Fasciola hepatica soluble antigens (FhAg) induce ovine PMN innate immune reactions and NET formation in vitro and in vivo.

Fasciola hepatica causes liver fluke disease, a worldwide neglected and re-emerging zoonotic disease, leading to hepatitis in humans and livestock. In the pathogenesis, flukes actively migrate through liver parenchyma provoking tissue damage. Here, parasites must confront leukocytes of the innate immune system in vivo. Polymorphonuclear neutrophils (PMN) are the most abundant granulocytes and first ones arriving at infection sites. PMN may display neutrophil extracellular traps (NETs), consisting of nuclear DNA, decorated with histones, enzymes, and antimicrobial peptides. We investigated for the first time whether F. hepatica soluble antigens (FhAg) can also trigger NETosis and innate immune reactions in exposed ovine PMN. Thus, isolated PMN were co-cultured with FhAg and NET formation was visualized by immunofluorescence and scanning electron microscopy analyses resulting in various phenotypes with spread NETs being the most detected in vitro. In line, NETs quantification via Picogreen®-fluorometric measurements revealed induction of anchored- and cell free NETs phenotypes. Live cell 3D-holotomographic microscopy revealed degranulation of stimulated PMN at 30 min exposure to FhAg. Functional PMN chemotaxis assays showed a significant increase of PMN migration (p = 0.010) and intracellular ROS production significantly increased throughout time (p = 0.028). Contrary, metabolic activities profiles of FhAg-exposed PMN did not significantly increase. Finally, in vivo histopathological analysis on F. hepatica-parasitized liver tissue sections of sheep showed multifocal infiltration of inflammatory cells within liver parenchyma, and further fluorescence microscopy analyses confirmed NETs formation in vivo. Overall, we hypothesized that NET-formation is a relevant host defence mechanism that might have a role in the pathogenesis of fasciolosis in vivo.

Mass Spectrometry Imaging of In Vitro Cryptosporidium parvum-Infected Cells and Host Tissue.

Cryptosporidium parvum is a zoonotic-relevant parasite belonging to the phylum Alveolata (subphylum Apicomplexa). One of the most zoonotic-relevant etiologies of cryptosporidiosis is the species C. parvum, infecting humans, cattle and wildlife. C. parvum-infected intestinal mucosa as well as host cells infected in vitro have not yet been the subject of extensive biochemical investigation. Efficient treatment options or vaccines against cryptosporidiosis are currently not available. Human cryptosporidiosis is currently known as a neglected poverty-related disease (PRD), being potentially fatal in young children or immunocompromised patients. In this study, we used a combination of atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) and liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) to determine and locate molecular biomarkers in in vitro C. parvum-infected host cells as well as parasitized neonatal calf intestines. Sections of C. parvum-infected and non-infected host cell pellets and infected intestines were examined to determine potential biomarkers. Human ileocecal adenocarcinoma cells (HCT-8) were used as a suitable in vitro host cell system. More than a thousand different molecular signals were found in both positive- and negative-ion mode, which were significantly increased in C. parvum-infected material. A database search in combination with HPLC-MS/MS experiments was employed for the structural verification of markers. Our results demonstrate some overlap between the identified markers and data obtained from earlier studies on other apicomplexan parasites. Statistically relevant biomarkers were imaged in cell layers of C. parvum-infected and non-infected host cells with 5 µm pixel size and in bovine intestinal tissue with 10 µm pixel size. This allowed us to substantiate their relevance once again. Taken together, the present approach delivers novel metabolic insights on neglected cryptosporidiosis affecting mainly children in developing countries.

Broad anti-pathogen potential of DEAD box RNA helicase eIF4A-targeting rocaglates.

