Besnoitia besnoiti-induced neutrophil clustering and neutrophil extracellular trap formation depend on P2X1 purinergic receptor signaling.

Bovine besnoitiosis is a re-emerging cattle disease caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti. Neutrophil extracellular trap (NET) formation represents an efficient innate immune mechanism of polymorphonuclear neutrophils (PMN) against apicomplexan parasites, including B. besnoiti. PMN purinergic signaling was proposed as a critical factor for NET formation. One important purinergic ligand is ATP, which is recognized as a danger signal and released into the extracellular space acting as an autocrine/paracrine signaling molecule. ATP-driven effects on PMN via the nucleotide P2 receptor family include chemotaxis, reactive oxygen species (ROS) production, and NET formation. So far, data on both PMN ATP concentrations and the role of ATP as a key modulator of purinergic signaling in B. besnoiti tachyzoite-triggered bovine NETosis is scarce. Current data showed that B. besnoiti tachyzoite exposure to bovine PMN neither changed total PMN ATP nor extracellular ATP quantities even though it significantly triggered NET formation. Moreover, B. besnoiti tachyzoite-exposed PMN revealed enhanced oxygen consumption rates (OCR) as quantified by the Seahorse metabolic analyzer. Exogenous supplementation of ATP or non-hydrolizable ATP (ATPγS) led to increased extracellular acidification rates (ECAR) but failed to alter tachyzoite-induced oxidative responses (OCR) in exposed PMN. In addition, exogenous supplementation of ATPγS, but not of ATP, boosted B. besnoiti tachyzoite-induced anchored NET formation. Referring to purinergic signaling, B. besnoiti tachyzoite-triggered anchored NET formation revealed P2X1 purinergic as receptor-dependent since it was blocked by the P2X1 inhibitor NF449 at an IC50 of 1.27 µM. In contrast, antagonists of P2Y2, P2Y6, P2X4, and P2X7 purinergic receptors all failed to affect parasite-driven NETosis. As an interesting finding, we additionally observed that B. besnoiti tachyzoite exposure induced PMN clustering in a P2X1-dependent manner. Thus, we identified P2X1 purinergic receptor as a pivotal molecule for both B. besnoiti tachyzoite-induced PMN clustering and anchored NET formation.

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

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

Novel 3D in situ visualization of seal heartworm (Acanthocheilonema spirocauda) larvae in the seal louse (Echinophthirius horridus) by X-ray microCT.

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.

Glycolysis, monocarboxylate transport, and purinergic signaling are key events in Eimeria bovis-induced NETosis.

The protozoan parasite Eimeria bovis is 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 by E. bovis stages. So far, the metabolic requirements of E. 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) in E. bovis sporozoite-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 studied via chemical 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 diminished via PMN 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 in E. bovis sporozoite-induced NETosis.

Octaarginine Improves the Efficacy of Nitazoxanide against Cryptosporidium parvum.

Cryptosporidiosis is an intestinal disease that affects a variety of hosts including animals and humans. Since no vaccines exist against the disease till date, drug treatment is the mainstay of disease control. Nitazoxanide (NTZ) is the only FDA-approved drug for the treatment of human cryptosporidiosis. However, its efficacy in immunocompromised people such as those with AIDS, in malnourished children, or those with concomitant cryptosporidiosis is limited. In the absence of effective drugs against cryptosporidiosis, improving the efficacy of existing drugs may offer an attractive alternative. In the present work, we have assessed the potential of the cell-penetrating peptide (CPP) octaarginine (R8) to increase the uptake of NTZ. Octaarginine (R8) was synthetically attached to NTZ in an enzymatically releasable manner and used to inhibit growth of Cryptosporidium parvum in an in vitro culture system using human ileocecal adenocarcinoma (HCT-8) cell line. We observed a significant concentration-dependent increase in drug efficacy. We conclude that coupling of octaarginine to NTZ is beneficial for drug activity and it represents an attractive strategy to widen the repertoire of anti-cryptosporidial therapeutics. Further investigations such as in vivo studies with the conjugate drug will help to further characterize this strategy for the treatment of cryptosporidiosis.

