Tamara Muñoz-Caro

Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato (s.l.), is a significant zoonotic disease affecting livestock and public health worldwide, particularly in endemic regions such as La Araucanía, Chile. This study evaluated the role of cattle in the transmission dynamics of E. granulosus sensu stricto (s.s.) by morphologically and molecular characterizing hydatid cysts (HC) collected from cattle, sheep, pigs, and goats. A total of 123 cysts were obtained from a local slaughterhouse, with cattle contributing the majority of samples (n = 94). Fertility was highest in sheep (76.2%) and low in cattle (3.2%), while cysts from pigs and goats were infertile. PCR amplification and sequencing of the cox1 gene confirmed the predominance of genotype G1 (98.1%), with two additional haplotypes (Eg^B and Eg^C) identified in cattle and sheep. Two cattle samples harbored genotype G3. Phylogenetic analyses grouped all sequences within the E. granulosus s.s. complex. The results corroborate the role of cattle as important sentinels for environmental surveillance of CE due to their exposure and traceability but highlight their lower competence in parasite transmission to definitive hosts compared with sheep. The genetic diversity observed aligns with previous findings in Chile, underscoring the epidemiological significance of E. granulosus s.s. and genotype G1 in the region.

Coccidiosis, caused by Eimeria species, remains a major challenge to the poultry industry. The parasite transmission relies on the environmental sporogony stage during which the parasite transforms from non-infectious to infectious oocysts. This host-independent metabolism is driven by the mannitol pathway, which operates in oocysts of Eimeria, Toxoplasma, and Cryptosporidium. Our previous in vitro study demonstrated that Piper betle L. essential oil (PBEO) exhibits anticoccidial activity through oocysticidal effects and sporulation inhibition (IC50 1.31% at 72 h). Here, we performed systematic in silico molecular docking to evaluate ten major PBEO phytochemicals against three key E. tenella mannitol pathway enzymes: mannitol-1-phosphate dehydrogenase (M1PDH), mannitol-1-phosphate phosphatase (M1Pase), and hexokinase. In hexokinase enzyme, 3-allyl-6-methoxyphenyl acetate showed the strongest binding affinity (-6.2 kcal/mol), stabilized by a conventional hydrogen bond with CYS161 and a π-sigma interaction with Leu58. For M1Pase, β-selinene and β-caryophyllene exhibited the highest affinities (-5.9 kcal/mol each) through hydrophobic alkyl networks. Next, the M1PDH receptor, β-selinene, achieved the most favourable binding (-7.4 kcal/mol), closely followed by the bicyclic sesquiterpene derivative (-7.1 kcal/mol). PBEO constituents potentially exert anti-sporulation effects by simultaneous inhibition of multiple mannitol pathway enzymes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping and analysis (map00051) confirms that these enzymes form a non-redundant metabolic unit essential for sporulation, with no bypass reactions annotated in the parasite genome. These findings support further bio-guided isolation and development of PBEO-derived compounds as anticoccidial agents targeting the environmental stage of coccidian parasites.

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.

