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Caprine coccidiosis represents a major concern in goat industry worldwide causing high economic losses, mainly due to poor weight gains, treatment costs and even death of kids. Therefore, analyses on risk factors involved in the establishment and outcome of this disease are important to improve infection status and herd management. In Portugal, approximately 400,000 goats are reared every year. The majority of them are allocated in the southern part of the country. In the current study, a representative number of animals (382 goats) and herds (37 herds) from Alentejo were monitored. Faecal samples from five young and five adult goats of each farm were collected rectally and examined individually by using the modified McMaster technique for oocyst counts (oocyst per gram of faeces - OPG). Additionally, a questionnaire on the husbandry conditions and anti-parasitic treatments was performed. Overall, a prevalence of Eimeria infections of 99% and 100% was detected on animal and herd level, respectively. Eimeria arloingi and E. ninakohlyakimovae as the two most pathogenic caprine species also constituted the most prevalent ones (34.91% and 29.90%, respectively), both in the young and the adult goat groups. As expected, OPG vales were significantly higher (p = 0.031) in young than in adult goats. Furthermore, risk factor analyses revealed young age, higher total number of animals on the farm and outdoor kidding as strongly associated with a higher risk of caprine coccidiosis. Overall, the high prevalence of pathogenic Eimeria species on Portuguese goat farms in combination with the occurrence of related risk factors strengthen the importance of coccidiosis control in caprine industry.

Eimeria zuernii and E. bovis are host-specific apicomplexan parasites of cattle causing haemorrhagic typhlocolitis in young animals worldwide. During first merogony, both Eimeria species form giant macromeronts (>300 μm) in host endothelial cells containing >120,000 merozoites I in vivo. During the massive intracellular replication of macromeronts, large amounts of cholesterol and fatty acids are indispensable for enormous merozoite I-derived membrane production. From a metabolic perspective, host endothelial cells might be of advantage to the parasite, as transcription of several genes involved in both, cholesterol de novo biosynthesis and low density lipoprotein (LDL)-mediated uptake, are up-regulated in Eimeria macromeront-carrying host endothelial cells. In order to analyse further influence of E. zuernii/E. bovis infections on the metabolism of cholesterol, fatty acids, and glycolysis of the host endothelial cells, suitable in vitro cell culture systems are necessary. So far, in vitro cell culture systems based on primary bovine umbilical vein endothelial cells (BUVEC) are available for E. bovis-macromeront I formation, but have not been evaluated for E. zuernii. A novel E. zuernii (strain A), initially isolated from naturally infected calves in Antioquia, Colombia, was used for sporozoite isolation. Primary BUVEC monolayers were concomitantly infected with E. zuernii- and E. bovis-sporozoites, resulting in large sized macromeronts whose morphological/morphometric characteristics were compared. BUVEC carrying E. zuernii-macromeronts resulted in the release of viable and highly motile merozoites I. Overall, E. zuernii-merozoites I differed morphologically from those of E. bovis. The new E. zuernii (strain A) will allow detailed in vitro investigations not only on the modulation of cellular cholesterol processing (i. e. cholesterol-25-hydroxylase and sterol O-acyltransferase) but also on the surface expression of LDL receptors during macromeront formation.

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 0.1 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.

Fatty and hydroxycarboxylic acids are one of the main intermediates of energy metabolism in ruminants and critical in the milk production of cattle. High production demands on a dairy farm can induce nutritional imbalances and metabolism disorders, which have been widely associated with the onset of sterile inflammatory processes and increased susceptibility to infections. The literature suggests that short-chain fatty acids (SCFA), long-chain fatty acids (LCFA) and hydroxycarboxylic acids are relevant modulators of the host innate inflammatory response. For instance, increased SCFA and lactate levels are associated with subacute ruminal acidosis (SARA) and the activation of pro-inflammatory processes mediated by diverse leukocyte and vascular endothelial cells. As such, free LCFA and the ketone body β-hydroxybutyrate are significantly increased in the plasma 1-2 weeks postpartum, coinciding with the time period in which cows are more susceptible to acquiring infectious diseases that the host innate immune system should actively oppose. Today, many of these pro-inflammatory responses can be related to the activation of specific G protein-coupled receptors, including GPR41/FFA3 and GPR43/FFA2 for SCFA; GPR40/FFA1 and GPR120/FFA4 for LCFA, GPR109A/HCA2 for ketone body β-hydroxybutyrate, and GPR81/HCA1 for lactate, all expressed in different bovine tissues. The activation of these receptors modulates the release of intracellular granules [e.g., metalloproteinase-9 (MMP-9) and lactoferrin], radical oxygen species (ROS) production, chemotaxis, and the production of relevant pro-inflammatory mediators. The article aimed to review the role of natural ligands and receptors and the resulting impact on the host innate immune reaction of cattle and, further, to address the most recent evidence supporting a potential connection to metabolic disorders.