New Insights into Gastrointestinal and Pulmonary Parasitofauna of Wild Eurasian lynx (Lynx lynx) in the Harz Mountains of Germany.

The Eurasian lynx (Lynx lynx) represents an endangered wild felid species. In Germany, it currently occurs in three isolated populations in and around the Harz Mountains, the Palatinate Forest and the Bavarian Forest. Lynx parasitic infections affect animal health and might have an influence on population performance. Therefore, we investigated the protozoan and helminth fauna of free-ranging Eurasian lynx of the Harz population with emphasis on zoonotic parasites. Individual scat samples (n = 24) were collected from wild animals between 2019 and 2021 in the Harz National Park and surrounding areas. In total, 15 taxa of endoparasites were detected, including seven nematodes (i.e., Aelurostrongylus abstrusus, Angiostrongylus spp., Uncinaria stenocephala, Toxascaris leonina, Toxocara cati, Cylicospirura spp. and Capillaria spp.), one cestode (Diphyllobothriidae) and one trematode (Heterophylidae) as well as six protozoans (i.e., Cystoisospora rivolta, Cystoisospora felis, Toxoplasma gondii/Hammondia spp., Sarcocystis spp., Giardia intestinalis and Cryptosporidium spp.). Moreover, first-stage larvae (L1) of spurious lungworm, Protostrongylus pulmonalis, originating from lagomorph preys were identified. This work represents the first report on patent A. abstrusus and Angiostrongylus spp. infections in wild German Eurasian lynxes. Some of the identified parasites represent relevant pathogens for lynxes, circulating between these carnivorous definitive hosts and a variety of mammalian and invertebrate intermediate hosts, e.g., Sarcocystis spp., T. gondii/Hammondia spp., T. cati, T. leonina, A. abstrusus and Angiostrongylus spp., while others are considered exclusively pathogenic for wild felids (e.g., Cylicospirura spp., C. rivolta, C. felis). This study provides insights in the occurrence of zooanthroponotically relevant metazoan (i.e., T. cati and U. stenocephala) and protozoan (i.e., G. intestinalis) species in free-ranging lynx. The present work should be considered as a baseline study for future monitoring surveys on endoparasites circulating in wild Eurasian lynx for appropriate management practices in lynx conservation strategies in Europe.

Intestinal Parasites of Neotropical Wild Jaguars, Pumas, Ocelots, and Jaguarundis in Colombia: Old Friends Brought Back from Oblivion and New Insights.

Neotropical wild felids (NWF) are obligate carnivore species present in Central and South America, and some are considered endangered due to constantly decreasing populations. NWF can become infected by a wide range of protozoan and metazoan parasites, some of them affecting their health conditions and others having anthropozoonotic relevance. Parasitological studies on NWF are still very scarce, and most data originated from dead or captive animals. On this account, the current study aimed to characterize gastrointestinal parasites of free-ranging jaguars (Panthera onca), pumas (Puma concolor), ocelots (Leopardus pardalis), and jaguarundis (Herpailurus yagouaroundi), i.e., four out of six NWF species endemic to Colombia. Fecal samples from jaguars (n = 10) and ocelots (n = 4) were collected between 2012 and 2017 as part of the Jaguar Corridor Initiative from six geographic locations in Colombia. In addition, cestode specimens were obtained during puma and jaguarundi necropsies. Scat samples were processed by standardized sodium acetate-acetic acid-formalin (SAF), sedimentation, and flotation techniques and by carbol fuchsin-stained fecal smears. Morphological evaluation of feces showed the presence of one cestode (Spirometra sp.), a nematode (Toxocara cati), an acanthocephalan (Oncicola sp.), and one cyst-forming coccidian (Cystoisospora-like oocysts). Feces oocysts were submitted to a Toxoplasma gondii-specific PCR for species identification, but no product was amplified. The cestodes isolated from a puma and jaguarundi were molecularly characterized by sequencing cytochrome c oxidase subunit I, identifying them as Taenia omissa and as a T. omissa sister lineage, respectively. These results collectively demonstrate the potential role of NWF as natural reservoir hosts for neglected zoonotic parasites (e.g., Spirometra sp., T. cati) and highlight their possible role in parasite transmission to human communities. Due to public health concerns, the occurrence of these parasites should be monitored in the future for appropriate zoonotic management practices in conservation strategies and wild felid health management programs.