Inhibition of eukaryotic initiation factor 4A has been proposed as a strategy to fight pathogens. Rocaglates exhibit the highest specificities among eIF4A inhibitors, but their anti-pathogenic potential has not been comprehensively assessed across eukaryotes. In silico analysis of the substitution patterns of six eIF4A1 aa residues critical to rocaglate binding, uncovered 35 variants. Molecular docking of eIF4A:RNA:rocaglate complexes, and in vitro thermal shift assays with select recombinantly expressed eIF4A variants, revealed that sensitivity correlated with low inferred binding energies and high melting temperature shifts. In vitro testing with silvestrol validated predicted resistance in Caenorhabditis elegans and Leishmania amazonensis and predicted sensitivity in Aedes sp., Schistosoma mansoni, Trypanosoma brucei, Plasmodium falciparum, and Toxoplasma gondii. Our analysis further revealed the possibility of targeting important insect, plant, animal, and human pathogens with rocaglates. Finally, our findings might help design novel synthetic rocaglate derivatives or alternative eIF4A inhibitors to fight pathogens.

Novel Insights Into Sterol Uptake and Intracellular Cholesterol Trafficking During Eimeria bovis Macromeront Formation.

Apicomplexan parasites are considered as defective in cholesterol synthesis. Consequently, they need to scavenge cholesterol from the host cell by either enhancing the uptake of extracellular cholesterol sources or by upregulating host cellular de-novo biosynthesis. Given that Eimeria bovis macromeront formation in bovine lymphatic endothelial host cells in vivo is a highly cholesterol-demanding process, we here examined host parasite interactions based on host cellular uptake of different low-density lipoprotein (LDL) types, i.e., of non-modified (LDL), oxidized (oxLDL), and acetylated LDL (acLDL). Furthermore, the expression of lipoprotein-oxidized receptor 1 (LOX-1), which mediates acLDL and oxLDL internalization, was monitored throughout first merogony, in vitro and ex vivo. Moreover, the effects of inhibitors blocking exogenous sterol uptake or intracellular transport were studied during E. bovis macromeront formation in vitro. Hence, E. bovis-infected primary bovine umbilical vein endothelial cells (BUVEC) were treated with inhibitors of sterol uptake (ezetimibe, poly-C, poly-I, sucrose) and of intracellular sterol transport and release from endosomes (progesterone, U18666A). As a read-out system, the size and number of macromeronts as well as merozoite I production were estimated. Overall, the internalization of all LDL modifications (LDL, oxLDL, acLDL) was observed in E. bovis-infected BUVEC but to different extents. Supplementation with oxLDL and acLDL at lower concentrations (5 and 10 µg/ml, respectively) resulted in a slight increase of both macromeront numbers and size; however, at higher concentrations (25-50 µg/ml), merozoite I production was diminished. LOX-1 expression was enhanced in E. bovis-infected BUVEC, especially toward the end of merogony. As an interesting finding, ezetimibe treatments led to a highly significant blockage of macromeront development and merozoite I production confirming the relevance of sterol uptake for intracellular parasite development. Less prominent effects were induced by non-specific inhibition of LDL internalization via sucrose, poly-I, and poly-C. In addition, blockage of cholesterol transport via progesterone and U18666A treatments resulted in significant inhibition of parasite development. Overall, current data underline the relevance of exogenous sterol uptake and intracellular cholesterol transport for adequate E. bovis macromeront development, unfolding new perspectives for novel drug targets against E. bovis.

A Preliminary Comparison on Faecal Microbiomes of Free-Ranging Large Baleen (Balaenoptera musculus, B. physalus, B. borealis) and Toothed (Physeter macrocephalus) Whales.