Obligate intracellular apicomplexan parasites are considered as deficient in cholesterol biosynthesis and scavenge cholesterol from their host cell in a parasite-specific manner. Compared to fast proliferating apicomplexan species producing low numbers of merozoites per host cell, (e. g. Toxoplasma gondii), the macromeront-forming protozoa Eimeria bovis is in extraordinary need for cholesterol for offspring production (≥ 170,000 merozoites I/macromeront). Interestingly, optimized in vitro E. bovis merozoite I production occurs under low foetal calf serum (FCS, 1.2%) supplementation. To analyze the impact of extensive E. bovis proliferation on host cellular sterol metabolism we here compared the sterol profiles of E. bovis-infected primary endothelial host cells grown under optimized (1.2% FCS) and non-optimized (10% FCS) cell culture conditions. Therefore, several sterols indicating endogenous de novo cholesterol synthesis, cholesterol conversion and sterol uptake (phytosterols) were analyzed via GC-MS-based approaches. Overall, significantly enhanced levels of phytosterols were detected in both FCS conditions indicating infection-triggered sterol uptake from extracellular sources as a major pathway of sterol acquisition. Interestingly, a simultaneous induction of endogenous cholesterol synthesis based on increased levels of distinct cholesterol precursors was only observed in case of optimized parasite proliferation indicating a parasite proliferation-dependent effect. Considering side-chain oxysterols, 25 hydroxycholesterol levels were selectively found increased in E. bovis-infected host cells, while 24 hydroxycholesterol and 27 hydroxycholesterol contents were not significantly altered by infection. Exogenous treatments with 25 hydroxycholesterol, 27 hydroxycholesterol, and 7 ketocholesterol revealed significant adverse effects on E. bovis intracellular development. Thus, the number and size of developing macromeronts and merozoite I production was significantly reduced indicating that these oxysterols bear direct or indirect antiparasitic properties. Overall, the current data indicate parasite-driven changes in the host cellular sterol profile reflecting the huge demand of E. bovis for cholesterol during macromeront formation and its versatility in the acquisition of cholesterol sources.

Both the immune response developed in ruminants against Eimeria spp. and the ability to bear patent infections seems to be dependent on the age of the host. In the present study we have evaluated the influence of the age in the development of protective immune responses against Eimeria ninakohlyakimovae. For this purpose, 3, 4 and 5-week-old goat kids were infected with sporulated oocysts and subjected to a homologous challenge 3 weeks later. Goat kids primary infected at 6, 7 and 8 weeks of age served as challenge controls, and uninfected animals were used as negative controls. The protective immunity was assessed by clinical, haematological, parasitological, immunological and pathological parameters. Altogether, the results demonstrate that goat kids of either 3, 4 or 5 weeks of age are able to develop patent infections and immunoprotective responses against E. ninakohlyakimovae, as all age groups: (i) released significantly less oocysts after challenge, which was associated to milder clinical signs; (ii) displayed a local immune response, with significant increase of numerous cellular populations; and (iii) had increased levels of IgG and IgM, and mainly of local IgA. Nevertheless, detailed analysis of the data showed some differences between the three age groups, related both to the Eimeria infection outcome and the resulting immune response, suggesting that youngest goat kids are not fully immunocompetent. This finding may be of interest for the design of immunoprophylactic approaches and/or prophylactic/methaphylactic treatments against goat coccidiosis.

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.

The gastropod-borne nematodes Angiostrongylus vasorum and Aelurostrongylus abstrusus are global causes of cardio/pulmonary diseases in dogs and cats. In the last decade, the number of reports on canine and feline lungworms has increased in several areas of Europe and North America. The unspecific clinical signs and prolonged course of these diseases often renders diagnosis challenging. Both infections are considered as emerging and underestimated causes of disease in domestic pets. In South America, little information is available on these diseases, apart from occasional reports proving the principle presence of A. vasorum and A. abstrusus. Thus, the purpose of this review is to summarize reports on infections in both domestic and wildlife animals in South America and to increase the awareness on gastropod-borne metastrongyloid parasites, which also include important zoonotic species, such as A. cantonensis and A. costaricensis. This review highlights the usefulness of diagnostic tools, such as the Baermann funnel technique, serology and PCR, and proposes to include these routinely on cases with clinical suspicion for lungworm infections. Future national epidemiological surveys are recommended to be conducted to gain a deeper insight into the actual epidemiological situation of gastropod-borne parasitoses in South America.

