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Neutrophil extracellular traps (NET) formation is one important host innate defense mechanism elicited by polymorphonuclear neutrophils (PMN). NETs are composed by chromatin and proteins with microbicidal and signaling activity. So far, there is one report on Toxoplasma gondii-triggered NETs in cattle, however, exact mechanisms, including signalling pathways and dynamics governing this reaction remain largely unknown. Recently, involvement of cell cycle proteins was demonstrated for phorbol myristate acetate (PMA)-triggered human PMN-derived NETs. Here, we studied the involvement of cell cycle proteins in T. gondii-induced NETs in exposed bovine PMN. Through confocal and transmission electron microscopy we discovered that Ki-67 and lamin B1 signals are upregulated and relocated during T. gondii-induced NETosis. Nuclear membrane disruption was also observed as a hallmark of NET formation in bovine PMN confronted with viable T. gondii tachyzoites, mimicking some steps of mitosis. However, we did not observe centrosome duplication as previously described for human PMN-derived NET formation stimulated with PMA.

M onoxenous Eimeria species are widespread enteropathogenic apicomplexan protozoa with a high economic impact on livestock. In cattle, tenacious oocysts shed by E. bovis-infected animals are ubiquitously found and making infection of calves almost inevitable. To become infectious oocysts, exogenous oxygen-dependent E. bovis sporogony must occur leading to the formation of sporulated oocysts containing four sporocysts each harboring two sporozoites. Investigations on sporogony by live cell imaging techniques of ruminant Eimeria species are still absent in literature as commonly used fluorescent dyes do not penetrate resistant oocyst bi-layered wall. Sporogonial oocysts were daily analyzed by a 3D Cell Explorer Nanolive microscope to explore ongoing aerobic-dependent sporogony as close as possible to an in vivo situation. Subsequently, 3D holotomographic images of sporulating E. bovis oocysts were digitally stained based on refractive indices (RI) of oocyst bi-layered wall and sub-compartments of circumplasm using STEVE software (Nanolive), and the cellular morphometric parameters were obtained. Overall, three different E. bovis sporogony phases, each of them divided into two sub-phases, were documented: (i) sporoblast/sporont transformation into sporogonial stages, (ii) cytokinesis followed by nuclear division, and finally (iii) formation of four sporocysts with two fully developed sporozoites. Approximately 60% of sporulating E. bovis oocysts accomplished aerobic sporogony in a synchronized manner. E. bovis sporogony was delayed (i.e., 6 days) when compared to an in vivo situation where 2–3 days are required but under optimal environmental conditions. Live cell 3D holotomography analysis might facilitate the evaluation of either novel disinfectants- or anti-coccidial drug-derived effects on ruminant/avian Eimeria sporogony in vitro as discrimination of sporogony degrees based on compactness, and dry mass was here successfully achieved. Main changes were observed in the oocyst area, perimeter, compactness, extent, and granularity suggesting those parameters as an efficient tool for a fast evaluation of the sporulation degree.

Neospora caninum represents an obligate intracellular parasite that belongs to the phylum Apicomplexa and is a major abortive agent in bovines. During merogony, N. caninum tachyzoites invade and proliferate in host cells in vivo, including endothelial cells of lymphatic and blood vessels. The egress at the end of the lytic cycle is tightly regulated in apicomplexans. Evidence in Toxoplasma gondii shows that Ca++ signalling governs tachyzoite egress. Much less is known on egress mechanisms of N. caninum. Here, we show, using 3D live cell holotomographic microscopy in fluo-4 AM-loaded N. caninum-infected BUVEC, that treatments with the calcium ionophore A23187 at 24- and 42-h post-infection (h p. i.) induced a fast and sustained increase in Ca++ signals in parallel to tachyzoite egress. A23187 treatments exclusively triggered tachyzoite release at 42-h p. i. but failed to do so at 24-h p. i. indicating a role for meront maturation in calcium-induced tachyzoite egress. Overall, we show that live cell 3D holotomographic analysis in combination with epifluorescence is a suitable tool to study calcium dynamics related to coccidian egress or other important cell functions.

