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.