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.

In silico prospection of Lactobacillus acidophilus strains with potential probiotic activity.

Lactic acid bacteria (LAB) are fermentative microorganisms and perform different roles in biotechnological processes, mainly in the food and pharmaceutical industries. Among the LAB, Lactobacillus acidophilus is a species that deserves to be highlighted for being used both in prophylaxis and in the treatment of pathologies. Most of the metabolites produced by this species are linked to the inhibition of pathogens. In this study, we utilized a pangenomic and metabolic annotation analysis using Roary and BlastKOALA, ML-based probiotic activity prediction with iProbiotic and whole-genome similarity using ANI to identify strains of L. acidophilus with potential probiotic activity. According to the results in BlastKOALA and iProbiotics, L. acidophilus NCTC 13721 had the greatest potential among the 64 strains tested, both in terms of its ability to be a Lactobacillus spp. probiotic, when in the amount of genes involved in the metabolism of organic acids and quorum sensing. In addition, DSM 20079 proved to be promising for prospecting new probiotic Lactobacillus from BlastKOALA analyses, as they presented similar results in the number of genes involved in the production of lactic acid, acetic acid, hydrogen peroxide, except for quorum sensing where the NCTC 13721 strain had 14 more genes. L. acidophilus NCTC 13721 and L. acidophilus La-5 strains showed greater ability to be Lactobacillus spp. probiotic capacity, showing 84.8% and 51.9% capacity in the iProbiotics tool, respectively. When analyzed in ANI, none of the evaluated strains showed genomic similarity with NCTC 13721. In contrast, the DSM 20079 strain showed genomic similarity with all evaluated strains except NCTC 13721. Furthermore, eight strains with characteristics with approximately 100% genomic similarity to La-5 were listed: S20_1, LA-5, FSI4, APC2845, LA-G80-111, DS1_1A, LA1, and BCRC 14065. Therefore, according to the findings in iProbiotics and BlastKoala, among the 64 strains evaluated, NCTC 13721 is the most promising strain to be used for future in vitro studies.

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.

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.

Evaluation of the immunosuppressive effect of cyclophosphamide and dexamethasone in mice with visceral toxocariasis.

Visceral toxocariasis is a serious public health problem with a cosmopolitan distribution. Children are susceptible due to their immature immune system and high risks of infection. Nevertheless, the few completed studies about immunosuppression have had controversial results. To evaluate the effect of two immunosuppressive drugs on the larval burden of Toxocara canis, four groups of ten Swiss strain mice each were inoculated on day 0 with 1,200 embryonated T. canis eggs. Fifteen days before the experimental infection, group 1 (control) was treated via intraperitoneal injection (IP) with sterile distilled water and groups 2 and 3 were treated with dexamethasone (DEX) at 1 and 5 mg/kg/day, respectively. Additionally, group 4 was treated IP with cyclophosphamide (CY) at 50 mg/kg at two times per week for 2 weeks. Sixty days following infection, the mice were euthanised to recover the larvae by means of the tissue digestion technique. The levels of antibodies detected by indirect ELISA were not associated with the larval burden. Administration of CY (50 mg/kg) and DEX (5 mg/kg) resulted in an increase of the larval burden of 162.1% and 50.8%, respectively, in relation to the control group. These two treatments, especially CY (50 mg/kg), promoted immunosuppression and the establishment of a significant larval burden, supporting its further utilisation in studies related to immunosuppression in visceral toxocariasis.