Selective inhibition of eukaryotic initiation factor 4A (eIF4A), an RNA helicase, has been proposed as a strategy to fight pathogens. Plant-derived rocaglates exhibit some of the highest specificities among eIF4A inhibitors. Sensitivity to rocaglates is determined by key amino acid (aa) residues mediating reversible clamping of the eIF4A:RNA complex. To date, no comprehensive assessment of eIF4A sensitivity to rocaglates across the eukaryotic tree of life has been performed to determine their anti-pathogenic potential.We performed an in silico analysis of the substitution patterns of six aa residues in eIF4A1 critical to rocaglate binding (human positions 158, 159, 163, 192, 195, 199), uncovering 35 pattern variants among 365 eIF4As sequenced to date. In silico molecular docking analysis of the eIF4A:RNA:rocaglate complexes of the 35 variants, modeled in a human eIF4A environment, and in vitro thermal shift assays with recombinantly expressed human eIF4A mutants, representing select natural and artificial variants, revealed that sensitivity to a natural or one of two synthetic rocaglates—silvestrol, CR-1-31-B, or zotatifin—was associated with lower inferred binding energies and higher melting temperature shifts. Helicase activities were comparable across variants and independent of sensitivity to rocaglates.In vitro testing with silvestrol validated predicted resistance based on position 163 substitutions in Caenorhabditis elegans and Leishmania amazonensis and predicted sensitivity in Aedes sp., Schistosoma mansoni, Trypanosoma brucei, Plasmodium falciparum, and Toxoplasma gondii.Our analysis shows resistance to rocaglates emerging in disparate eukaryotic clades pointing to resistance being a selective neutral trait except in rocaglate-producing Aglaia plants and their fungal parasite Ophiocordyceps. The analysis further revealed the possibility of targeting important insect, plant, animal, and human pathogens including Galleria mellonella, Ustilago maydis, Babesia ovata, and Cryptosporidium sp., with rocaglates. Finally, combined docking and thermal shift analyses might help design novel synthetic rocaglate derivatives or alternative eIF4A inhibitors to fight pathogens.Author SummaryIn the ongoing search for novel ways to fight non-viral and non-bacterial pathogens, targeting translation—the universal process of protein synthesis—to inhibit growth and cell proliferation has emerged as an attractive strategy. Here, we focused on the potential of rocaglates, a group of plant-derived compounds, to inhibit an early step in translation mediated by a RNA helicase called eIF4A. We performed a comprehensive analysis of eIF4A sequence variants to determine their potential sensitivities to rocaglates, especially in pathogens of prokaryotic, fungal, or animal origin. We complemented this in silico analysis with enzyme-based in vitro and whole pathogen in vivo experiments to confirm the sensitivity or resistance to rocaglates of specific variants of eIF4A. Our analysis provides the first comprehensive picture of rocaglate sensitivity among pathogens and establishes targeting important insect, plant, animal, and human pathogens such as wax moth larvae, a major parasite of honey bees, corn smut, a widely distributed fungal disease, Babesia, a livestock parasite that causes anemia and babesiois, and Cryptosporidium, the causative organism of cryptosporidiosis in humans, with rocaglates as a viable anti-pathogen strategy.

Infectious vaginitis is a microbiological syndrome of great importance in public health that affects millions of women worldwide. However, no studies have explored the phenomenon of the production of the neutrophil extracellular traps (NETs) that are released into the female reproductive tract in these pathologies. This study aimed to determine the presence of NETosis in vaginal discharges of women with bacterial vaginosis, candidiasis, and trichomoniasis by characterizing NETs. Extracellular DNA with neutrophil elastase and citrullinated histones was identified to confirm the NET components (n = 10). The concentration, phenotypes of NETs, and number of NETotic cells were determined. The results showed an increase in NETotic cells in women with Candida albicans (CA) and Trichomonas vaginalis (TV) and an increase in NETs in TV-induced vaginitis. Samples of CA- and TV-infected women showed different NET phenotypes (diffNETs, sprNETs, and aggNETs); diffNETs were found in high concentrations in samples with CA and were increased in three types of NETs in TV infections. Samples with intermediate microbiota and bacterial vaginosis showed increased NETotic cells while the intermediate microbiota presented a higher concentration of NETs. Therefore, alterations in the microbiota and the presence of fungal and parasitic infections are important stimuli for the activation and induction of NETosis, and their cytotoxic effects could enhance tissue damage.

The protozoan parasiteEimeria bovisis the causative agent of bovine coccidiosis, an enteric disease of global importance that significantly affects cattle productivity. Previous studies showed that bovine NETosis—an important early host innate effector mechanism of polymorphonuclear neutrophil (PMN)—is elicited byE. bovisstages. So far, the metabolic requirements ofE. bovis-triggered NET formation are unknown. We here studied early glycolytic and mitochondrial responses of PMN as well as the role of pH, distinct metabolic pathways, P2 receptor-mediated purinergic signaling, and monocarboxylate transporters 1 and 2 (MCT1, MCT2) inE. bovissporozoite-induced NET formation. Seahorse-based experiments revealed a rapid induction of both neutrophil oxygen consumption rate (OCR) and early glycolytic responses, thereby reflecting immediate PMN activation and metabolic changes upon confrontation with sporozoites. The impact of these metabolic changes on NET formation was studiedviachemical inhibition experiments targeting glycolysis and energy generation by the use of 2-fluor-2-deoxy-D-glucose (FDG), 6-diazo-5-oxo-L-norleucin (DON), sodium dichloroacetate (DCA), oxythiamine (OT), sodium oxamate (OXA), and oligomycin A (OmA) to block glycolysis, glutaminolysis, pyruvate dehydrogenase kinase, pyruvate dehydrogenase, lactate dehydrogenase, and mitochondrial ATP-synthase, respectively. Overall, sporozoite-induced NET formation was significantly diminishedviaPMN pretreatments with OmA and OXA, thereby indicating a key role of ATP- and lactate-mediated metabolic pathways. Consequently, we additionally studied the effects of extracellular pH, MCT1, MCT2, and purinergic receptor inhibitors (AR-C141900, AR-C155858, theobromine, and NF449, respectively). Pretreatment with the latter inhibitors led to blockage of sporozoite-triggered DNA release from exposed bovine PMN. This report provides first evidence on the pivotal role of carbohydrate-related metabolic pathways and purinergic receptors being involved inE. bovissporozoite-induced NETosis.

