The in vitro and in vivo anti-virulent effect of organic acid mixtures against Eimeria tenella and Eimeria bovis

Eimeria tenella and Eimeria bovis are complex parasites responsible for the condition of coccidiosis, that invade the animal gastrointestinal intestinal mucosa causing severe diarrhoea, loss of appetite or abortions, with devastating impacts on the farming industry. The negative impacts of these parasitic infections are enhanced by their role in promoting the colonisation of the gut by common foodborne pathogens. The aim of this study was to test the anti-Eimeria efficacy of maltodextrin, sodium chloride, citric acid, sodium citrate, silica, malic acid, citrus extract, and olive extract individually, in vitro and in combination, in vivo. Firstly, in vitro infection models demonstrated that antimicrobials reduced (p < 0.05), both singly and in combination (AG), the ability of E. tenella and E. bovis to infect MDBK and CLEC-213 epithelial cells, and the virulence reduction was similar to that of the anti-coccidial drug Robenidine. Secondly, using an in vivo broiler infection model, we demonstrated that AG reduced (p = 0.001) E. tenella levels in the caeca and excreted faeces, reduced inflammatory oxidative stress, improved the immune response through reduced ROS, increased Mn-SOD and SCFA levels. Levels of IgA and IgM were significantly increased in caecal tissues of broilers that received 0.5% AG and were associated with improved (p < 0.0001) tissue lesion scores. A prophylactic approach increased the anti-parasitic effect in vivo, and results indicated that administration from day 0, 5 and 10 post-hatch reduced tissue lesion scores (p < 0.0001) and parasite excretion levels (p = 0.002). Conclusively, our in vitro and in vivo results demonstrate that the natural antimicrobial mixture (AG) reduced parasitic infections through mechanisms that reduced pathogen virulence and attenuated host inflammatory events.

Eimeria bovis Macromeront Formation Induces Glycolytic Responses and Mitochondrial Changes in Primary Host Endothelial Cells

Eimeria bovis is an intracellular apicomplexan parasite that causes considerable economic losses in the cattle industry worldwide. During the first merogony, E. bovis forms large macromeronts with &gt;140,000 merozoites I in host endothelial cells. Because this is a high-energy demanding process, E. bovis exploits the host cellular metabolism to fulfill its metabolic requirements. We here analyzed the carbohydrate-related energetic metabolism of E. bovis–infected primary bovine umbilical vein endothelial cells during first merogony and showed that during the infection, E. bovis–infected culture presented considerable changes in metabolic signatures, glycolytic, and mitochondrial responses. Thus, an increase in both oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were found in E. bovis–infected host cells indicating a shift from quiescent to energetic cell status. Enhanced levels of glucose and pyruvate consumption in addition to increased lactate production, suggesting an important role of glycolysis in E. bovis–infected culture from 12 days p.i. onward. This was also tested by glycolytic inhibitors (2-DG) treatment, which reduced the macromeront development and diminished merozoite I production. As an interesting finding, we observed that 2-DG treatment boosted sporozoite egress. Referring to mitochondrial activities, intracellular ROS production was increased toward the end of merogony, and mitochondrial potential was enhanced from 12 d p. i. onward in E. bovis–infected culture. Besides, morphological alterations of membrane potential signals also indicated mitochondrial dysfunction in macromeront-carrying host endothelial culture.

The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation

Background: Bovine polymorphonuclear neutrophils (PMN) constitutively express the Toll-like receptors (TLRs) TLR2 and TLR4 and have been shown to generate Neutrophil extracellular traps (NETs) upon exposure to Eimeria bovis. The present work investigated the role of TLR2 and TLR4 in the recognition and uptake of E. bovis sporozoites, IL-8 production and neutrophil extracellular trap (NET) formation. Methods: TLR expression was performed by flow cytometric analysis on PMN exposed to live carboxyfluorescein succinimidyl ester (CFSE)-stained sporozoites. Supernatants of PMN exposed to different E. bovis sporozoite preparations and antigens in the absence or presence of TLR antibodies were assessed for IL-8 secretion. Cells were exposed to sporozoite preparations and assessed for the activation of transcription factor NF-κB using a luciferase reporter assay. Immunofluorescence analysis was done to investigate TLR2 and TLR4 surface expression and NET formation on bovine PMN exposed to vital sporozoites. Results: we observed significantly increased TLR2 and TLR4 expression with a mean increase in expression that was greater for TLR2 than TLR4. This upregulation neither inhibited nor promoted sporozoite phagocytosis by bovine PMN. Live sporozoites together with anti-TLR2 mAb resulted in a significant enhancement of IL-8 production. NF-κB activation was more strongly induced in TLR2-HEK cells than in TLR4/MD2-HEK cells exposed to heat-killed sporozoites and antigens. Immunofluorescence analysis showed TLR-positive signals on the surface of PMN and concomitant NET formation. Conclusions: This is the first report on E. bovis-induced concomitant TLR2 and TLR4 expression during bovine PMN-derived NETosis.

Eimeria bovis infections induce G1 cell cycle arrest and a senescence-like phenotype in endothelial host cells

Apicomplexan parasites are well-known to modulate their host cells at diverse functional levels. As such, apicomplexan-induced alteration of host cellular cell cycle was described and appeared dependent on both, parasite species and host cell type. As a striking evidence of species-specific reactions, we here show that Eimeria bovis drives primary bovine umbilical vein endothelial cells (BUVECs) into a senescence-like phenotype during merogony I. In line with senescence characteristics, E. bovis induces a phenotypic change in host cell nuclei being characterized by nucleolar fusion and heterochromatin-enriched peripheries. By fibrillarin staining we confirm nucleoli sizes to be increased and their number per nucleus to be reduced in E. bovis-infected BUVECs. Additionally, nuclei of E. bovis-infected BUVECs showed enhanced signals for HH3K9me2 as heterochromatin marker thereby indicating an infection-induced change in heterochromatin transition. Furthermore, E. bovis-infected BUVECs show an enhanced β-galactosidase activity, which is a well-known marker of senescence. Referring to cell cycle progression, protein abundance profiles in E. bovis-infected endothelial cells revealed an up-regulation of cyclin E1 thereby indicating a cell cycle arrest at G1/S transition, signifying a senescence key feature. Similarly, abundance of G2 phase-specific cyclin B1 was found to be downregulated at the late phase of macromeront formation. Overall, these data indicate that the slow proliferative intracellular parasite E. bovis drives its host endothelial cells in a senescence-like status. So far, it remains to be elucidated whether this phenomenon indeed reflects an intentionally induced mechanism to profit from host cell-derived energy and metabolites present in a non-dividing cellular status.