Sarcocystis species are apicomplexan protozoa infecting a wide range of domestic and wild animals, including cattle, in which several species are of zoonotic relevance. This study reports, for the first time, the detection and molecular identification of pathogenic and zoonotic Sarcocystis hominis in slaughtered cattle from Central Chile. A total of 200 muscle samples (100 = myocardium, 100 = diaphragm) were examined by macroscopic inspection and tissue homogenization. Selected samples were additionally analyzed by histology, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and multiplex real-time PCR targeting the 18S rDNA. No macroscopic sarcocysts were observed, nonetheless microscopic sarcocysts were detected in 56% of assessed samples, with higher infection rates in the heart (91%) than in the diaphragm (21%). SEM and TEM analyses revealed thin-walled sarcocysts with finger-like protrusions in the diaphragm, as well as flattened hair-like projections in the myocardium. Molecular analysis identified Sarcocystis cruzi in all positive samples and detected additional DNA of Sarcocystis bovifelis/Sarcocystis rommeli and for the first time the zoonotic species S. hominis. These findings confirm the coexistence of canine-, feline-, and human-transmitted Sarcocystis species in Chilean cattle and highlight potential public health implications associated with consumption of raw or undercooked S. hominis-carrying beef meat. This constitutes the first molecular evidence of S. hominis in Chile, emphasizing the need for further surveillance and control measures in the meat production chain. These novel data on human S. hominis infections in Chile confirm the importance of initiating investigations on human sarcocystosis as this enteric parasitic disease is still sparsely considered by local public health authorities.

Fasciola hepatica is a parasitic trematode that causes fasciolosis in sheep, provoking a decrease in their reproductive capacity, weight gain, meat and milk production, and wool quality. In the pathogenesis of F. hepatica, the penetration and migration of parasitic stages through the liver provoke intense inflammatory immune responses and tissue damage. The aim of this study was to investigate the cytotoxic effects of Fascila hepatica-induced ovine NETs in exposed hepatocytes in vitro, and to analyze whether F. hepatica antigens (FhAg) trigger the release of ovine NETs under hypoxic conditions as well as the roles of matrix metalloproteinase-9 (MMP-9) and CD11b in this cellular process in vitro. Here, isolated ovine PMNs were co-cultured with FhAg under hypoxia (5% O₂) and NETs were visualized via immunofluorescence analyses, confirming their classical characteristics. The quantification of NETs in response to FhAg in hypoxic conditions significantly enhanced the formation of anchored and cell-free NETs (p < 0.01), and NADPH oxidase (NOX) inhibitor diphenylene iodonium (DPI) significantly reduced their production (p < 0.05). Furthermore, the cytotoxic effect of NETs on hepatic cells was determined by using a live/dead-staining with Sytox Orange, thereby demonstrating that FhAg-induced NETs are cytotoxic for hepatic cells (p = 0.001). We additionally analyzed PMN supernatants to determine the enzymatic activity of MMP-9, observing that FhAg exposure enhances MMP-9 release in ovine PMNs (p < 0.05) but not in bovine PMNs. Interestingly, by using flow cytometric analysis, we determined that the exposure of PMNs to FhAg does not increase the CD11b surface expression of ovine PMNs. This could be an effect of the activation of other surface receptors or transcription factors involved in F. hepatica-induced NETosis. Consequently, we hypothesize that F. hepatica-induced NETs play a role in the pathogenesis of fasciolosis, contributing to liver tissue damage if released in an uncontrolled manner.