Neospora caninum represents a major cause of abortive disease in bovines and small ruminants worldwide. As a typical obligate intracellular apicomplexan parasite, N. caninum needs to modulate its host cell for successful replication. In the current study, we focused on parasite-driven interference with host cell cycle progression. By performing DNA content-based cell cycle phase analyses in N. caninum-infected primary bovine umbilical vein endothelial cells (BUVEC), a parasite-driven S-phase arrest was detected at both 24 and 32 h p. i., being paralleled by fewer host cells experiencing the G0/G1 cell cycle phase. When analyzing S-subphases, proliferation cell nuclear antigen (per PCNA)-based experiments showed a reduced population of BUVEC in the late S-phase. Analyses on key molecules of cell cycle regulation documented a significant alteration of cyclin A2 and cyclin B1 abundance in N. caninum-infected host endothelial cells, thereby confirming irregularities in the S-phase and S-to-G2/M-phase transition. In line with cell cycle alterations, general nuclear parameters revealed smaller nuclear sizes and morphological abnormalities of BUVEC nuclei within the N. caninum-infected host cell layer. The latter observations were also confirmed by transmission electron microscopy (TEM) and by analyses of lamin B1 as a marker of nuclear lamina, which illustrated an inhomogeneous nuclear lamin B1 distribution, nuclear foldings, and invaginations, thereby reflecting nuclear misshaping. Interestingly, the latter finding applied to both non-infected and infected host cells within parasitized BUVEC layer. Additionally, actin detection indicated alterations in the perinuclear actin cap formation since typical nucleo-transversal filaments were consistently lacking in N. caninum-infected BUVEC, as also documented by significantly decreased actin-related intensities in the perinuclear region. These data indicate that N. caninum indeed alters host cell cycle progression and severely affects the host cell nuclear phenotype in primary bovine endothelial host cells. In summary, these findings add novel data on the complex N. caninum-specific modulation of host cell and nucleus, thereby demonstrating clear differences in cell cycle progression modulation driven by other closely related apicomplexans like Toxoplasma gondii and Besnotia besnoiti.

Coccidia are obligate apicomplexan parasites that affect humans and animals. In fast replicating species, in vitro merogony takes only 24–48 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.

Human spermatozoa activate neutrophil extracellular traps (NETs) in vitro. NETosis is an efficient mechanism through which polymorphonuclear neutrophils (PMN) capture sperm in vitro. The objective of this study was to establish the role of store-operated Ca+2 entry (SOCE) in human sperm-triggered NETs and its impact on sperm integrity and oocyte binding capacity. PMN isolated from donors were exposed to spermatozoa isolated from normozoospermic donors using the swim-up technique and were divided into the following groups: (1) sperm, (2) PMN, (3) PMN + sperm, (4) PMN (pretreated with 2-APB, SOCE inhibitor) + sperm, (5) (PMN + DNase) + sperm, and (6) (PMN + PMA) + sperm (positive control). NETs were quantified using PicoGreen® and visualised by scanning electron microscopy and immunofluorescence of extracellular DNA and neutrophil elastase. Plasma membrane, acrosome, and DNA integrity were analysed by flow cytometry, and oocyte binding was evaluated using the hemizona pellucida assay. Sperm-triggered NETosis negatively affected the sperm membrane and acrosome integrity and decreased the oocyte binding capacity. These effects were negated by an SOCE inhibitor, thus improving sperm function and achieving high oocyte binding capacity. The SOCE inhibitor significantly reduced NET formation compared with that in control PMN/sperm (P < 0.05). Collectively, these results advance the knowledge about the role of PMN in reproduction and will allow the development of strategies to block NET formation in situations of reduced fertilisation success.

During artificial insemination in bovine, the deposition of semen into the uterus results in an immune reaction which is based on polymorphonuclear neutrophils activity, including the formation of neutrophil extracellular traps. The formation of neutrophil extracellular traps as a reaction of neutrophils to spermatozoa was recently described. However, it is not completely clear which components of the semen are responsible for this reaction. The objective of this study was to quantify and compare the formation of neutrophil extracellular traps following in vitro incubation of bovine polymorphonuclear neutrophils with semen and extenders of different origins and conditions. We investigated the interactions between bovine polymorphonuclear neutrophils and different semen extenders, various seminal plasma concentrations from young and old bulls as well as sexed and non-sexed semen and their corresponding extenders. Three different semen extenders from two companies in fresh and frozen-thawed conditions were compared. Fresh semen extenders showed higher neutrophil extracellular traps induction than did frozen-thawed ones. The formation of neutrophil extracellular traps were also dependent on the presence of seminal plasma. We could show that seminal plasma alone, without any sperm cells, induced the reaction and that the addition of at least 1% seminal plasma already resulted in the formation of neutrophil extracellular traps. Furthermore, seminal plasma from young bulls led to significant higher neutrophil extracellular traps induction. No difference between non-sex-sorted and sex-sorted sperm and its extenders was observed. Taken together, different semen extenders as well as the amount and origin of seminal plasma influence neutrophil extracellular traps formation, whereas sex-sorted sperm did not affect the reaction.

