Eimeria ninakohlyakimovae casts NOX-independent NETosis and induces enhanced IL-12, TNF-α, IL-6, CCL2 and iNOS gene transcription in caprine PMN.

Eimeria ninakohlyakimovae represents a highly pathogenic coccidian parasite causing severe haemorrhagic typhlocolitis in goat kids worldwide. NETosis was recently described as an efficient defense mechanism of polymorphonuclear neutrophils (PMN) acting against different parasites in vitro and in vivo. In vitro interactions of caprine PMN with parasitic stages of E. ninakohlyakimovae (i. e. oocysts and sporozoites) as well as soluble oocyst antigens (SOA) were analyzed at different ratios, concentrations and time spans. Extracellular DNA staining was used to illustrate classical molecules induced during caprine NETosis [i. e. histones (H3) and neutrophil elastase (NE)] via antibody-based immunofluorescence analyses. Functional inhibitor treatments with DPI and DNase I were applied to unveil role of NADPH oxidase (NOX) and characterize DNA-backbone composition of E. ninakohlyakimovae-triggered caprine NETosis. Scanning electron microscopy (SEM)- and immunofluorescence-analyses demonstrated that caprine PMN underwent NETosis upon contact with sporozoites and oocysts of E. ninakohlyakimovae, ensnaring filaments which firmly entrapped parasites. Detailed co-localization studies of E. ninakohlyakimovae-induced caprine NETosis revealed presence of PMN-derived DNA being adorned with nuclear H3 and NE corroborating molecular characteristics of NETosis. E. ninakohlyakoimovae-induced caprine NETosis was found to be NOX-independent since DPI inhibition led to a slight decrease of NETosis. Exposure of caprine PMN to vital E. ninakohlyakimovae sporozoites as well as SOA resulted in up-regulation of IL-12, TNF-α, IL-6, CCL2 and iNOS gene transcription in stimulated PMN. Since vital E. ninakohlyakimovae-sporozoites induced caprine NETosis, this effective entrapment mechanism might reduce initial sporozoite epithelial host cell invasion during goat coccidiosis ultimately resulting in less macromeront formation and reduced merozoites I production.

Occurrence of anthropozoonotic parasitic infections and faecal microbes in free-ranging sperm whales (Physeter macrocephalus) from the Mediterranean Sea.

Sperm whales (Physeter macrocephalus) are the largest toothed whales and only living member of family Physeteridae. Present survey represents first report on cultivable faecal microbes and gastrointestinal helminths and protozoans infecting free-ranging sperm whales inhabiting Mediterranean Sea waters surrounding Balearic Archipelago, Spain. Twenty-five individual sperm whale scat samples, including one calf, were collected without disturbance of animals during the summer of 2016. Parasitological diagnostic methods, such as sodium acetate acetic formalin (SAF) method, carbol fuchsin-stained faecal smears, Giardia/Cryptosporidium coproantigen ELISAs and an Anisakis-specific PCR were applied for further identification. Five bacterial genera, i.e. Acinetobacter, Clostridium, Enterococcus, Staphylococcus and Streptococcus, and one fungus namely Cladosporium were identified. Parasitological infections included seven different parasite species with some of them bearing anthropozoonotic potential. Thus, four of these parasites were zoonotic, i.e. Anisakis, Balantidium, Diphyllobothriidae gen. sp. and Giardia. Additionally, Zalophotrema curilensis eggs, spirurid-like eggs and Cystoisospora-like oocysts were identified. Molecular characterization identified Anisakis physeteris as the species infecting these whales. This survey provides first records on occurrence of two zoonotic enteropathogenic protozoan parasites (Giardia and Balantidium) and of facultative pathogenic bacteria (Clostridium and Enterococcus) in sperm whales. Presented data should be considered as a baseline study for future monitoring surveys on anthropozoonotic pathogens affecting free-living sperm whale populations and enhance investigations on possible impact on public health as well as on isolated Mediterranean sperm whale subpopulation.

Parasite prevalence in Mycobacterium spp. infected dairy goats in the region of Murcia (south-east Spain).

