence of transmammary transmission in mice with chronic toxocariasis. BALB/c mice in groups 1 (G1) and 3 (G3) were inoculated with 1200 T. canis eggs 60days before mating, whereas those of group 2 (G2) were not infected. After delivery, the G1 neonates were transferred to G2 females to be nursed, and vice versa. Thus, the mice generated by G2 females and breastfed by G1 females could be infected only during lactation. In the G3 group, offspring were not exchanged. The search for T. canis larvae in the bodies of the lactating females and their offspring was performed after weaning and at 60days old, respectively. The frequency of transmammary infection in the mice generated by G2 uninfected females and breastfed by G1 infected females was 19.8%, which was similar to that observed (19.6%) in the mice bred and fed by G3 females. The frequency of infection in the mice generated by G1 females and breastfed by G2 females was only 4.2%, which was lower than that of G1 (p=0.0064) and G3 (p=0.0062) groups. Transmammary infection by mice with chronic toxocariasis was found to be more prevalent than congenital infection.

Protective effect of the probiotic Saccharomyces boulardii in Toxocara canis infection is not due to direct action on the larvae.

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.

Evaluation of the initial and chronic phases of toxocariasis after consumption of liver treated by freezing or cooling.

Human toxocariasis is a neglected parasitic zoonosis of worldwide distribution. The consumption of raw or undercooked meat and offal from paratenic hosts of the Toxocara canis nematode can cause infection in humans, but there have been a lack of studies examining specific prophylactic measures to combat this mode of transmission. The aim of this study was to evaluate the establishment of infection by T. canis larvae at the initial and chronic phases of visceral toxocariasis after the consumption of mouse liver subjected to cold treatment. This study was divided into two stages using groups (G) of five donor mice inoculated with 2,000 eggs of T. canis. Two days post-inoculation, the livers of donor mice in G1 and G2 were kept at -20 °C and between 0 and 4 °C, respectively, for 10 days. In the first stage of the study, the livers of mice from G1, G2, and G3 (control) were subjected to a tissue digestion technique and found to be positive for infection. In the second stage, which evaluated infection in mice that had consumed livers from donor mice, receiver mice of G4 and G7 were fed with livers of donor mice from G1 (freezing), receiver mice of G5 and G8 were fed with livers of donor mice from G2 (cooling), and receiver mice of G6 and G9 with livers from G3 (control). Then, the tissue digestion technique was performed for recovering larvae from organs and carcasses of mice, at 2 days (G4, G5, and G6) and 60 days after liver consumption (G7, G8, and G9). It was observed that freezing inhibited the viability of 100 % of the larvae, while cooling promoted 87.7 and 95.7 % reductions in the intensity of infection at 2 and 60 days after liver consumption, respectively. Under the studied conditions, cold treatment shows great potential to help control this parasitosis, both in the initial and chronic phases of toxocariasis.

Saccharomyces boulardii reduces infection intensity of mice with toxocariasis.

Several studies have shown the benefit of the use of probiotics in the prevention and treatment of diseases; however, few of them have investigated the effect of probiotics on parasitosis. In this study, the effect of Saccharomyces boulardii on the intensity of infection of mice with toxocariasis was evaluated. The animals were fed with a diet supplemented with S. boulardii for 15 days before inoculation with Toxocara canis eggs and for 2 or 60 days post-inoculation. S. boulardii promoted a reduction of approximately 36% in the average number of recovered T. canis larvae, suggesting that it can be used as an alternative to help control toxocariasis.