The anthropozoonotic metastrongyloid nematodes Angiostrongylus cantonensis and Angiostrongylus costaricensis, as well as Angiostrongylus vasorum, Crenosoma vulpis, Aelurostrongylus abstrusus and Troglostrongylus brevior are currently considered as emerging gastropod-borne parasites and have gained growing scientific attention in the last years. However, the knowledge on invertebrate immune responses and on how metastrongyloid larvae are attacked by gastropod immune cells is still limited. This work aims to describe an in vitro system to investigate haemocyte-derived innate immune responses of terrestrial gastropods induced by vital axenic metastrongyloid larvae. We also provide protocols on slug/snail management and breeding under standardized climate conditions (circadian cycle, temperature and humidity) for the generation of parasite-free F0 stages which are essential for immune-related investigations. Adult slug species (Arion lusitanicus, Limax maximus) and giant snails (Achatina fulica) were maintained in fully automated climate chambers until mating and production of fertilized eggs. Newly hatched F0 juvenile specimens were kept under parasite-free conditions before experimental use. An improved protocol for gastropod haemolymph collection and haemocyte isolation was established. Giemsa-stained haemolymph preparations showed adequate haemocyte isolation in all three gastropod species. Additionally, a protocol for the production of axenic first and third stage larvae (L1, L3) was established. Haemocyte functionality was tested in haemocyte-nematode-co-cultures. Scanning electron microscopy (SEM) and light microscopy analyses revealed that gastropod-derived haemocytes formed clusters as well as DNA-rich extracellular aggregates catching larvae and decreasing their motility. These data confirm the usefulness of the presented methods to study haemocyte-mediated gastropod immune responses to better understand the complex biology of gastropod-borne diseases.

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.

Coccidiosis caused by Eimeria species is an important disease worldwide, particularly in ruminants and poultry. Eimeria infection can result in significant economic losses due to costs associated with treatment and slower growth rates, or even with mortality of heavily infected individuals. In goat production, a growing industry due to increasing demand for caprine products worldwide, coccidiosis is caused by several Eimeria species with E. arloingi and E. ninakohlyakimovae the most pathogenic. The aims of this study were genetic characterization of a newly isolated European E. arloingi strain (A) and determination of phylogenetic relationships with Eimeria species from other ruminants. Therefore, a DNA sequence of E. arloingi strain (A) containing 2290 consensus nucleotides (the majority of 18S rDNA, complete ITS-1 and 5.8S sequences, and the partial ITS-2) was amplified and phylogenetic relationship determined with the most similar sequences available on GenBank. The phylogenetic tree presented a branch constituted by bovine Eimeria species plus E. arloingi, and another one exclusively populated by ovine Eimeria species. Moreover, E. arloingi, E. bovis and E. zuernii, which all replicate in host intestinal endothelial cells of the lacteals, were found within the same cluster. This study gives new insights into the evolutionary phylogenetic relationships of this newly described caprine Eimeria strain and confirmed its close relationship to other highly pathogenic ruminant Eimeria species characterized by macromeront formation in host endothelial cells of the central lymph capillaries of the small intestine.

During the first schizogony, the goat coccidia Eimeria ninakohlyakimovae develops macroschizonts in lacteal duct endothelial cells, whose rupture leads to severe ileal damage and clinical signs during the prepatent period. The immune response elicited against early stages of the parasite development still requires to be investigated. In the present study we have evaluated immune reactions in goat kids primary- and challenged-infected with Eimeria ninakohlyakimovae, and sacrificed during prepatency (7 days after challenge). The oocyst output during the primary infection, body weight and clinical condition of all the animals were examined and, at the end of the experiment, all the goat kids were euthanized and subjected to necropsy. Samples were taken from different sections of the ileum, colon and mesenteric lymph nodes (MLN) of primary- and challenged E. ninakohlyakimovae-infected animals. Intestinal leukocyte subpopulations were characterized in E. ninakohlyakimovae-infected mucosa and counts of lymphocytes, eosinophils, polymorphonuclear neutrophils (PMN), globular leukocytes and mast cells were recorded. Additionally, gene expression of caprine IL-2, IL-4, IL-10 and INFγ of ileal, colonic and MLN tissues were performed, as well as the immunohistochemical characterization of immune cells. The E. ninakohlyakimovae primary infection resulted in moderate to severe enteritis with different degrees of diarrhoea and was accompanied by high OPG counts and an increase of most immune cells analyzed when compared to uninfected control animals. Furthermore, eosinophil-, lymphocyte-, globular leukocyte- and mast cell-counts were significantly higher in the challenge group compared to the primary infected animals, whilst the opposite was true for PMN counts. The challenge infection was also associated with moderate increased levels of local mucosal IgA. Interestingly, the number of immature schizonts found at the ileal mucosa was statistically higher in the challenge infected group compared to the challenged control animals. Furthermore, in the challenged E. ninakohlyakimovae-infected animals a significantly higher number of mucosal CD4+ and CD8+ lymphocytes were observed, indicating that these T cell subpopulations might be involved in protective host immune response elicited against early stages of parasite development. The immune response was however very complex, as antigen presenting cells and other effector cell populations of the innate immune system, as well as certain cytokines, were involved. In summary, the results of this study contribute to the better understanding of local cellular and humoral immune responses against caprine E. ninakohlyakimovae, particularly during the prepatency.