Eighty-four Murciano-Granadina dairy goats slaughtered in the region of Murcia (South-East Spain) were necropsied to evaluate parasitic infections. The majority of the animals (94.0%) were parasitised and multiple infections were present. Twenty-one parasite species were found, including 18 nematode species (Muellerius capillaris, Dictyocaulus filaria, Neostrongylus linearis, Cystocaulus ocreatus, Teladorsagia circumcincta, T. occidentalis, T. trifurcata, Marshallagia marshalli, Camelostrongylus mentulatus, Trichostrongylus capricola, Nematodirus abnormalis, N. filicollis, N. spathiger, T. vitrinus, T. colubriformis, Trichuris spp., Chabertia ovina and Skrjabinema ovis); one trematode species (Dicrocoelium dendriticum); one arthropod species (Oestrus ovis); and one protozoa genus (Eimeria spp.). Additionally, 17.85% of the animals were Mycobacterium spp. positive. Therefore, comparison between parasite prevalence, intensity and abundance in tuberculosis-positive and -negative animals was performed. Statistically significant differences between the prevalence of lungworms and gastrointestinal nematodes in Mycobacterium spp. infected and free goats were found. The paper discusses this co-infection between Mycobacteriaceae and endoparasites.

Une étude a été conduite sur 84 chèvres laitières de race Murciano­Granadina abattues dans la région de Murcia (sud-est de l'Espagne) afin d'évaluer la présence d'infections parasitaires. Dans leur grande majorité (94,0 %), les chèvres étaient infestées de parasites et le nombre de pluri-infections était important. Au total, 21 espèces de parasites ont été observées, dont 18 espèces de nématodes (Muellerius capillaris, Dictyocaulus filaria, Neostrongylus linearis, Cystocaulus ocreatus, Teladorsagia circumcincta, T. occidentalis, T. trifurcata, Marshallagia marshalli, Camelostrongylus mentulatus, Trichostrongylus capricola, Nematodirus abnormalis, N. filicollis, N. spathiger, T. vitrinus, T. colubriformis, Trichuris spp., Chabertia ovina et Skrjabinema ovis), une espèce de trématode (Dicrocoelium dendriticum), une espèce d'arthropode (Oestrus ovis) et des membres d'un genre de protozoaire (Eimeria spp.). En outre, la présence de Mycobacterium spp. a été détectée chez 17,85 % des chèvres examinées. Par conséquent, une étude comparative a été réalisée pour déterminer la prévalence, l'intensité et l'abondance des parasites chez les animaux ayant donné des résultats respectivement positifs et négatifs à la détection de la tuberculose. Des différences significatives au plan statistique ont été constatées entre les chèvres infectées et indemnes de Mycobacterium spp., respectivement, pour ce qui concerne la prévalence des vers pulmonaires et des nématodes gastro-intestinaux. Les auteurs font le point sur cette co-infection par des mycobactéries et des endoparasites.

Tras ser sacrificadas, ochenta y cuatro cabras lecheras murciano­granadinas de la región de Murcia (sudeste de España) fueron sometidas a necropsia para determinar la presencia de eventuales parasitosis. La mayoría de los animales (un 94,0%) presentaban parásitos y múltiples infecciones. Se descubrieron veintiuna especies de parásito: 18 especies de nemátodo (Muellerius capillaris, Dictyocaulus filaria, Neostrongylus linearis, Cystocaulus ocreatus, Teladorsagia circumcincta, T. occidentalis, T. trifurcata, Marshallagia marshalli, Camelostrongylus mentulatus, Trichostrongylus capricola, Nematodirus abnormalis, N. filicollis, N. spathiger, T. vitrinus, T. colubriformis, Trichuris spp., Chabertia ovina y Skrjabinema ovis); una especie de tremátodo (Dicrocoelium dendriticum); una especie de artrópodo (Oestrus ovis) y un género de protozoo (Eimeria spp.). Además, el 17,85% de los ejemplares eran positivos para Mycobacterium spp. A partir de ahí se procedió a comparar la prevalencia, intensidad y abundancia de parásitos en los animales positivos para la tuberculosis con respecto a los negativos. Se observaron diferencias estadísticamente significativas entre las cabras que estaban infectadas por Mycobacterium spp. y las que no lo estaban en cuanto a la prevalencia de nemátodos pulmonares y gastrointestinales. Los autores examinan esta infección concomitante entre micobacteriáceas y endoparásitos.