Large baleen and toothed whales play crucial ecological roles in oceans; nonetheless, very little is known about their intestinal microbiomes. Based on striking differences in natural history and thus in feeding behaviours, it can be expected that intestinal microbiomes of large baleen whales and toothed whales are different. To test this hypothesis, the phylogenetic composition of faecal microbiomes was investigated by a 16S rRNA gene amplicon sequence-based approach for Bacteria and Archaea. Faecal samples from free-ranging large whales collected off the Azores Archipelago (Portugal) were used, comprising 13 individual baleen whales (one sei, two blue and ten fin whales) and four sperm whales. The phylogenetic composition of the Bacteria faecal microbiomes of baleen and toothed whales showed no significant differences at the phylum level. However, significant differences were detected at the family and genus levels. Most abundant phyla were Firmicutes, Bacteroidetes, Proteobacteria, Tenericutes and Spirochaeta. Few highly abundant bacterial genera were identified as key taxa with a high contribution to differences among baleen and toothed whales microbiomes. Only few archaeal sequences were detected, primarily Methanomassiliicoccales representing potential methanogenic Archaea. This is the first study that directly compares the faecal bacterial and archaeal microbiomes of free-ranging baleen and toothed whales which represent the two parvorders of Cetacea which members are fully aquatic large mammals which were evolutionary split millions of years ago.

Atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging of Neospora caninum-infected cell monolayers.

Neospora caninum is an obligate intracellular protozoan parasite of the phylum Alveolata (subphylum Apicomplexa) which has not been studied extensively in a biochemical context. N. caninum is a primary cause of reproductive disorders causing mummification and abortion not only in cattle but also in other small ruminant species resulting in a substantial economic impact on the livestock industry. In canids, which are the final hosts of N. caninum, clinical disease includes neuromuscular symptoms, ataxia, and ascending paralysis. Fatal outcomes of neosporosis have also been reported depending on the host species, age and immune status, however, its zoonotic potential is still uncertain. Therefore, N. caninum should be thoroughly investigated. Matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry (MS) and MS imaging (MSI) were used, combined with high-performance liquid chromatography (HPLC) to investigate these intracellular parasites. The aim of this study was to identify molecular biomarkers for N. caninum tachyzoite-infected host cells and to further clarify their functions. By atmospheric-pressure scanning microprobe MALDI MS(I), sections of N. caninum-infected and non-infected host cell pellets were examined in order to determine potential markers. In vivo, N. caninum infects different types of nucleated cells, such as endothelial cells which represent a highly immunoreactive cell type. Therefore, primary bovine umbilical vein endothelial cells were here used as a suitable infection system. For comparison, the permanent MARC-145 cell line was used as an additional, simplified in vitro cell culture model. HPLC-tandem MS (HPLC-MS/MS) experiments combined with database search were employed for structural verification of markers. The statistically relevant biomarkers found by MS and identified by HPLC-MS/MS measurements were partly also found in infected monolayers. Marker signals were imaged in cell layers of N. caninum-infected and non-infected host cells at 5 µm lateral resolution.

The Cellular Innate Immune Response of the Invasive Pest Insect Drosophila suzukii against Pseudomonas entomophila Involves the Release of Extracellular Traps.

Drosophila suzukii is a neobiotic invasive pest that causes extensive damage to fruit crops worldwide. The biological control of this species has been unsuccessful thus far, in part because of its robust cellular innate immune system, including the activity of professional phagocytes known as hemocytes and plasmatocytes. The in vitro cultivation of primary hemocytes isolated from D. suzukii third-instar larvae is a valuable tool for the investigation of hemocyte-derived effector mechanisms against pathogens such as wasp parasitoid larvae, bacteria, fungi and viruses. Here, we describe the morphological characteristics of D. suzukii hemocytes and evaluate early innate immune responses, including extracellular traps released against the entomopathogen Pseudomonas entomophila and lipopolysaccharides. We show for the first time that D. suzukii plasmatocytes cast extracellular traps to combat P. entomophila, along with other cell-mediated reactions, such as phagocytosis and the formation of filopodia.

Thiosemicarbazone Copper Chelator BLT-1 Blocks Apicomplexan Parasite Replication by Selective Inhibition of Scavenger Receptor B Type 1 (SR-BI).