Background Aedes aegypti is the main vector of several arthropod-borne viral infections in the tropics profoundly affecting humans, such as dengue fever (DF), West Nile (WN), chikungunya and more recently Zika. Eradication of Aedes still largely depends on insecticides, which is the most cost-effective strategy, and often inefficient due to resistance development in exposed Aedes populations. We here conducted a study of Ae. aegypti resistance towards several insecticides regularly used in the city of Denpasar, Bali, Indonesia. Methods Aedes aegypti egg samples were collected with ovitraps and thereafter hatched in the insectary of the Gadjah Mada University. The F0 generation was used for all bioassay-related experiments and knockdown resistance (kdr) assays. Results Results clearly showed resistance development of Ae. aegypti against tested insecticides. Mortalities of Ae. aegypti were less than 90% with highest resistance observed against 0.75% permethrin. Mosquitoes from the southern parts of Denpasar presented high level of resistance pattern in comparison to those from the western and northern parts of Denpasar. Kdr analysis of voltage-gated sodium channel (Vgsc) gene showed significant association to S989P and V1016G mutations linked to resistance phenotypes against 0.75% permethrin. Conversely, Ae. aegypti F1534C gene mutation did not result in any significant correlation to resistance development. Conclusions Periodically surveillance of insecticide resistances in Ae. aegypti mosquitoes will help local public health authorities to set better goals and allow proper evaluation of on-going mosquito control strategies. Initial detection of insecticide resistance will contribute to conduct proper actions in delaying mosquito resistance development such as insecticide rotation or combination of compounds in order to prolong chemical efficacy in combating Ae. aegypti vectors in Indonesia.

Although cellular immune reactions seem to be crucial for protective immune responses in Eimeria spp. infections, there are also evidences on an active involvement of the humoral counterpart. In the present study, we have analyzed the humoral response of goat kids subjected to primary and challenge infections with Eimeria ninakholyakimovae. Specific levels of IgG and IgM in serum samples and IgA in the ileal mucus were estimated. In infected kids, significantly increased levels of IgG were observed from 3 weeks post infection onwards in addition to an enhancement of specific IgM and secretory IgA levels. A wide range of peptides of sporulated oocyst antigen (SOA) was recognized by specific IgG as determined by immunoblotting. However, no correlations were found between immunoglobulin levels and OPG counts after challenge infection. Overall, these data indicate a significant specific humoral response of E. ninakohlyakimovae-infected goat kids that does not seem to convey immunoprotection. Further studies should be addressed to clarify if the lack of correlation might be associated to the type of antigen used for the immunoenzimatic assays, the age of the animals or other factors.

The European wolf (Canis lupus) is a large carnivore species present in limited areas of Europe with sev- eral 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 ecol- ogy 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 sequenc- ing 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%), Diphyl- lobothrium 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 pro- vides first records on the occurrence of the two relevant anthropozoonotic parasites, Giardia spp. and Cryptosporidium spp., in wild wolves from Croatia.