Eimeria ninakohlyakimovae induces NADPH oxidase-dependent monocyte extracellular trap formation and upregulates IL-12 and TNF-α, IL-6 and CCL2 gene transcription.

Extracellular trap (ET) formation has been demonstrated as novel effector mechanism against diverse pathogens in polymorphonuclear neutrophils (PMN), eosinophils, mast cells, macrophages and recently also in monocytes. In the current study, we show that E. ninakohlyakimovae triggers the deliverance of monocyte-derived ETs in vitro. Fluorescence illustrations as well as scanning electron microscopy (SEM) analyses showed that monocyte-derived ET formation was rapidly induced upon exposure to viable sporozoites, sporocysts and oocysts of E. ninakohlyakimovae. Classical features of monocyte-released ETs were confirmed by the co-localization of extracellular DNA adorned with myeloperoxidase (MPO) and histones (H3) in parasite-entrapping structures. The treatment of caprine monocyte ET structures with NADPH oxidase inhibitor diphenylene iodondium (DPI) significantly reduced ETosis confirming the essential role of reactive oxygen species (ROS) in monocyte mediated ETs formation. Additionally, co-culture of monocytes with viable sporozoites and soluble oocyst antigen (SOA) induced distinct levels of cytokine and chemokine gene transcription. Thus, the transcription of genes encoding for IL-12 and TNF-α was significantly upregulated after sporozoite encounter. In contrast IL-6 and CCL2 gene transcripts were rather weakly induced by parasites. Conversely, SOA only induced the up-regulation of IL-6 and CCL2 gene transcription, and failed to enhance transcripts of IL-12 and TNF-α in vitro. We here report on monocyte-triggered ETs as novel effector mechanism against E. ninakohlyakimovae. Our results strongly suggest that monocyte-mediated innate immune reactions might play an important role in early host immune reactions against E. ninakohlyakimovae in goats.

Metabolic signatures of Besnoitia besnoiti-infected endothelial host cells and blockage of key metabolic pathways indicate high glycolytic and glutaminolytic needs of the parasite.

Besnoitia besnoiti is an obligate intracellular and emerging coccidian parasite of cattle with a significant economic impact on cattle industry. During acute infection, fast-proliferating tachyzoites are continuously formed mainly in endothelial host cells of infected animals. Given that offspring formation is a highly energy and cell building block demanding process, the parasite needs to exploit host cellular metabolism to meet its metabolic demands. Here, we analyzed the metabolic signatures of B. besnoiti-infected endothelial host cells and aimed to influence parasite proliferation by inhibitors of specific metabolic pathways. The following inhibitors were tested: fluoro 2-deoxy-D-glucose and 2-deoxy-D-glucose (FDG, DG; inhibitors of glycolysis), 6-diazo-5-oxo-L-norleucin (DON; inhibitor of glutaminolysis), dichloroacetate (DCA; inhibitor of pyruvate dehydrogenase kinase which favorites channeling of glucose carbons into the TCA cycle) and adenosine-monophosphate (AMP; inhibitor of ribose 5-P synthesis). Overall, B. besnoiti infections of bovine endothelial cells induced a significant and infection rate-dependent increase of glucose, lactate, glutamine, glutamate, pyruvate, alanine, and serine conversion rates which together indicate a parasite-triggered up-regulation of glycolysis and glutaminolysis. Thus, addition of DON, FDG, and DG into the cultivation medium of B. besnoiti infected endothelial cells led to a dose-dependent inhibition of parasite replication (4 µM DON, 99.5 % inhibition; 2 mM FDG, 99.1 % inhibition; 2 mM DG, 93 % inhibition; and 8 mM DCA, 71.9 % inhibition). In contrast, AMP had no significant effects on total tachyzoite production up to a concentration of 20 mM. Together, these data may open new strategies for the development of therapeutics for B. besnoiti infections.

Targeting essential Eimeria ninakohlyakimovae sporozoite ligands for caprine host endothelial cell invasion with a phage display peptide library.