Neutrophil extracellular traps (NETs) are a striking defense mechanism of innate immunity. Upon activation, neutrophils release web-like structures composed of DNA scaffolds coated with various proteins, which immobilize and kill pathogens (1,2). However, NET formation comes at a cost, and excessive NET release can contribute to endothelial damage, as well as to autoimmune, inflammatory, and numerous other diseases in which NETs have been implicated (3). While NETs are a conserved defense strategy across species, their underlying mechanisms vary (4). Despite significant advances, questions remain regarding the role of NETs in infections caused by protozoa and helminths. This editorial introduces a collection of articles that expand our knowledge of NETs induced by parasites. Topics include their pathological impact in amoebic liver abscesses induced by Entamoeba histolytica, the role of extracellular vesicles from Besnoitia besnoiti in NET formation and cytokine secretion, and the weak NET-inducing effect of the trematode Calicophoron daubneyi. A bibliometric review further highlights key research trends and future directions in the field.NETs in infection-associated tissue damage NETs can have both friend and foe effects on infections: they provide an important defense against invading pathogens, but excessive NET formation or impaired clearance can lead to tissue damage. In malaria, for example, a correlation has been established between disease severity and endothelial injury (5).The study by Jorge-Rosas et al. shed light on the cytotoxic effects of NETs in Entamoeba histolytica infections, an underexplored aspect of amebiasis. While NETs are well known for their antimicrobial functions, this study reveals their detrimental side, showing that they contribute to tissue damage during invasive amebiasis. Using in vitro and ex vivo models, authors show that E. histolytica-induced NETs are highly cytotoxic to colonic (HCT 116) and hepatic (Hep G2) cells via serine proteases and MPO, respectively. The study also identifies NET-like structures in hamster liver abscesses, linking them to necrotic areas and implicating them in disease pathology.These findings reinforce the idea that NETs can act as a double-edged sword in parasitic infectionstrapping pathogens but also promoting tissue injury. Targeting NET components may offer new therapeutic approaches to limit tissue damage in amebiasis, a major health issue in tropical regions.Extracellular vesicles (EVs) are membrane-bound structures involved in various biological processes, contributing to both physiological mechanismssuch as cell-cell communicationand pathological conditions, including cancer and autoimmune diseases (6). Recent evidence indicates that EVs are present within NETs and can modulate their release (7,8).In The study concluded that the field of NETs in parasitology has grown rapidly and is likely to keep expanding. It emphasized advances in understanding NET induction by parasites and their dual role in host defense and pathology. Future directions include therapeutic modulation of NETs to enhance antiparasitic responses while reducing host damage, targeting parasite evasion mechanisms such as DNase secretion, and conducting comparative studies across host species and parasite taxa.The collection of studies presented in this Research Topic underscores the complexity and dual nature of NETs in parasitic infections. By exploring both pathogenic and regulatory roles of NETs, from tissue damage in amebiasis to immune evasion by helminths, these contributions advance our understanding of how neutrophils shape the outcome of protozoan and helminth infections. Importantly, they reveal the influence of parasite species, extracellular vesicles, and host signaling pathways in modulating NET responses. We anticipate that these findings will not only inspire deeper investigations into parasite-induced NETs, but also support the development of targeted therapies that modulate NET activity to benefit the host. The integration of bibliometric insights further provides a roadmap for future research directions in this rapidly evolving field.

Introduction: Parasitic diseases pose a significant global public health challenge, affecting billions of people and causing substantial economic losses in livestock and poultry. In the fight against these infections, neutrophils play a crucial role, employing various strategies, including the release of neutrophil extracellular traps (NETs). Recent studies have made significant progress in understanding NETs triggered by protozoa and helminths. However, a comprehensive bibliometric analysis that compiles these findings and identifies research hotspots and trends in this field is still lacking.Methods: We utilized the Web of Science Core Collection and Scopus databases to retrieve original articles on NETs induced by protozoa and helminths. After screening, the data was transferred to the visualization tools VOSviewer, Biblioshiny, and CiteSpace for analysis.Results: Our study included 159 original articles published in 69 journals, involving 909 authors from 270 institutions across 41 countries. Germany and Brazil have made the most significant contributions to the research on NETs and parasites, accounting for 45 and 32 publications, and 1,495 and 1,342 citations, respectively. Carlos Hermosilla and Anja Taubert (Justus Liebig University Giessen, Germany), and Elvira Saraiva (Federal University of Rio de Janeiro, Brazil), are leaders in the field, both in terms of publication output and citations. Frontiers in Immunology has consistently and significantly impacted the field, and an article in the Proceedings of the National Academy of Sciences of the United States of America providing the first direct evidence of NETs release in response to a parasite is by far the most cited. Current research focuses on malaria, Toxoplasma gondii, Besnoitia besnoiti, nematodes, and the mechanisms of NETs production and their effects on parasites and host cells. Emerging trends include therapeutic targeting of NETs and comparative studies across different host and parasite species.Conclusion: This study offers a comprehensive overview and visual analysis of NETs and parasites, highlighting key areas for future research.