The seal louse (Echinophthirius horridus) in the Dutch Wadden Sea: investigation of vector-borne pathogens.

BACKGROUND: Belonging to the anopluran family Echinophthiriidae, Echinophthirius horridus, the seal louse, has been reported to parasitise a broad range of representatives of phocid seals. So far, only a few studies have focused on the vector function of echinophthiriid lice, and knowledge about their role in pathogen transmission is still scarce. The current study aims to investigate the possible vector role of E. horridus parasitising seals in the Dutch Wadden Sea. METHODS: E. horridus seal lice were collected from 54 harbour seals (Phoca vitulina) and one grey seal (Halichoerus grypus) during their rehabilitation period at the Sealcentre Pieterburen, The Netherlands. DNA was extracted from pooled seal lice of individual seals for molecular detection of the seal heartworm Acanthocheilonema spirocauda, the rickettsial intracellular bacterium Anaplasma phagocytophilum, and the cell wall-less bacteria Mycoplasma spp. using PCR assays. RESULTS: Seal lice from 35% of the harbour seals (19/54) and from the grey seal proved positive for A. spirocauda. The seal heartworm was molecularly characterised and phylogenetically analysed (rDNA, cox1). A nested PCR was developed for the cox1 gene to detect A. spirocauda stages in seal lice. A. phagocytophilum and a Mycoplasma species previously identified from a patient with disseminated 'seal finger' mycoplasmosis were detected for the first time, to our knowledge, in seal lice. CONCLUSIONS: Our findings support the potential vector role of seal lice in the transmission of A. spirocauda and reveal new insights into the spectrum of pathogens occurring in seal lice. Studies on vector competence of E. horridus, especially for bacterial pathogens, are essentially needed in the future as these pathogens might have detrimental effects on the health of seal populations. Furthermore, studies on the vector role of different echinophthiriid species infecting a wide range of pinniped hosts should be conducted to extend the knowledge of vector-borne pathogens.

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

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

Mitochondria-derived ATP participates in the formation of neutrophil extracellular traps induced by platelet-activating factor through purinergic signaling in cows.

Neutrophil extracellular trap (NET) formation eliminates/prevents the spread of infectious agents. Platelet activating factor (PAF) is involved in infectious diseases of cattle because it recruits and activates neutrophils. However, its ability to induce NET release and the role of metabolism in this process is not known. We investigated if inhibition of glycolysis, mitochondrial-derived adenosine triphosphate (ATP) synthesis and purinergic signaling though P2X1 purinoceptors interfered with NET formation induced by PAF. We inhibited bovine neutrophils with 2-deoxy-d-glucose, rotenone, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and NF449 to evaluate PAF-mediated NET extrusion. PAF induced mitochondrial hyperpolarization and triggered extracellular ATP release via pannexin-1. Inhibition of mitochondrial metabolism prevented extracellular ATP release. Inhibition of glycolysis, complex-I activity and oxidative phosphorylation prevented NET formation induced by PAF. Inhibition of P2X1 purinergic receptors inhibited mitochondrial hyperpolarization and NET formation. We concluded that PAF-induced NET release is dependent upon glycolysis, mitochondrial ATP synthesis and purinergic signaling.

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

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

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

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

Antarctophthirus microchir infestation in synanthropic South American sea lion (Otaria flavescens) males diagnosed by a novel non-invasive method.

Antarctophthirus microchir is a sucking louse species belonging to the family Echinophthiriidae and has been reported to parasitize all species of the subfamily Otariinae, the sea lions. Former studies on this ectoparasite mainly required fixation, immobilization, or death of host species and especially examinations of adult male sea lions are still very rare. Between March and May 2018, adult individuals of a unique "urban" bachelor group of South American sea lions (Otaria flavescens) living directly in the city of Valdivia, Chile, were studied regarding their ectoparasite infestation status. For first time, a non-invasive method in the form of a lice comb screwed on a telescopic rod and grounded with adhesive tape was used for sample taking process. Overall, during combing different stages of A. microchir were detected in 4/5 O. flavescens individuals, especially at the junction between the back and hind flippers. Our findings represent the first report of A. microchir infesting individuals of this synanthropic colony and fulfilling complete life cycle in a sea lion group despite inhabiting freshwater and in absence of females/pups. Our "telescopic lice comb apparatus" offers a new strategy to collect different stages of ectoparasites and a range of epidermal material, such as fur coat hair and superficial skin tissue for a broad spectrum of research fields in wildlife sciences in an unmolested and stress reduced manner.