Eimeria ninakohlyakimovae is an important coccidian parasite of goats which causes severe diarrhoea in young animals. Specific molecules that mediate E. ninakohlyakimovae host interactions and molecular mechanisms involved in the pathogenesis are still unknown. Although strong circumstantial evidence indicates that E. ninakohlyakimovae sporozoite interactions with caprine endothelial host cells (ECs) are specific, hardly any information is available about the interacting molecules that confer host cell specificity. In this study, we describe a novel method to identify surface proteins of caprine umbilical vein endothelial cells (CUVEC) using a phage display library. After several panning rounds, we identified a number of peptides that specifically bind to the surface of CUVEC. Importantly, caprine endothelial cell peptide 2 (PCEC2) and PCEC5 selectively reduced the infection rate by E. ninakohlyakimovae sporozoites. These preliminary data give new insight for the molecular identification of ligands involved in the interaction between E. ninakohlyakimovae sporozoites and host ECs. Further studies using this phage approach might be useful to identify new potential target molecules for the development of anti-coccidial drugs or even new vaccine strategies.

Modulation of the pro-inflammatory molecules E-selectin and TNF-α gene transcription in Eimeria ninakohlyakimovae-infected primary caprine host endothelial cells.

Eimeria ninakohlyakimovae is an important coccidian parasite of goats which causes severe hemorrhagic typhlocolitis in young animals, thereby leading to high economic losses in goat industry worldwide. The first merogony of E. ninakohlyakimovae occurs within host endothelial cells (ECs) of the lacteal capillaries of the villi of the distal ileum resulting in the formation of macromeronts (up to 170 µm) within 10-12 days post-infection (p.i.) and releasing >120,000 merozoites I. The E. ninakohlyakimovae-macromeront formation within highly immunoreactive host endothelial cells (ECs) should rely on several regulatory processes to fulfill this massive replication. Here host EC-parasite interactions were investigated to determine the extent of modulation carried out by E. ninakohlyakimovae in primary caprine umbilical vein endothelial cells (CUVEC) during the first merogony. Gene transcription of the adhesion molecule E-selectin and the cytokine TNF-α were significantly enhanced in the first hours and days p.i. in E. ninakohlyakimovae-infected CUVEC. The activation of CUVEC was also demonstrated by enhanced chemokine CCL2 and cytokine GM-CSF gene transcription, whereas no differences of the eNOS gene transcription were observed in E. ninakohlyakimovae-infected CUVEC when compared to un-infected controls. The data presented here suggest that infection of caprine host ECs by E. ninakohlyakimovae results in EC activation associated with enhanced gene transcription encoding for pro-inflammatory as well as immunomodulatory molecules, which might be important for the defense against this intracellular parasite.

[Multidisciplinary health care needs of psychologically distressed cancer patients in a Comprehensive Cancer Center]. / Multidisziplinärer Versorgungsbedarf psychisch belasteter Tumorpatienten an einem Comprehensive Cancer Center.

BACKGROUND AND OBJECTIVE: Considering the prolonged life-expectancies and the resulting demands that are placed on cancer patients and their relatives, the importance of specific counseling and support services including psycho-oncology, social services, nutritional, and exercise counseling has profoundly increased. The main focus of the current study was to evaluate the multidisciplinary health care needs of emotionally distressed cancer patients whoe were treated in a Comprehensive Cancer Center. METHODS AND STUDYGROUP: 831 out-patients were evaluated with regard to their psychological distress level and their multidisciplinary health care needs for specialist services of psycho-oncology, social services, nutritional, and exercise counseling using a tablet-PC assisted screening questionnaire. Separate analyses were completed for patients with and without psychological distress. RESULTS: One third of the screened patients showed clinically relevant psychological distress. Health care needs for all specialist services were significantly greater among these patients compared to patients without psychological distress (all p-values < 0.005). The higher needs were foremost presented by the number of needed specialist services (p < 0.001): two thirds of the psychologically distressed patients demonstrated, besides the need for a psycho-oncological service, a need for two or three further specialist services, whereas among patients without psychological distress more than 70% showed a need for at most one specialist service. CONCLUSION: Multidisciplinary health care needs of psychologically distressed cancer patients should be systematically addressed in a Comprehensive Cancer Center, and patients should be offered a coordinated and integrated health care program.