Coccidian parasites are obligate intracellular pathogens that affect humans and animals. Apicomplexans are defective in de novo synthesis of cholesterol, which is required for membrane biosynthesis and offspring formation. In consequence, cholesterol has to be scavenged from host cells. It is mainly taken up from extracellular sources via LDL particles; however, little is known on the role of HDL and its receptor SR-BI in this process. Here, we studied effects of the SR-BI-specific blocker BLT-1 on the development of different fast (Toxoplasma gondii, Neospora caninum, Besnoitia besnoiti) and slow (Eimeria bovis and Eimeria arloingi) replicating coccidian species. Overall, development of all these parasites was significantly inhibited by BLT-1 treatment indicating a common SR-BI-related key mechanism in the replication process. However, SR-BI gene transcription was not affected by T. gondii, N. caninum and B. besnoiti infections. Interestingly, BLT-1 treatment of infective stages reduced invasive capacities of all fast replicating parasites paralleled by a sustained increase in cytoplasmic Ca++ levels. Moreover, BLT1-mediated blockage of SR-BI led to enhanced host cell lipid droplet abundance and neutral lipid content, thereby confirming the importance of this receptor in general lipid metabolism. Finally, the current data suggest a conserved role of SR-BI for successful coccidian infections.

First Metabolic Insights into Ex Vivo Cryptosporidium parvum-Infected Bovine Small Intestinal Explants Studied under Physioxic Conditions.

The apicomplexan Cryptosporidium parvum causes thousands of human deaths yearly. Since bovines represent the most important reservoir of C. parvum, the analysis of infected bovine small intestinal (BSI) explants cultured under physioxia offers a realistic model to study C. parvum-host cell-microbiome interactions. Here, C. parvum-infected BSI explants and primary bovine small intestinal epithelial cells were analysed for parasite development and metabolic reactions. Metabolic conversion rates in supernatants of BSI explants were measured after infection, documenting an immediate parasite-driven metabolic interference. Given that oxygen concentrations affect cellular metabolism, measurements were performed at both 5% O2 (physiological intestinal conditions) and 21% O2 (commonly used, hyperoxic lab conditions). Overall, analyses of C. parvum-infected BSI explants revealed a downregulation of conversion rates of key metabolites-such as glucose, lactate, pyruvate, alanine, and aspartate-at 3 hpi, followed by a rapid increase in the same conversion rates at 6 hpi. Moreover, PCA revealed physioxia as a driving factor of metabolic responses in C. parvum-infected BSI explants. Overall, the ex vivo model described here may allow scientists to address pending questions as to how host cell-microbiome alliances influence intestinal epithelial integrity and support the development of protective intestinal immune reactions against C. parvum infections in a realistic scenario under physioxic conditions.

Ezetimibe blocks Toxoplasma gondii-, Neospora caninum- and Besnoitia besnoiti-tachyzoite infectivity and replication in primary bovine endothelial host cells.

Coccidia are obligate apicomplexan parasites that affect humans and animals. In fast replicating species, in vitro merogony takes only 24­48 h. In this context, successful parasite proliferation requires nutrients and other building blocks. Coccidian parasites are auxotrophic for cholesterol, so they need to obtain this molecule from host cells. In humans, ezetimibe has been applied successfully as hypolipidaemic compound, since it reduces intestinal cholesterol absorption via blockage of Niemann−Pick C-1 like-1 protein (NPC1L1), a transmembrane protein expressed in enterocytes. To date, few data are available on its potential anti-parasitic effects in primary host cells infected with apicomplexan parasites of human and veterinary importance, such as Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti. Current inhibition experiments show that ezetimibe effectively blocks T. gondii, B. besnoiti and N. caninum tachyzoite infectivity and replication in primary bovine endothelial host cells. Thus, 20 µm ezetimibe blocked parasite proliferation by 73.1−99.2%, via marked reduction of the number of tachyzoites per meront, confirmed by 3D-holotomographic analyses. The effects were parasitostatic since withdrawal of the compound led to parasite recovery with resumed proliferation. Ezetimibe-glucuronide, the in vivo most effective metabolite, failed to affect parasite proliferation in vitro, thereby suggesting that ezetimibe effects might be NPC1L1-independent.