Anticoccidial efficacy of Canary rue (Ruta pinnata) extracts against the caprine apicomplexan Eimeria ninakohlyakimovae.

Continuous use of anticoccidial treatments against Eimeria infections has resulted in the development of drug resistance. This study aimed to evaluate the anticoccidial efficacy of a methanolic extract derived from the endemic Canary rue (Ruta pinnata) plant of the Canary Islands, Spain, against Eimeria ninakohlyakimovae using in vitro assays. Freshly unsporulated oocysts were exposed to different concentrations of R. pinnata extract and thereafter evaluated for sporulation inhibition. Additionally, anticoccidial activity was examined by testing the viability of the E. ninakohlyakimovae sporozoites and their ability to infect bovine colonic epithelial cells after incubation with different concentrations of R. pinnata plant extract. The inhibition of oocyst sporulation by the extract was both time and concentration dependent, with certain combinations affording the same levels of sporulation inhibition as formaldehyde used as positive control (P < 0.001). Moreover, concentrations >0.1 mg/mL also affected not only the viability of the sporozoites but also their cell invasion capacity (P < 0.001). Altogether, these results show that methanolic fruit extracts from R. pinnata have important anticoccidial activity against oocysts and sporozoites of Eimeria. The potential efficacy of the extracts against other animal/human parasites remains to be elucidated, and further studies are needed to better understand its mode of action against coccidian parasites.

Bovine macrophage-derived extracellular traps act as early effectors against the abortive parasite Neospora caninum.

Macrophages are multipurpose phagocytes and are considered to be irreplaceable during the early host innate immune response against microbial and parasitic pathogens. However, no report has investigated the novel anti-parasitic mechanism of macrophage-derived extracellular traps (ETs) against the abortive apicomplexan parasite Neospora caninum (N. caninum) in cattle. Scanning electron microscopy (SEM) was used to visualize and characterize N. caninum tachyzoite-induced macrophage-triggered ETs in exposed bovine macrophages. Fluorescence confocal microscopy was used to confirm the classical backbone structure of DNA embedded with histone 3 (H3) and myeloperoxidase (MPO) in N. caninum tachyzoite-induced macrophage-derived ETs. Furthermore, the lactate dehydrogenase (LDH) levels in the supernatants of parasite-exposed macrophages were detected by a LDH Cytotoxicity Assay® kit. The results clearly demonstrated that N. caninum tachyzoites triggered bovine macrophage-derived ET-like structures. Inhibiting assays revealed that N. caninum tachyzoite-induced macrophage-mediated ET formation may be an ERK 1/2- and p38 MAPK-dependent cell death process. In conclusion, the present study is the first report on the formation of ETs in bovine macrophages against N. caninum tachyzoites and adds new data on the possible role of macrophages in vivo infection by capturing invasive stages and exposing them to other leukocytes.

Antiparasitic Efficacy of Curcumin Against Besnoitia besnoiti Tachyzoites in vitro.