The seal heartworm Acanthocheilonema spirocauda (Nematoda: Onchocercidae) parasitizes the heart and pulmonary arteries of various phocid seals of the Northern Hemisphere. Over many decades, potential vectors of this parasite have been discussed, and to this date, the life cycle is not fully known. The seal louse Echinophthirius horridus (Anoplura: Echinophthiriidae) is an obligatory, permanent and haematophagous ectoparasite of phocids that has been hypothesized to function as obligate intermediate host for A. spirocauda. We examined 11 adult E. horridus specimens collected from stranded harbour seals (Phoca vitulina) in rehabilitation at the Sealcentre Pieterburen by X-ray microCT imaging, aiming to illustrate larval A. spirocauda infection sites in situ. In three of these specimens, thread-like larvae were detected in insect organs. Detailed imaging of the most infected louse revealed a total of 54 A. spirocauda larvae located either in fat bodies or the haemocoel. Histological analysis of the same specimen illustrated nematode cross-sections, confirming X-ray microCT data. The current data strongly suggest that E. horridus is a natural intermediate host for A. spirocauda. Moreover, we demonstrate the potential of X-ray microCT-based imaging as a non-destructive method to analyze host-parasite interactions, especially in the neglected field of marine mammal parasitology.

Ethylene glycol (EG) is obtained by a novel, two-step approach combining a biotechnological and a heterogeneously catalyzed step. First, microalgae are cultivated to photobiocatalytically yield glycolic acid (GA) by means of photosynthesis from CO

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.

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.

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.

Alongside exotic reptiles, amphibians, such as toads, frogs, salamanders, and newts, are nowadays considered popular pets worldwide. As reported for other exotic pet animals, amphibians are known to harbor numerous gastrointestinal parasites. Nonetheless, very little data are available on captive amphibian parasitic diseases. In this study, we applied direct saline fecal smears (DSFS) to examine in total 161 stool samples from 41 different amphibian species belonging to the orders Anura and Caudata. In addition, carbolfuchsin-smear (CFS) staining (n = 74 samples) was used to detect amphibian Cryptosporidium oocysts. Also, complete dissections of deceased amphibians (n = 107) were performed to specify parasite infections and to address parasite-associated pathogenicity. Overall, examined amphibian fecal samples contained 12 different parasite taxa. The order Rhabditida with the species Rhabdias spp. and Strongyloides spp. were the most prevalent nematode species (19.3%), followed by flagellated protozoans (8.7%), Amphibiocapillaria spp./Neocapillaria spp. (7.5%), Oswaldocruzia spp. (4.3%), Blastocystis spp. (3.1%), Cosmocerca spp. (3.1%), oxyurids (Pharyngonoidae) (3.1%), spirurids (1.2%), un-sporulated coccidian oocysts (0.6%), Tritrichomonas spp. (0.6%), Karotomorpha spp. (0.6%), and Cryptosporidium spp. (0.6%). One CFS-stained fecal sample (1.4%) was positive for Cryptosporidium oocysts. Within dissected amphibians, 31 (48.4%) of the anurans and 11 (26.2%) of the salamanders were infected with gastrointestinal parasites. One cutaneous Pseudocapillaroides xenopi infection was diagnosed in an adult African clawed frog (Xenopus laevis). Etiologically, 17 (15.9%) of them died due to severe parasitic and/or bacterial infections (e.g., Chryseobacterium indologenes, Citrobacter freudii, Sphingobacterium multivorum, Klebsiella pneumoniae). High prevalence and pathological findings of several clinical amphibian parasitoses call for more detailed investigation on gastrointestinal parasite-derived molecular mechanisms associated with detrimental lesions or even death.

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.

Besnoitia besnoiti is an important obligate intracellular parasite of cattle which primarily infects host endothelial cells of blood vessels during the acute phase of infection. Similar to the closely related parasite Toxoplasma gondii, B. besnoiti has fast proliferating properties leading to rapid host cell lysis within 24–30 h p.i. in vitro. Some apicomplexan parasites were demonstrated to modulate the host cellular cell cycle to successfully perform their intracellular development. As such, we recently demonstrated that T. gondii tachyzoites induce G2/M arrest accompanied by chromosome missegregation, cell spindle alteration, formation of supernumerary centrosomes, and cytokinesis impairment when infecting primary bovine umbilical vein endothelial cells (BUVEC). Here, we follow a comparative approach by using the same host endothelial cell system for B. besnoiti infections. The current data showed that—in terms of host cell cycle modulation—infections of BUVEC by B. besnoiti tachyzoites indeed differ significantly from those by T. gondii. As such, cyclin expression patterns demonstrated a significant upregulation of cyclin E1 in B. besnoiti–infected BUVEC, thereby indicating parasite-driven host cell stasis at G1-to-S phase transition. In line, the mitotic phase of host cell cycle was not influenced since alterations of chromosome segregation, mitotic spindle formation, and cytokinesis were not observed. In contrast to respective T. gondii–related data, we furthermore found a significant upregulation of histone H3 (S10) phosphorylation in B. besnoiti–infected BUVEC, thereby indicating enhanced chromosome condensation to occur in these cells. In line to altered G1/S-transition, we here additionally showed that subcellular abundance of proliferating cell nuclear antigen (PCNA), a marker for G1 and S phase sub-stages, was affected by B. besnoiti since infected cells showed increased nuclear PCNA levels when compared with that of control cells.