P-Glycoprotein Inhibitors Differently Affect Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti Proliferation in Bovine Primary Endothelial Cells.

Apicomplexan parasites are obligatory intracellular protozoa. In the case of Toxoplasma gondii, Neospora caninum or Besnoitia besnoiti, to ensure proper tachyzoite production, they need nutrients and cell building blocks. However, apicomplexans are auxotrophic for cholesterol, which is required for membrane biosynthesis. P-glycoprotein (P-gp) is a transmembrane transporter involved in xenobiotic efflux. However, the physiological role of P-gp in cholesterol metabolism is unclear. Here, we analyzed its impact on parasite proliferation in T. gondii-, N. caninum- and B. besnoiti-infected primary endothelial cells by applying different generations of P-gp inhibitors. Host cell treatment with verapamil and valspodar significantly diminished tachyzoite production in all three parasite species, whereas tariquidar treatment affected proliferation only in B. besnoiti. 3D-holotomographic analyses illustrated impaired meront development driven by valspodar treatment being accompanied by swollen parasitophorous vacuoles in the case of T. gondii. Tachyzoite and host cell pre-treatment with valspodar affected infection rates in all parasites. Flow cytometric analyses revealed verapamil treatment to induce neutral lipid accumulation. The absence of a pronounced anti-parasitic impact of tariquidar, which represents here the most selective P-gp inhibitor, suggests that the observed effects of verapamil and valspodar are associated with mechanisms independent of P-gp. Out of the three species tested here, this compound affected only B. besnoiti proliferation and its effect was much milder as compared to verapamil and valspodar.

Metabolic Signatures of Cryptosporidium parvum-Infected HCT-8 Cells and Impact of Selected Metabolic Inhibitors on C. parvum Infection under Physioxia and Hyperoxia.

Cryptosporidium parvum is an apicomplexan zoonotic parasite recognized as the second leading-cause of diarrhoea-induced mortality in children. In contrast to other apicomplexans, C. parvum has minimalistic metabolic capacities which are almost exclusively based on glycolysis. Consequently, C. parvum is highly dependent on its host cell metabolism. In vivo (within the intestine) infected epithelial host cells are typically exposed to low oxygen pressure (1-11% O2, termed physioxia). Here, we comparatively analyzed the metabolic signatures of C. parvum-infected HCT-8 cells cultured under both, hyperoxia (21% O2), representing the standard oxygen condition used in most experimental settings, and physioxia (5% O2), to be closer to the in vivo situation. The most pronounced effect of C. parvum infection on host cell metabolism was, on one side, an increase in glucose and glutamine uptake, and on the other side, an increase in lactate release. When cultured in a glutamine-deficient medium, C. parvum infection led to a massive increase in glucose consumption and lactate production. Together, these results point to the important role of both glycolysis and glutaminolysis during C. parvum intracellular replication. Referring to obtained metabolic signatures, we targeted glycolysis as well as glutaminolysis in C. parvum-infected host cells by using the inhibitors lonidamine [inhibitor of hexokinase, mitochondrial carrier protein (MCP) and monocarboxylate transporters (MCT) 1, 2, 4], galloflavin (lactate dehydrogenase inhibitor), syrosingopine (MCT1- and MCT4 inhibitor) and compound 968 (glutaminase inhibitor) under hyperoxic and physioxic conditions. In line with metabolic signatures, all inhibitors significantly reduced parasite replication under both oxygen conditions, thereby proving both energy-related metabolic pathways, glycolysis and glutaminolysis, but also lactate export mechanisms via MCTs as pivotal for C. parvum under in vivo physioxic conditions of mammals.