Gastrointestinal parasites of free-living Indo-Pacific bottlenose dolphins (Tursiops aduncus) in the Northern Red Sea, Egypt.

The present study represents the first report on the gastrointestinal parasite fauna infecting the free-living and alive Indo-Pacific bottlenose dolphins (Tursiops aduncus) inhabiting waters of the Red Sea at Hurghada, Egypt. A total of 94 individual faecal samples of the examined bottlenose dolphins were collected during several diving expeditions within their natural habitats. Using classical parasitological techniques, such as sodium acetate acetic acid formalin method, carbol fuchsin-stained faecal smears, coproantigen ELISA, PCR and macroscopical analyses, the study revealed infections with 21 different parasite species belonging to protozoans and metazoans with some of them bearing zoonotic and/or pathogenic potential. Four identified parasite species are potential zoonotic species (Giardia spp., Cryptosporidium spp., Diphyllobothrium spp., Ascaridida indet.); three of them are known to have high pathogenic potential for the examined dolphin species (Nasitrema attenuata, Zalophotrema spp. and Pholeter gastrophilus) and some appear to be directly associated with stranding events. In detail, the study indicates stages of ten protozoan species (Giardia spp., Sarcocystis spp., Isospora (like) spp., Cystoisospora (like) spp., Ciliata indet. I and II, Holotricha indet., Dinoflagellata indet., Hexamita (like) spp., Cryptosporidium spp.), seven trematode species (N. attenuata, Nasitrema spp. I and II, Zalophotrema curilensis, Zalophotrema spp., Pholeter gastrophilus, Trematoda indet.), one cestode species (Diphyllobothrium spp.), two nematode species (Ascaridida indet, Capillaria spp.) and one crustacean parasite (Cymothoidae indet.). Additionally, we molecularly identified adult worms of Anisakis typica in individual dolphin vomitus samples by molecular analyses. A. typica is a common parasite of various dolphin species of warmer temperate and tropical waters and has not been attributed as food-borne parasitic zoonoses so far. Overall, these parasitological findings include ten new host records for T. aduncus (i.e. in case of Giardia spp., Sarcocystis spp., Cryptosporidium spp., Nasitrema spp., Zalophotrema spp., Pholeter gastrophilus, A. typica, Capillaria spp., Diphyllobothrium spp. and Cymothoidae indet.). The present results may be used as a baseline for future monitoring studies targeting the impact of climate or other environmental changes on dolphin's health conditions and therefore contribute to the protection of these fascinating marine mammals.

A novel type of silver nanoparticles and their advantages in toxicity testing in cell culture systems.

Silver nanoparticles (SNPs) are among the most commercialized nanoparticles worldwide. Often SNP are used because of their antibacterial properties. Besides that they possess unique optic and catalytic features, making them highly interesting for the creation of novel and advanced functional materials. Despite its widespread use only little data exist in terms of possible adverse effects of SNP on human health. Conventional synthesis routes usually yield products of varying quality and property. It thus may become puzzling to compare biological data from different studies due to the great variety in sizes, coatings or shapes of the particles applied. Here, we applied a novel synthesis approach to obtain SNP of well-defined colloidal and structural properties. Being stabilized by a covalently linked small peptide, these particles are nicely homogenous, with narrow size distribution, and form monodisperse suspensions in aqueous solutions. We applied these peptide-coated SNP in two different sizes of 20 or 40 nm (Ag20Pep and Ag40Pep) and analyzed responses of THP-1-derived human macrophages while being exposed against these particles. Gold nanoparticles of similar size and coating (Au20Pep) were used for comparison. The cytotoxicity of particles was assessed by WST-1 and LDH assays, and the uptake into the cells was confirmed via transmission electron microscopy. In summary, our data demonstrate that this novel type of SNP is well suited to serve as model system for nanoparticles to be tested in toxicological studies in vitro.

Bovine recombinant IFNgamma induces endothelial cell gene transcription of immunoregulatory molecules and upregulates PMN and PBMC adhesion on bovine endothelial cells.