Aedes aegypti represents one of the main vectors of at least five relevant arthropod-borne viral infections in humans (i.e., Rift Valley fever, Dengue fever, Zika, chikungunya, and yellow fever) worldwide. Ae. aegypti control strategies are mostly based on using chemical insecticides (i.e., organophosphates, pyrethroids, carbamates, and organochlorines) and reducing larval sources. Furthermore, monitoring the growth activity and mapping the geographical distribution of insecticide resistance are mandatory, as recommended by the WHO. Accordingly, we conducted a study on the possible mechanism by which Ae. aegypti develops resistance to several frequently used chemical insecticides (i.e., λ-cyhalothrin, bendiocarb, cyfluthrin, deltamethrin, malathion, and permethrin) in the city of Makassar, Sulawesi, Indonesia. The results showed the progression of resistance toward the examined insecticides in Ae. aegypti populations in Makassar. The mortality rate of Ae. aegypti was less than 90%, with the highest resistance recorded against 0.75% permethrin. The molecular evaluation of the voltage-gated sodium channel gene (VGSC) showed a significant correlation of the V1016G gene mutation in the tested 0.75% permethrin-resistant Ae. aegypti phenotypes. Nevertheless, the F1534C point mutation in the VGSC gene of Ae. aegypti did not show a significant correlation with the phenotype exhibiting insecticide resistance to 0.75% permethrin. These results indicate that Ae. aegypti mosquitoes in Makassar City have developed resistance against the frequently used insecticide permethrin, which might spread to less-populated regions of Sulawesi. Therefore, we call for further entomological monitoring of insecticide resistance not only on Sulawesi but also on other closely located islands of the Indonesian archipelago to delay the spread of Ae. aegypti insecticide resistance.

Nowadays, snakes established as domestic exotic pets, harboring numerous (zoonotic) gastrointestinal parasites. In this parasitological survey, we used direct saline fecal smears (DSFS) to examine 586 stool samples from 71 different snake species either kept as pets in households or in zoological gardens in Germany. In addition to DSFS, carbol-fuchsin-fecal smears (n = 296), coproantigen ELISA tests (n = 98), and immunofluorescence assays (IFA; n = 77) for the detection reptile Cryptosporidium infections were conducted. Complete dissections of deceased snakes (n = 63) were also performed in order to gain data on endoparasite species burdens affecting domestic snakes. Overall, examined fecal samples contained 20 different parasite taxa: Ancylostomatid Kalicephalus spp. were the most prevalent nematode species (3.3%), followed by Strongyloides/Rhabdias (2.6%), flagellated protozoan trophozoites (e. g., Proteromonadida, Reteromonadida) (2.3%), Monocercomonas spp. (1.9%), Entamoeba spp. (1.4%), unsporulated coccidian oocysts (1.4%), Kapsulotaenia spp. (0.9%), Capillaria spp. (0.7%), indet. trematodes (0.5%), pentastomids (0.5%), spirurids (0.4%), Eimeria spp. (0.4%), ascarids (0.4%), Blastocystis sp. (0.2%), heterakids (0.2%), cestodes (Proteocephalidae) (0.2%), Plagiorchis spp. (0.2%), Cryptosporidium spp. (0.2%), Caryospora epicratesi (0.2%), and Sarcocystis spp. (0.2%). For Cryptosporidium, four carbol-fuchsin-stained smears (1.4%), 12 (12.2%) coproantigen ELISA-examined samples and 5.2% of examined samples were diagnosed with IFA. Fourteen (22.2%) of dissected snakes showed infections with various pathogenic nematode genera and 8 of them (12.7%) died due to protozoan parasitic infections. High prevalences of intestinal protozoan parasites resulting in severe pathological findings observed in dissected snakes call for more detailed investigations on gastrointestinal parasites.

Neutrophil extracellular traps (NETs) play a key role in fertilisation by eliminating microorganisms and entrapping spermatozoa in the female reproductive tract (FRT). The deleterious effects of NETs on spermatozoa have been previously described; however, individual exposure to NET-derived components in bull spermatozoa has not been explored. The aim of this study was to evaluate the effects of the main NET-derived proteins, histone 2A (H2A), neutrophil elastase (ELA), myeloperoxidase (MPO), pentraxin 3 (PTX), cathepsin G (Cat-G), and cathelicidin LL37 (LL-37), at concentrations of 1, 10, and 30 μg/mL, on sperm parameters. Sperm were selected and incubated with different NET-derived proteins for 4 h. Membrane and acrosome integrity, lipoperoxidation, and membrane phospholipid disorders were also evaluated. Bovine polymorphonuclear neutrophil (PMN)/sperm co-cultures were evaluated by scanning electron microscopy and immunofluorescence. All NET-derived proteins/enzymes resulted in a reduction in membrane integrity, acrosome integrity, and lipoperoxidation at a concentration of 30 μg/mL. Bovine PMN/sperm co-cultures showed marked NET formation in the second hour. In conclusion, all NET-derived proteins/enzymes exerted cytotoxic effects on bull sperm, and this effect should be considered in future investigations on the uterine microenvironment and the advancement of spermatozoa in the FRT.

Cryptosporidiosis is a zoonotic intestinal disease that affects humans, wildlife, and neonatal cattle, caused by