The Oesophageal Squamous Cell Carcinoma Cell Line COLO-680N Fails to Support Sustained Cryptosporidium parvum Proliferation.

Cryptosporidium parvum is an important diarrhoea-associated protozoan, which is difficult to propagate in vitro. In 2017, a report described a continuous culture of C. parvum Moredun strain, in the oesophageal squamous cell carcinoma cell line COLO-680N, as an easy-to-use system for C. parvum propagation and continuous production of oocysts. Here, we report that-using the Köllitsch strain of C. parvum-even though COLO-680N cells, indeed, allowed parasite invasion and early asexual parasite replication, C. parvum proliferation decreased after the second day post infection. Considering recurring studies, reporting on successful production of newly generated Cryptosporidium oocysts in the past, and the subsequent replication failure by other research groups, the current data stand as a reminder of the importance of reproducibility of in vitro systems in cryptosporidiosis research. This is of special importance since it will only be possible to develop promising strategies to fight cryptosporidiosis and its ominous consequences for both human and animal health by a continuous and reliable methodological progress.

Pterygodermatites nycticebi infections in golden lion tamarins (Leontopithecus rosalia rosalia) and aye-ayes (Daubentonia madagascariensis) from a German zoo.

In a golden lion tamarin (Leontopithecus rosalia rosalia) colony kept indoors in a German zoo, two animals presented a sudden onset of reduced general condition, lethargy, and diarrhea. At animal capture for clinical examination, adult nematode stages were observed after stress-induced defecation. Despite treatment, two golden lion tamarins died in the following 2 days. At necropsy, spirurid stages were found in the lungs and intestine. Additionally, adult Pterygodermatites spp. were identified in histopathological samples of intestine and pancreas, confirming the previous diagnosis. Upon diagnosis, all animals were treated with ivermectin (0.2 mg/kg; SC). Thereafter, the general condition of the golden lion tamarins improved, whereby some of them excreted spirurid nematodes over 3 days. Four weeks after treatment, 20 fecal samples from the colony were examined and proved negative for parasitic stages. Given that common German cockroaches (Blattella germanica) are suitable intermediate hosts of Pterygodermatites nycticebi, 30 specimens were collected from seven different locations around the golden lion tamarins housing. Third-stage larvae of Pterygodermatites spp. were recovered from those cockroaches. Regular anthelmintic treatments, coprological screenings, and controls for intermediate hosts were recommended. More than 2 years later, P. nycticebi infection was diagnosed again histopathologically in an aye-aye (Daubentonia madagascariensis) which suddenly died. Coprological analysis confirmed the presence of spirurid eggs. Due to prosimian primates' cockroach-eating habits and given that total cockroach eradication proved impossible, continuous cockroach control strategies and regular treatments of primates are currently performed to prevent further P. nycticebi infections.

Fasciola hepatica induces weak NETosis and low production of intra- and extracellular ROS in exposed bovine polymorphonuclear neutrophils.

Fasciola hepatica is the causative agent of fasciolosis, a worldwide distributed zoonotic disease, leading to hepatitis in humans and livestock. Newly excysted juveniles (NEJ) of F. hepatica are the first invasive stages to encounter leukocytes of host innate immune system in vivo. Among leukocytes, polymorphonuclear neutrophils (PMN) are the most abundant granulocytes of blood system and first ones to migrate into infection sites. PMN are able to cast neutrophil extracellular traps (NETs), also known as NETosis, consisting of nuclear DNA, decorated with histones, enzymes and antimicrobial peptides, which can entrap and eventually kill invasive parasites. Given that only few large parasitic helminths have been identified as potent NETosis inducers, here we studied for first time whether different F. hepatica stages can also trigger NETosis. Therefore, isolated bovine PMN were co-cultured with viable F. hepatica-NEJ, -metacercariae, -eggs and soluble antigen (FhAg). Interestingly, all stages failed to induce considerable levels of NETosis as detected by immunofluorescence- and scanning electron microscopy (SEM) analyses. NEJ remained motile until the end of incubation period. In line, NETosis quantification via nuclear area expansion (NAE) analysis revealed NEJ as weak NETosis inducers. However, bovine PMN frequently displaced towards motile NEJ and were found attached to NEJ surfaces. Functional PMN chemotaxis assays using vital F. hepatica-NEJ revealed a slight increase of PMN migration when compared to non-exposed controls. Additional experiments on intra- and extracellular reactive oxygen species (ROS) production revealed that soluble FhAg failed to induce ROS production of exposed PMN. Finally, mitochondrial oxygen consumption rates (OCR), extracellular acidification rates (ERAC) and proton production rates (PPR) were not significantly increased in FhAg-stimulated PMN. In summary, data suggest that F. hepatica might effectively evade PMN activation and NETosis by secreting parasite-specific molecules to either resolve NETs or to impair NETosis signaling pathways. We call for future molecular analysis not only on F. hepatica-derived NETosis modulation but also on its possible role in fasciolosis-associated pathology in vivo.