Besnoitia besnoiti is the causative agent of bovine besnoitiosis. B. besnoiti infections lead to reduced fertility and productivity in cattle causing high economic losses, not only in Europe, but also in Asia and Africa. Mild to severe clinical signs, such as anasarca, oedema, orchitis, hyperkeratosis, and characteristic skin and mucosal cysts, are due to B. besnoiti tachyzoite and bradyzoite replication in intermediate host tissues. So far, there are no commercially available effective drugs against this parasite. Curcumin, a polyphenolic compound from Curcuma longa rhizome is well-known for its antioxidant, anti-inflammatory, immunomodulatory and also anti-protozoan effects. Hence, the objective of this study was to evaluate the effects of curcumin on viability, motility, invasive capacity, and proliferation of B. besnoiti tachyzoites replicating in primary bovine umbilical vein endothelial cells (BUVEC) in vitro. Functional inhibition assays revealed that curcumin treatments reduce tachyzoite viability and induce lethal effects in up to 57% of tachyzoites (IC50 in 5.93 µM). Referring to general motility, significant dose-dependent effects of curcumin treatments were observed. Interestingly, curcumin treatments only dampened helical gliding and twirling activities whilst longitudinal gliding motility was not significantly affected. In addition, curcumin pretreatments of tachyzoites resulted in a dose-dependent reduction of host cell invasion as detected by infections rates at 1 day p. i. These findings demonstrate feeding cattle with Curcuma longa rhizomes may represent a new strategy for besnoitiosis treatment.

Protozoan and helminth parasite fauna of free-living Croatian wild wolves (Canis lupus) analyzed by scat collection.

The European wolf (Canis lupus) is a large carnivore species present in limited areas of Europe with several small populations still being considered as endangered. Wolves can be infected by a wide range of protozoan and metazoan parasites with some of them affecting free-living wolf health condition. On this account, an epidemiological survey was conducted to analyze the actual parasite fauna in Croatian wild wolves. In total, 400 individual faecal samples were collected during field studies on wolf ecology in the years 2002-2011. Parasite stages were identified by the sodium acetate acetic acid formalin (SAF)-technique, carbolfuchsin-stained faecal smears and Giardia/Cryptosporidium coproantigen-ELISAs. A subset of taeniid eggs-positive wolf samples was additionally analyzed by PCR and subsequent sequencing to identify eggs on Echinococcus granulosus/E. multilocularis species level. In total 18 taxa of parasites were here detected. Sarcocystis spp. (19.1%) occurred most frequently in faecal samples, being followed by Capillaria spp. (16%), ancylostomatids (13.1%), Crenosoma vulpis (4.6%), Angiostrongylus vasorum (3.1%), Toxocara canis (2.8%), Hammondia/Neospora spp. (2.6 %), Cystoisospora ohioensis (2.1%), Giardia spp. (2.1%), Cystoisospora canis (1.8%), Cryptosporidium spp. (1.8%), Trichuris vulpis (1.5%), Taenia spp. (1.5%), Diphyllobothrium latum (1.5%), Strongyloides spp. (0.5%), Opisthorchis felineus (0.5%), Toxascaris leonina (0.3%), Mesocestoides litteratus (0.3%) and Alaria alata (0.3%). Some of the here identified parasites represent relevant pathogens for wolves, circulating between these carnivorous definitive hosts and a variety of mammalian intermediate hosts, e. g. Taenia spp. and Sarcocystis spp., while others are considered exclusively pathogenic for canids (e.g. A. vasorum, C. vulpis, T. vulpis, Cystoisospora spp.). This study provides first records on the occurrence of the two relevant anthropozoonotic parasites, Giardia spp. and Cryptosporidium spp., in wild wolves from Croatia.

Endoparasite survey of free-swimming baleen whales (Balaenoptera musculus, B. physalus, B. borealis) and sperm whales (Physeter macrocephalus) using non/minimally invasive methods.

A number of parasitic diseases have gained importance as neozoan opportunistic infections in the marine environment. Here, we report on the gastrointestinal endoparasite fauna of three baleen whale species and one toothed whale: blue (Balaenoptera musculus), fin (Balaenoptera physalus), and sei whales (Balaenoptera borealis) and sperm whales (Physeter macrocephalus) from the Azores Islands, Portugal. In total, 17 individual whale fecal samples [n = 10 (B. physalus); n = 4 (P. macrocephalus); n = 2 (B. musculus); n = 1 (B. borealis)] were collected from free-swimming animals as part of ongoing studies on behavioral ecology. Furthermore, skin biopsies were collected from sperm whales (n = 5) using minimally invasive biopsy darting and tested for the presence of Toxoplasma gondii, Neospora caninum, and Besnoitia besnoiti DNA via PCR. Overall, more than ten taxa were detected in whale fecal samples. Within protozoan parasites, Entamoeba spp. occurred most frequently (64.7%), followed by Giardia spp. (17.6%) and Balantidium spp. (5.9%). The most prevalent metazoan parasites were Ascaridida indet. spp. (41.2%), followed by trematodes (17.7%), acanthocephalan spp., strongyles (11.8%), Diphyllobotrium spp. (5.9%), and spirurids (5.9%). Helminths were mainly found in sperm whales, while enteric protozoan parasites were exclusively detected in baleen whales, which might be related to dietary differences. No T. gondii, N. caninum, or B. besnoiti DNA was detected in any skin sample. This is the first record on Giardia and Balantidium infections in large baleen whales.