Zoonotic larvae of the family Anisakidae found in several fish species represent a serious risk in public health since they may cause food-borne anisakidosis in humans. Chile has culinary preferences including eating raw fish in many traditional preparations. In the present study, a total of 180 fish specimens representing three different fish species, i.e., Chilean hake (Merluccius gayi), snoek (Thyrsites atun), and sea bream (Brama australis), were caught at central coast of Chile. Parasitological examination was performed on musculature and abdominal cavity for subsequent extraction and quantification of anisakid larvae. Estimation of infection parameters, such as prevalence, was performed indicating 100% (CI: 0.94–1.0) prevalence of anisakid L3 in Chilean hakes and snoeks. Moreover, sea breams reached a prevalence of 35% (CI: 0.23–0.48). Prevalence of anisakid larvae in muscle was also analyzed showing values of 18.6% (CI: 0.097–0.309) in Chilean hakes, 15% (CI: 0.07–0.26) in snoeks, and 1.7% (CI: 0–0.089) in sea breams. Meanwhile, prevalence of anisakid larvae in internal organs showed highest values for peritoneum (100% and 83.3%) for snoeks and Chilean hakes, respectively, for liver (96.7%) and gonads (86.6%) in Chilean hakes, and for intestine (98.3%) in snoeks. Molecular analysis of collected anisakid L3 unveiled presence of two potentially zoonotic nematode species, i.e., Pseudoterranova cattani and Anisakis pegreffii. P. cattani was found in Chilean hakes and snoeks being the first molecular host species report for Chilean snoeks. Besides, A. pegreffii was also identified in these species being the first molecular report on this regard. These findings are relevant for better understanding of epidemiology of anisakiasis in Chilean coasts and for public health issues considering potential risk of human population due to its culinary preferences in eating raw fish.

Angiostrongylus vasorum is a cardiopulmonary nematode, causing several clinical manifestations in dogs, e.g., severe respiratory signs, coagulopathy, and gastrointestinal or neurological signs. In the last decades, this parasite has been described to spread and emerge in Europe and North America. Scant studies on A. vasorum occurrence in South America exist. Recently, A. vasorum was detected in gastropod intermediate hosts in Colombia, where data on definitive host prevalence, e.g., dogs and wild canids, are still limited. Therefore, the sera of 955 dogs, varying in age and breed from seven different departments all over Colombia, were collected and analysed for A. vasorum antigens and parasite-specific antibodies by ELISA. In total, 1.05 % (n = 10; 95 % CI 0.40–1.69) of the samples were antigen-positive and 2.62 % (n = 25; 95 % CI 1.61–3.63) were antibody-positive. These results confirm the presence of A. vasorum in Colombia, although positive results in antigen and antibody reactions in the same dog were not detected. This study is the first large-scale survey on A. vasorum seroprevalences in dogs from Colombia.

Various studies have shown that the transmission and passage of alien and native pathogens play a critical role in the establishment process of an invasive species and its further spread. Egyptian geese (Alopochen aegyptiaca) are neozotic birds on various continents. They live not only in the countryside near fresh water bodies but also in urban habitats in Central Europe with close contact to humans and their pets. Although their rapid distribution in Europe is widely debated, scientific studies on the anthropozoonotic risks of the population and studies on the present endoparasites in Egyptian geese are rare worldwide. In the present study, 114 shot Egyptian geese and 148 non-invasively collected faecal samples of wild Egyptian geese from 11 different Federal States in Germany were examined. A total of 13 metazoan endoparasite species in 12 different genera were identified. The main endoparasites found were Hystrichis tricolor, Polymorphus minutus, and, in lesser abundance, Cloacotaenia sp. and Echinuria uncinata. Adult stages of Echinostoma revolutum, an anthropozoonotic heteroxenic trematode, were found in 7.9% of the animals examined postmortem. This species was additionally identified by molecular analysis. Although Egyptian geese live in communities with native waterfowl, it appears that they have a lower parasitic load in general. The acquisition of generalistic parasites in an alien species and the associated increased risk of infection for native species is known as “spill-back” and raises the question of impacts on native waterfowl. Differences between animals from rural populations and urban populations were observed. The present study represents the first large-scale survey on gastrointestinal parasites of free-ranging Egyptian geese.

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

Strategies to control goat coccidiosis traditionally rely on the use of management practices combined with anticoccidial treatments, and limited effort has been made, so far, to address immunological control of caprine Eimeria infections. Previously, we showed that monospecific immunization with X-Rad-attenuated Eimeria ninakohlyakimovae oocysts induced considerable immunoprotection upon challenge. In the present study, we conducted a similar vaccination trial but using a mixture of caprine Eimeria species typically present in natural infected goats. For immunization, sporulated oocysts were attenuated by X irradiation (20 kilorad). All infections were performed orally applying 105 sporulated oocysts of mixed Eimeria spp. per animal. In total, 18 goat kids were grouped as follows: (G1) immunized + challenge infected; (G2) primary + challenge infected; (G3) challenge infection control; and (G4) non-immunized/non-infected control. Overall, goat kids infected with attenuated oocysts (= immunized) shed less oocysts in the faeces and showed a lower degree of clinical coccidiosis than animals infected with non-attenuated oocysts. Animals of both challenge groups (G1 and G2) showed partial immunoprotection upon reinfection when compared to challenge infection control (G3). However, the degree of immunoprotection was less pronounced than recently reported for monospecific vaccination against Eimeria ninakohlyakimovae, most probably due to the complexity of the pathogenesis and related immune responses against mixed Eimeria spp. infections. Nevertheless, the data of the present study demonstrate that immunization with attenuated Eimeria spp. oocysts may be worth pursuing as a strategy to control goat coccidiosis.

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.