Intracellular Parasites Toxoplasma gondii and Besnoitia besnoiti, Unveiled in Single Host Cells Using AP-SMALDI MS Imaging.

The obligate intracellular apicomplexan parasites Toxoplasma gondii and Besnoitia besnoiti are important causes of disease in both humans and cattle. To date, effective specific treatments are lacking for both infections. To counteract severe symptoms leading to, e.g., disabilities and even abortion in the case of human toxoplasmosis and bovine besnoitiosis, novel targets are required for development of drugs and vaccines. A promising emerging technique for molecular characterization of organisms is high-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) which enables semiquantitative visualization of metabolite distributions. MSI was here used to trace and characterize lipid metabolites in primary bovine umbilical vein endothelial cells (BUVECs) upon infection with tachyzoites, an early and pathogenic fast-replicating life stage of T. gondii and B. besnoiti. A cell bulk, derived from noninfected controls and parasite-infected cell pellets, was analyzed by AP-SMALDI MSI in technical and biological triplicates. Multivariate statistical analysis including hierarchical clustering and principle component analysis revealed infection-specific metabolites in both positive- and negative-ion mode, identified by combining database search and LC-MS2 experiments. MSI analyses of host cell monolayers were conducted at 5 µm lateral resolution, allowing single apicomplexan-infected cells to be allocated. This is the first mass spectrometry imaging study on intracellular T. gondii and B. besnoiti infections and the first detailed metabolomic characterization of B. besnoiti tachyzoites. MSI was used here as an efficient tool to discriminate infected from noninfected cells at the single-cell level in vitro.

Optimized excystation protocol for ruminant Eimeria bovis- and Eimeria arloingi-sporulated oocysts and first 3D holotomographic microscopy analysis of differing sporozoite egress.

Successful excystation of sporulated Eimeria spp. oocysts is an important step to acquire large numbers of viable sporozoites for molecular, biochemical, immunological and in vitro experiments for detailed studies on complex host cell-parasite interactions. An improved method for excystation of sporulated oocysts and collection of infective E. bovis- and E. arloingi-sporozoites is here described. Eimeria spp. oocysts were treated for at least 20 h with sterile 0.02 M L-cysteine HCl/0.2 M NaHCO3 solution at 37 °C in 100% CO2 atmosphere. The last oocyst treatment was performed with a 0.4% trypsin 8% sterile bovine bile excystation solution, which disrupted oocyst walls with consequent activation of sporozoites within oocyst circumplasm, thereby releasing up to 90% of sporozoites in approximately 2 h of incubation (37 °C) with a 1:3 (oocysts:sporozoites) ratio. Free-released sporozoites were filtered in order to remove rests of oocysts, sporocysts and non-sporulated oocysts. Furthermore, live cell imaging 3D holotomographic microscopy (Nanolive®) analysis allowed visualization of differing sporozoite egress strategies. Sporozoites of both species were up to 99% viable, highly motile, capable of active host cell invasion and further development into trophozoite- as well as macroment-development in primary bovine umbilical vein endothelial cells (BUVEC). Sporozoites obtained by this new excystation protocol were cleaner at the time point of exposure of BUVEC monolayers and thus benefiting from the non-activation status of these highly immunocompetent cells through debris. Alongside, this protocol improved former described methods by being is less expensive, faster, accessible for all labs with minimum equipment, and without requirement of neither expensive buffer solutions nor sophisticated instruments such as ultracentrifuges.

