Visceral leishmaniasis is a neglected vector-borne tropical disease caused by Leishmania donovani and Leishmania infantum that is endemic not only in East African countries, but also in Asia, regions of South America and the Mediterranean Basin. For the pharmacological control of this disease, there is a limited number of old and, in general, poorly adherent drugs, with a multitude of adverse effects and low oral bioavailability, which favor the emergence of resistant pathogens. Pentavalent antimonials are the first-line drugs, but due to their misuse, resistant Leishmania strains have emerged worldwide. Although these drugs have saved many lives, it is recommended to reduce their use as much as possible and replace them with novel and more friendly drugs. From a commercial collection of anti-infective drugs, we have recently identified nifuratel—a nitrofurantoin used against vaginal infections—as a promising repurposing drug against a mouse model of visceral leishmaniasis. In the present work, we have tested combinations of miltefosine—the only oral drug currently used against leishmaniasis—with nifuratel in different proportions, both in axenic amastigotes from bone marrow and in intracellular amastigotes from infected Balb/c mouse spleen macrophages, finding a potent synergy in both cases. In vivo evaluation of oral miltefosine/nifuratel combinations using a bioimaging platform has revealed the potential of these combinations for the treatment of this disease.

One of the major drawbacks of current treatments for neglected tropical diseases is the low safety of the drugs used and the emergence of resistance. Leishmaniasis is a group of neglected diseases caused by protozoa of the trypanosomatidae family that lacks preventive vaccines and whose pharmacological treatments are scarce and unsafe. Combination therapy is a strategy that could solve the above-mentioned problems, due to the participation of several mechanisms of action and the reduction in the amount of drug necessary to obtain the therapeutic effect. In addition, this approach also increases the odds of finding an effective drug following the repurposing strategy. From the previous screening of two collections of repositioning drugs, we found that pyrvinium pamoate had a potent leishmanicidal effect. For this reason, we decided to combine it separately with two clinically used leishmanicidal drugs, miltefosine and paromomycin. These combinations were tested in axenic amastigotes of Leishmania infantum obtained from bone marrow cells and in intramacrophagic amastigotes obtained from primary cultures of splenic cells, both cell types coming from experimentally infected mice. Some of the combinations showed synergistic behavior, especially in the case of the combination of pyrvinium pamoate with paromomycin, and exhibited low cytotoxicity and good tolerability on intestinal murine organoids, which reveal the potential of these combinations for the treatment of leishmaniasis.

New therapeutic molecules for farm animals are needed to address worldwide problems in the food industry, like the rise of resistance among ruminant parasites and pathogenic microbes. Since in vivo testing would involve an excessive number of animals, with consequent ethical and economic issues, the generation of sheep intestinal organoids represents a promising close-to-reality in vitro model for veterinary drug development; however, the characterization and application of such organoids remain limited. In this study, ovine intestinal organoids were generated from adult LGR5+ stem cells from the intestinal crypts of freshly slaughtered lambs, and developed in an in vitro culture system. Morphological analysis via brightfield microscopy and immunocytochemical staining revealed a pseudostratified epithelium with multiple cell types, and distinct apical–basal polarity, while RNA sequencing validated the preservation of the physiological characteristics of the original organ. The development and characterization of a robust and reproducible protocol for culturing sheep duodenum intestinal organoids in a high-throughput screening (HTS) compatible format demonstrated reliability in HTS applications, with Z’-factor tests indicating robust assay performance. Dose–response studies using pre-identified compounds showed comparable pharmacodynamic profiles between mouse and sheep organoids. These findings establish sheep intestinal organoids as an innovative tool for veterinary pharmacology and toxicology, offering a cost-effective and sustainable platform to address challenges such as drug resistance and improve livestock health.

The development of new alternative models is essential to overcome the limitations of traditional two-dimensional (2D) cell cultures and animal models. Three-dimensional (3D) models, such as organoids, better mimic the structural and functional complexity of mammalian organs, thereby reducing the ethical and economic issues related to animal experimentation. These systems provide more physiologically relevant environments, improving the accuracy of disease modeling and drug response prediction. In this context, we have developed mouse hepatic organoids from livers of adult BALB/c mice and characterized them by microscopy and transcriptional analysis. This model was applied to a robust and reproducible high-throughput screening (HTS) platform for testing cytotoxicity at the preclinical stage of drug discovery. In addition, mouse hepatic organoids were co-cultured with amastigotes of Leishmania donovani parasites to establish a model of host–parasite interaction, which was characterized by RNA-seq linked to differential expression analysis and cytokine production by the hepatic organoids. The findings provided in this work establish mouse hepatic organoids as an alternative model for drug discovery and pathogenesis studies.

Gastrointestinal nematode infections (GINs) in ruminants are a major constraint to efficient livestock production worldwide. Currently, only a limited number of anthelmintic drugs are available for the control of these infections, but their widespread use in preventive deworming campaigns and the incorrect administration of the drugs are responsible for the emergence of resistance. Therefore, new anthelmintic drugs are urgently needed. However, drug discovery methods for new anthelmintics based on GINs isolated from ruminants often have low throughput. In this study, a screening of five commercial collections of chemical compounds, including one collection of anti-infective drugs, three plant-based natural product collections, and one collection from the FDA-approved Chinese Pharmacopoeia, with a total of 2228 molecules, have been carried out in a high-throughput format. In the single slot screen, 32 compounds (1.44% success rate) achieved a >70% motility inhibition rate. Of these, 10 are known anthelmintic drugs, while the remaining 22 were tested against Haemonchus contortus and a resistant strain of Teladorsagia circumcincta. Four compounds (two flavonoids, chalcone and trans-chalcone), and two anti-infectives (octenidine and tolfenpyrad), showed anthelmintic activity with EC₅₀ values below 20 µM, and were further tested for their safety against HepG2 spheroids and mouse intestinal organoids. Trans-chalcone and chalcone emerged as promising candidates for future development, showing selective indexes > 5, while tolfenpyrad and octenidine require careful evaluation due to their toxicity profiles.

Ovine colostrum exosomes obtained from nutritionally programmed dairy ewes (F0) may present modifications in microRNAs, thus having consequences for the intestinal barrier function and immunity parameters of lambs (F1). To test this hypothesis, colostrum exosomes from two ewe groups [F0-MET (nutritionally programmed ewes being fed methionine during early life) and F0-CTRL (ewes not supplemented with methionine during early life)] were sequenced to compare differences in the miRNAome. In addition, these exosomes were added to an in vitro co-culture in a Transwell chamber system consisting of ovine duodenum intestinal organoids and macrophages to assess the expression of genes encoding tight junction proteins in organoids and immunity parameters in macrophages. Finally, the concentrations of cytokines (e.g., IL-12 and IL-6) were assessed by ELISA kits in the supernatants of the chamber containing macrophages. According to the miRNAome, the expression of two miRNAs (e.g., oar_miR_376c_3p and oar_miR_432) was reduced in the colostrum exosomes obtained from dairy ewes nutritionally programmed with dietary supplementation of methionine during early life (F0-MET ewes). These changes did not seem to modify the expression of intestinal barrier and immune response marker genes when these exosomes were added to a co-culture of ovine intestinal organoids and macrophages. However, the levels of IL-12 produced by macrophages were reduced (p < 0.05), which suggests the inhibition of inflammatory pathways. Further studies using ovine colostrum exosomes obtained from nutritionally programmed ewes will help to clarify their potential to improve the health of suckling lambs.