Besnoitia besnoiti is the causative agent of bovine besnoitiosis, a disease affecting both, animal welfare and cattle productivity. NETosis represents an important and early host innate effector mechanism of polymorphonuclear neutrophils (PMN) that also acts against B. besnoiti tachyzoites. So far, no data are available on metabolic requirements of B. besnoiti tachyzoite-triggered NETosis. Therefore, here we analyzed metabolic signatures of tachyzoite-exposed PMN and determined the relevance of distinct PMN-derived metabolic pathways via pharmacological inhibition experiments. Overall, tachyzoite exposure induced a significant increase in glucose and serine consumption as well as glutamate production in PMN. Moreover, tachyzoite-induced cell-free NETs were significantly diminished via PMN pre-treatments with oxamate and dichloroacetate which both induce an inhibition of lactate release as well as oxythiamine, which inhibits pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and transketolase, thereby indicating a key role of pyruvate- and lactate-mediated metabolic pathways for proper tachyzoite-mediated NETosis. Furthermore, NETosis was increased by enhanced pH conditions; however, inhibitors of MCT-lactate transporters (AR-C141900, AR-C151858) failed to influence NET formation. Moreover, a significant reduction of tachyzoite-induced NET formation was also achieved by treatments with oligomycin A (inhibitor of ATP synthase) and NF449 (purinergic receptor P2X1 antagonist) thereby suggesting a pivotal role of ATP availability for tachyzoite-mediated NETosis. In summary, the current data provide first evidence on carbohydrate-related metabolic pathways and energy supply to be involved in B. besnoiti tachyzoite-induced NETosis.

A large-scale cross-sectional epidemiological study was conducted to evaluate prevalence, species diversity, and associated risk factors of Eimeria infections in 55 cattle farms across seven states of Colombia, including subtropical and tropical regions. In total, 1333 fecal samples from young animals (< 1 year of age) were examined at a single sampling date from August 2016 to December 2016. Flotation and McMaster techniques were conducted for parasitological investigation. Excreted Eimeria oocysts were allowed to sporulate in vitro and thereafter identified to species level based on morphological and morphometric characteristics. The overall Eimeria prevalence was 75.5% (1006/1333), with no difference observed between age categories. In total, 13 different Eimeria species were identified. The most prevalent species was E. bovis (33.5%), followed by E. auburnensis (12.5%) and E. zuernii (11.9%). Analysis of extrinsic associated risk factors revealed the floor type, feeding system, watering system, and herd size as significant (p < 0.05) risk factors for Eimeria spp. infections. Based on these data, it can be assumed that bovine coccidiosis infections occur ubiquitously in the country and might play an important role especially in its subclinical form by affecting production parameters in conventional cattle management systems.

The presence of bacteria and/or leukocytes can alter semen quality resulting in low sperm quality and infertility. Inflammation or infection increases the numbers of PMN or macrophages/monocytes in male genital tract. Release of extracellular traps (ETs) by leukocytes has been recognized as a novel mechanism of early host innate immunity, in response to invasive pathogens. This is the first work that evaluated the mechanism of triggered ETs in monocytes co-incubated with spermatozoa or bacteria and the effect on sperm function. Selected spermatozoa and human monocytes isolated from peripheral blood were obtained by healthy donors. Two experimental models were developed, one aseptic (non-infectious) incubating spermatozoa and monocytes, and septic models (infectious) incubating spermatozoa with monocytes and uropathogenic Escherichia coli (E. coli). ETs of monocytes (METs) (DNA, global histone and citrullinated histones) were visualized by scanning electron microscopy (SEM) and immunofluorescence analyses. Progressive motility was performed at 0, 10, 30, 60, and 180 min after co-incubation with CASA system. SEM- and immunofluorescence-analyses revealed human spermatozoa alone or in the presence of E. coli as strong inducers METs. In aseptic model, the motility decreased to 65.2 ± 3.5% at 10 min of incubation and 29.3 ± 3.3% at 30 min (p < 0.001). In septic model, motility decreased to 44.5 ± 5.9% (10 min) and 12.7 ± 2.2% (30 min) (p < 0.001). MET-derived small spermatozoa aggregations were observed in both models. METs might physically block spermatozoa and decrease motility after a brief contact. This may impair male fertility, especially in patients with genital tract infections or chronic inflammation. Abbreviations: PMN: polymorphonuclear; ETs: extracellular traps; E. coli: Escherichia coli; METs: ETs of monocytes; SEM: scanning electron microscopy; NE: neutrophil elastase; MPO: myeloperoxidase; MAGI: male accessory gland infection; PBMC: peripheral blood mononuclear cells; RT: room temperature; CFU: colony forming units; CASA: computer-aided sperm analysis; H4Cit3: histone H4 citrullinated 3.

Summary Glycolate is produced in autotrophic cells under high temperatures and Ci‐limitation via oxygenation of ribulose‐1,5‐bisphosphate. In unicellular algae, glycolate is lost via excretion or metabolized via the C2 cycle by consuming reductants, ATP and CO 2 emission (photorespiration). Therefore, photorespiration is an inhibitory process for biomass production. However, cells can be manipulated in a way that they become glycolate‐producing ‘cell factories’, when the ratio carboxylation/oxygenation is 2. If under these conditions the C2 cycle is blocked, glycolate excretion becomes the only pathway of photosynthetic carbon flow. The study aims to proof the biotechnological applicability of algal‐based glycolate excretion as a new biotechnological platform. It is shown that cells of Chlamydomonas can be cultivated under specific conditions to establish a constant and long‐term stable glycolate excretion during the light phase. The cultures achieved a high efficiency of 82% of assimilated carbon transferred into glycolate biosynthesis without losses of function in cell vitality. Moreover, the glycolate accumulation in the medium is high enough to be directly used for microbial fermentation but does not show toxic effects to the glycolate‐producing cells.