NADPH oxidase, MPO, NE, ERK1/2, p38 MAPK and Ca2+ influx are essential for Cryptosporidium parvum-induced NET formation.

Cryptosporidium parvum causes a zoonotic infection with worldwide distribution. Besides humans, cryptosporidiosis affects a wide range of animals leading to significant economic losses due to severe enteritis in neonatal livestock. Neutrophil extracellular trap (NET) formation has been demonstrated as an important host effector mechanism of PMN acting against several invading pathogens. In the present study, C. parvum-mediated NET formation was investigated in human and bovine PMN in vitro. We here demonstrate that C. parvum sporozoites indeed trigger NET formation in a time-dependent manner. Thereby, the classical characteristics of NETs were demonstrated by co-localization of extracellular DNA with histones, neutrophil elastase (NE) and myeloperoxidase (MPO). A significant reduction of NET formation was measured following treatments of PMN with NADPH oxidase-, NE- and MPO-inhibitors, confirming the key role of these enzymes in C. parvum-induced NETs. Additionally, sporozoite-triggered NETosis revealed as dependent on intracellular Ca(++) concentration and the ERK 1/2 and p38 MAPK-mediated signaling pathway. Moreover, sporozoite-triggered NET formation led to significant parasite entrapment since 15% of the parasites were immobilized in NET structures. Consequently, PMN-pre-exposed sporozoites showed significantly reduced infectivity for epithelial host cells confirming the capability of NETs to prevent active parasite invasion. Besides NETs, we here show that C. parvum significantly up-regulated CXCL8, IL6, TNF-α and of GM-CSF gene transcription upon sporozoite confrontation, indicating a pivotal role of PMN not only in the bovine and human system but most probably in other final hosts for C. parvum.

Suitable in vitro culture of Eimeria bovis meront II stages in bovine colonic epithelial cells and parasite-induced upregulation of CXCL10 and GM-CSF gene transcription.

We here established a suitable in vitro cell culture system based on bovine colonic epithelial cells (BCEC) for the development of Eimeria bovis merozoites I and the characterization of early parasite-induced innate epithelial host cell reactions as gene transcription of proinflammatory molecules. Both primary and permanent BCEC (BCEC (rim) and BCEC(perm)) were suitable for E. bovis merozoite I invasion and subsequent development of meronts II leading to the release of viable merozoites II. E. bovis merozoite II failed to develop any further neither into gamont nor oocyst stages in BCEC in vitro. E. bovis merozoite I induced innate epithelial host cell reactions at the level of CXC/CCL chemokines (CXCL1, CXCL8, CXCL10, CCL2), IL-6, and GM-CSF gene transcription. Overall, both BCEC types were activated by merozoite I infections since they showed significantly enhanced gene transcript levels of the immunomodulatory molecules CXCL10 and GM-CSF. However, gene transcription profiles of BCEC(prim) and BCEC(perm) revealed different reaction patterns in response to merozoite I infection with regard to quality and kinetics of chemokine/cytokine gene transcription. Although both BCEC types equally showed most prominent responses for CXCL10 and GM-CSF, the induction of CXCL1, CXCL8, CCL2, and IL-6 gene transcripts varied qualitatively and quantitatively. Our results demonstrate that BCEC seem capable to respond to E. bovis merozoite I infection by the upregulation of CXCL10 and GM-CSF gene transcription and therefore probably contribute to host innate effector mechanisms against E. bovis.