Besnoitia besnoiti bradyzoite stages induce suicidal- and rapid vital-NETosis.

Besnoitia besnoiti is an obligate intracellular apicomplexan protozoan parasite, which causes bovine besnoitiosis. Recently increased emergence within Europe was responsible for significant economic losses in the cattle industry due to the significant reduction of productivity. However, still limited knowledge exists on interactions between B. besnoiti and host innate immune system. Here, B. besnoiti bradyzoites were successfully isolated from tissue cysts located in skin biopsies of a naturally infected animal, and we aimed to investigate for the first time reactions of polymorphonuclear neutrophils (PMN) exposed to these vital bradyzoites. Freshly isolated bovine PMN were confronted to B. besnoiti bradyzoites. Scanning electron microscopy (s.e.m.)- and immunofluorescence microscopy-analyses demonstrated fine extracellular networks released by exposed bovine PMN resembling suicidal NETosis. Classical NETosis components were confirmed via co-localization of extracellular DNA decorated with histone 3 (H3) and neutrophil elastase (NE). Live cell imaging by 3D holotomographic microscopy (Nanolive®) unveiled rapid vital NETosis against this parasite. A significant increase of autophagosomes visualized by specific-LC3B antibodies and confocal microscopy was observed in B. besnoiti-stimulated bovine PMN when compared to non-stimulated group. As such, a significant positive correlation (r = 0.37; P = 0.042) was found between B. besnoiti-triggered suicidal NETosis and autophagy. These findings suggest that vital- as well as suicidal-NETosis might play a role in early innate host defence mechanisms against released B. besnoiti bradyzoites from tissue cysts, and possibly hampering further parasitic replication. Our data generate first hints on autophagy being associated with B. besnoiti bradyzoite-induced suicidal NETosis and highlighting for first time occurrence of parasite-mediated vital NETosis.

Besnoitia besnoiti infection alters both endogenous cholesterol de novo synthesis and exogenous LDL uptake in host endothelial cells.

Besnoitia besnoiti, an apicomplexan parasite of cattle being considered as emergent in Europe, replicates fast in host endothelial cells during acute infection and is in considerable need for energy, lipids and other building blocks for offspring formation. Apicomplexa are generally considered as defective in cholesterol synthesis and have to scavenge cholesterol from their host cells for successful replication. Therefore, we here analysed the influence of B. besnoiti on host cellular endogenous cholesterol synthesis and on sterol uptake from exogenous sources. GC-MS-based profiling of cholesterol-related sterols revealed enhanced cholesterol synthesis rates in B. besnoiti-infected cells. Accordingly, lovastatin and zaragozic acid treatments diminished tachyzoite production.  Moreover, increased lipid droplet contents and enhanced cholesterol esterification was detected and inhibition of the latter significantly blocked parasite proliferation. Furthermore, artificial increase of host cellular lipid droplet disposability boosted parasite proliferation. Interestingly, lectin-like oxidized low density lipoprotein receptor 1 expression was upregulated in infected endothelial hostcells, whilst low density lipoproteins (LDL) receptor was not affected by parasite infection. However, exogenous supplementations with non-modified and acetylated LDL both boosted B. besnoiti proliferation. Overall, current data show that B. besnoiti simultaneously exploits both, endogenous cholesterol biosynthesis and cholesterol uptake from exogenous sources, during asexual replication.