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.

Coccidiosis poses a significant threat to the poultry industry, with synthetic antibiotics and disinfectants being the primary tools for control. This study investigated the potential of Piper betle L essential oil (PBEO) as a natural alternative against Eimeria tenella, one of the most pathogenic Eimeria species affecting poultry. Our findings revealed that PBEO exhibits significant anticoccidial effects through two primary mechanisms: (i) oocysticidal activity by disintegrating oocyst walls and (ii) inhibition of the sporulation process. PBEO demonstrated oocysticidal activities ranging from 8.67 to 95.33% across concentrations from 0.04 to 40%. Notably, at 72 h post-incubation, a 0.04% PBEO concentration significantly reduced the number of sporulated oocysts (P ≤ 0.05) to 71.67%, showing effects comparable to those of formalin. PBEO reduced 50% of oocyst sporulation (IC₅₀) in the concentration of 1.31% at 72 h. Gas chromatography-mass spectrometry (GC-MS) identified the primary constituents of PBEO, including eugenol, beta-caryophyllene, and other key compounds, collectively constituting 96% of the oil. This research underscores the potential of PBEO as a natural anticoccidial agent and lays the groundwork for further studies aimed at identifying, isolating, and developing active compounds that may specifically target the sporogony process in coccidian parasites.

Parasitic infections are among the most important causes of morbidity and mortality in horses worldwide. In the Andean mountainous regions of Chile, horses are a highly valued specie, playing a key role as working animals also providing support in cattle transhumance grazing into high mountainous areas during dry season. The breeding and maintenance of horses in the mountainous region of La Araucanía in Chile is a key issue allowing to the subsistence economy of communities with a strong ethnic component represented by Mapuche-Pehuenche origin families. However, the health status of these animals is unknown. Therefore, the objective of this study was to determine endoparasitic infections in horses from this area and the main factors that may influence parasitism. Thus, a cross-sectional study performed in 126 working horses from Andean region was achieved by using different coproparasitologycal analyses, resulting in a 97,6% of prevalence of endoparasitism with the strongyle eggs showing the highest prevalence of infection in analysed horses (96%) followed by Oxyuris equi (27%), Parascaris equorum (17,5%) and protozoans including ciliates (11,1%), Blastocystis spp (5,6%), Giardia spp (2,4%) and Cryptosporidium spp (1,6%) highlighting zoonotic relevance of some of these species. Concerning risk factors, the high prevalence suggests that exposure was high regardless of age or sex. The higher proportion of infected horses were grazing compared to those stabled, likewise drinking non-potable water, being these the main risks factors linked to the high prevalence of horse infections in Andean communities here studied.

The presence of gastrointestinal parasites (PGI) was determined in meat sheep from farms in the foothills of the commune of San Clemente Chile through convenience sampling (n=100) between August and November 2022. Faecal samples were collected from the rectum. and were processed using the modified Dennis sedimentation technique for the determination of F. hepatica eggs and the Sheather flotation technique for the determination of nematode and coccidia eggs. In addition, the modified McMaster quantitative technique was performed on the samples. A higher prevalence of parasites from the Trichostrongylidae family (68%) was found, followed by F. hepatica (47%), Nematodirus sp (25%) and Eimeria sp (43%). In addition, the epidemiological variables age, sex and health status were evaluated. The age group was significant (p=0.003) for the age variable in Trichostrongylidae, and the type of health management affected the prevalence of Eimeria (p=0.020); Trichostrongylidae (p=0.001); Nematodirus sp (p=0.04) and F. hepatica (p=0.020). Moderate parasite loads were detected in all cases. Likewise, cases of biparasitism (26%) and multi-parasitism (32%) were detected.