Interferon gamma (IFNgamma) is an important modulator of immune responses acting on multiple cell types, such as lymphocytes, macrophages or endothelial cells. We investigated the effects of recombinant bovine IFNgamma on bovine umbilical vein endothelial cells (BUVEC) for the level of polymorphonuclear neutrophil cell (PMN)- and peripheral blood mononuclear cell (PBMC)-adhesion as well as the gene transcription of endothelial cell-derived adhesion molecules (E-selectin, P-selectin, VCAM-1, ICAM-1), chemokines (CXCL1, CXCL8, CXCL10, CCL2, CCL5), GM-CSF, iNOS and COX-2 in comparison to TNFalpha-stimulation. IFNgamma strongly induced PMN and PBMC adhesion on BUVEC involving CD4(+), CD8(+) and gammadelta-TCR(+) (WC1(+)) lymphocytes. Furthermore, IFNgamma-stimulation led to a strong upregulation in the transcription of VCAM-1, ICAM-1, CXCL10 and CCL2 genes and to a low to moderate increase in the E- and P-selectin, CXCL1, CXCL8, CCL5, COX-2 and iNOS gene transcripts, but failed to enhance GM-CSF gene transcription. These results indicate that IFNgamma can be considered an important activator of endothelial cells in the bovine system, most probably by influencing the outcome of inflammatory responses through selective upregulation of immunoregulatory molecules.

Estrogen receptor beta (ERbeta) is expressed differently from ERalpha in bovine placentomes.

The functions of placental estrogens in cattle are still poorly understood. In order to investigate a putative role as local regulators of placental growth, differentiation and functions via estrogen receptor beta (ERbeta), the expression of this ER isoform was determined in bovine placentomes obtained from cows at midgestation (days 110-150; n=7), late gestation (days 180-280; n=9), from prepartal cows (sampling immediately after the prepartal decline in maternal progesterone blood levels became obvious; n=3) and from cows at normal term (n=5) on the protein and mRNA level. By means of immunohistochemistry using a monoclonal antibody against human ERbeta (clone PPG5/10), nuclear signals were found in numerous cell types of the fetal (cotyledon) and maternal (caruncle) component of the placentomes with highest intensities in mature trophoblast giant cells (TGC), fetal and maternal vascular cells and caruncular stromal cells, in which the percentage of positive cells increased from 58.9+/-2.3 and 59.1+/-3.3 at mid- and late gestation to 65.1+/-4.9 and 69.4+/-4.2 in prepartal and parturient cows, resp. (p<0.0001). Clear staining of uninucleated trophoblast cells was only observed in prepartal and parturient animals. Conventional RT-PCR confirmed ERbeta expression in caruncles and cotyledons at all phases under investigation. With semi-quantitative real-time RT-PCR higher levels of ERbeta-specific mRNA were measured in cotyledons compared to caruncles (p<0.01). The results show that in contrast to ERalpha, which is only expressed in the caruncle, ERbeta is also extensively expressed in the cotyledon and suggest a role of ERbeta primarily in TGC differentiation and placental angiogenesis and/or vascular functions.

Molecular characterization of a Litomosoides sigmodontis protein involved in the development of the microfilarial sheath during embryogenesis.

A cDNA clone Ls110 was isolated from a female Litomosoides sigmodontis expression library using an antiserum raised against the microfilarial sheath. The complete cDNA encodes a protein (Ls110) of 382 amino acids. Southern and PCR analyses revealed the presence of Ls110 in L. sigmodontis as a single copy gene. The transcription of the Ls110 gene was limited to female worms. In these worms the transcription was confined to the epithelial cells of the uterus. The protein Ls110 was detected not only in the epithelial layer of the uterus but also secreted in the lumen of the uterus. All the intra-uterine embryonic stages showed the protein bound to their egg shell/sheath, except the early multicellular embryonic stages and fully developed microfilariae. The transient occurrence of Ls110 on these structures of intra-uterine stages besides the presence of a cysteine-rich N-terminal region (SXC-like domain) suggest that the protein may play a role in the formation of the microfilarial sheath during embryogenesis.