Gurltia paralysans is a neglected and re-emerging metastrongyloid angio-neurotropic nematode causing severe chronic meningomyelitis in domestic cats (Felis catus) as well as in free-ranging small wild felids such as kodkods (Leopardus guigna), margays (Leopardus wiedii) and the northern tiger cat (Leopardus triginus) in South America. Within these definitive hosts (DH), adult males and females of G. paralysans parasitize the leptomeningeal veins of the subarachnoid space and/or the meningeal veins of spinal cord parenchyma, inducing vascular alterations. Feline gurltiosis has been associated with progressive thrombophlebitis of the meningeal veins, resulting in ambulatory paraparesis, paraplegia, ataxia, hindlimb proprioceptive deficit, uni- or bilateral hyperactive patellar reflexes, faecal and urinary incontinence, and tail paralysis. The complete life cycle of G. paralysans has not been elucidated yet, but most probably involves gastropods as obligate intermediate hosts (IH). In terms of epidemiology, G. paralysans infections in domestic and wild felids are scattered around various South American countries, with hyperendemic areas in southern parts of Chile. Etiological diagnosis of G. paralysans still represents a challenge for clinicians due to a lack of evidence of the excretion of either eggs or larvae in faeces or in other body fluids. Diagnosis is based on clinical neurological signs, imaging findings through computed tomography (CT), myelography, magnetic resonance imaging (MRI), and post mortem examination. Nonetheless, novel diagnostic tools have been developed, including semi-nested PCR for detecting circulating G. paralysans DNA in the cerebrospinal fluid, serum and blood samples as well as in serological diagnostic kits detecting parasite-derived antigens, but these need validation for routine usage. The hypothetical life cycle of G. paralysans is addressed in this article, including the exogenous stages (i.e., eggs, and first- (L1), second- (L2) and third-stage (L3) larvae) and obligate gastropod IH and/or paratenic hosts (PH), and we propose possible anatomical migration routes of infective L3 that reach the leptomeningeal veins in vivo. Finally, the pro-inflammatory endothelium- and leukocyte-derived innate immune reactions of the host against G. paralysans, which most likely result in thrombophlebitis and meningomyelitis, are briefly touched on.

Canine angiostrongylosis is a gastropod-borne parasitosis caused by the cosmopolitan nematode Angiostrongylus vasorum. It is associated with cardiorespiratory disorders and eventually fatal coagulopathies. A 6-months-old female dog previously diagnosed with bronchopneumonia and pneumothorax, treated accordingly without success, was hospitalized for further treatment. Complementary diagnostics confirmed previous findings plus pneumomediastinum, thoracic exudate, leucocytosis, thrombocytosis, hypercoagulability, and hyperglobulinemia. Coprological analysis showed a severe patent A. vasorum infection. After the third daily fenbendazole administration, all A. vasorum larvae were found dead in follow-up coprological analyses while in intensive care. Upon release, the dog was monitored until full clinical recovery 3 months later. Up to date, A. vasorum infection-related pneumothorax was rarely described, particularly with full recovery of such a young patient. Thus, a parasitological aetiology should be considered for dogs with severe bronchopneumonia or pneumothorax, especially in Europe and South America where increasing numbers of canine angiostrongylosis cases call for enhanced awareness of veterinary practitioners.

Simple Summary Angiostrongylus vasorum is a cardiopulmonary nematode that affects canids, residing in the pulmonary artery and right atrium/ventricle. Due to its location, the parasite will have a close interaction with the different components of the innate immune system, including endothelial cells and polymorphonuclear neutrophils (PMN). Here we evaluated the expression of adhesion molecules of canine aortic endothelial cells (CAEC), and NETs formation by co-culture of freshly isolated canine PMN with A. vasorum L3. Overall, we found distinct inter-donor variations in adhesion molecule expression among CAEC isolates. Additionally, PMN and A. vasorum co-culture induced NETs release without affecting larval viability. Abstract Due to its localization in the canine blood stream, Angiostrongylus vasorum is exposed to circulating polymorphonuclear neutrophils (PMN) and the endothelial cells of vessels. NETs release of canine PMN exposed to A. vasorum infective stages (third stage larvae, L3) and early pro-inflammatory immune reactions of primary canine aortic endothelial cells (CAEC) stimulated with A. vasorum L3-derived soluble antigens (AvAg) were analyzed. Expression profiles of the pro-inflammatory adhesion molecules ICAM-1, VCAM-1, P-selectin and E-selectin were analyzed in AvAg-stimulated CAEC. Immunofluorescence analyses demonstrated that motile A. vasorum L3 triggered different NETs phenotypes, with spread NETs (sprNETs) as the most abundant. Scanning electron microscopy confirmed that the co-culture of canine PMN with A. vasorum L3 resulted in significant larval entanglement. Distinct inter-donor variations of P-selectin, E-selectin, ICAM-1 and VCAM-1 gene transcription and protein expression were observed in CAEC isolates which might contribute to the high individual variability of pathological findings in severe canine angiostrongylosis. Even though canine NETs did not result in larval killing, the entanglement of L3 might facilitate further leukocyte attraction to their surface. Since NETs have already been documented as involved in both thrombosis and endothelium damage events, we speculate that A. vasorum-triggered NETs might play a critical role in disease outcome in vivo.

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.