Dairy cows undergo metabolic disturbances in the peripartum period, during which infectious inflammatory diseases and detrimental polymorphonuclear leukocytes (PMN) functions, such as radical oxygen species (ROS) production, are observed. Platelet-activating factor (PAF) is a key pro-inflammatory mediator that increases PMN ROS production. To date, the role of glycolysis and mitochondria in PAF-induced ROS production in bovine PMN has not been known. The aim of this study was to assess whether inhibition of glycolysis and disruption of mitochondrial function alter the oxidative response induced by PAF. We isolated PMN from non-pregnant Holstein Friesian heifers and pre-incubated them with 2-deoxy-d-glucose (2-DG; 2 mM, 30 min), carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 5 μM, 5 min), oligomycin (10 μM, 30 min) or rotenone (10 μM, 30 min). Respiratory burst was measured by luminol-chemiluminescence assay, while mitochondrial ROS (mtROS) were evaluated by MitoSOX probe and flow cytometry. Also, we detected the presence of mitochondria by MitoTracker Deep Red FM probe and changes in mitochondrial membrane potential (Δψm) were assessed by JC-1 probe and flow cytometry. We observed that all inhibitors separately were able to reduce PAF-induced ROS production. Presence of mitochondria was detected and PAF increased the Δψm, while CCCP reduced it. 2-DG and rotenone reduced the mtROS production induced by PAF. CCCP did not alter the mtROS and oligomycin administered independently increased mtROS production. We concluded that PAF-induced ROS production is glycolysis- and mitochondria-dependent. Bovine PMN have a functional mitochondrion and PAF induced mtROS via glycolysis and mitochondrial complex-I activity. Our results highlight an important modulation of cellular metabolism in the oxidative response induced by proinflammatory agents, which could contribute to PMN disfunction during peripartum in cattle.

Besnoitia besnoiti tachyzoites infect and develop in bovine endothelial cells in vivo and trigger the release of neutrophil extracellular traps (NETs) from bovine polymorphonuclear neutrophils (PMN). The purpose of this study was to analyze if pure B. besnoiti tachyzoite-triggered NETs would damage endothelial host cells and subsequently influence intracellular development and proliferation of B. besnoiti tachyzoites in primary bovine endothelial cells. For comparison purposes, isolated A23187-induced NETs were also used. Thus, we here evaluated endothelial host cell damage triggered by histone 2A (H2A) and B. besnoiti tachyzoite-induced NET preparations and furthermore estimated the effects of PMN floating over B. besnoiti-infected endothelium under physiological flow conditions on endothelial host cell viability. Overall, all treatments (H2A, B. besnoiti-triggered NETs and floating PMN) induced endothelial cell death of B. besnoiti-infected host cells. However, though host cell damage led to significantly altered intracellular parasite development with respect to parasitophorous vacuole diameter and numbers, the total proliferation of the parasite over time was not significantly affected by these treatments thereby denying any direct effect of NETs on intracellular B. besnoiti replication.

Deposition of sperm during artificial insemination in the bovine female reproductive tract results in early host innate immune reactions of polymorphonuclear neutrophils (PMNs). Furthermore, sperm-mediated neutrophil extracellular trap (NET) formation (NETosis) was recently reported to occur in different mammalian species, including humans. We, here, investigated the interactions of bovine PMN with different semen-derived samples and analyzed in more depth molecular aspects of this effector mechanism. Overall, confrontation of PMN with sperm/cell preparation (SCP) resulted in a rapid and dose-dependent NET formation leading to effective spermatozoa entrapment. Thereby, spermatozoa induced different phenotypes of NETs. Immunostaining analyses revealed the presence of histones (H3), neutrophil elastase (NE), and pentraxin (PTX) in sperm-triggered NET structures. Fresh SCP strongly induced NETosis than frozen-thawed ones. The level of NETosis was not related to spermatozoa viability. SCP as well as purified sperm cells (SCs) and supernatant (SN) induce NETosis, although the reaction in SC was lower. Enhanced levels of oxygen consumption and proton leak in PMN revealed sperm SNs but not purified SCs as PMN activators. Functional inhibition experiments revealed sperm-triggered NETosis as an NADPH oxidase- and peptidylarginine deiminase 4-dependent process and proved to be dependent on intra- and extracellular Ca++ influxes while myeloperoxidase activity and as ERK1/2- and PI3K-related signaling pathways did not seem to play a pivotal role in this effector mechanism. From these findings, we speculate that sperm-derived NETosis might also occur in vivo during artificial insemination and might therefore play a role related to reduced fertility.

Leucocytospermia has been associated with loss of sperm function. Extracellular traps (ETs) of leucocytes are produced during innate immune response. ETs can be activated by spermatozoa in contact with polymorphonuclear (in vitro), inducing sperm entrapment and decrease motility. In this pilot study, we describe the results of ETosis ex vivo, in seminal fluid (SF) smear of infertile patients, associating ETs with leucocytospermia and bacteriospermia. In 21 infertile patients, semen parameters (WHO, 2010), microbiological study, leucocytospermia and presence of ETs in SF were determined. Leucocytes (CD45, CD15 and CD68) were evaluated by immunostaining in SF smears. Indirect immunofluorescence (global histone and H4-citrullinated 3) and scanning electron microscopy (SEM) were used to determine ETs morphology. In 28.6% of patients presented leucocytospermia without bacteriospermia, all of them presented a large number of ETs in the SF smears examined. About 76.6% of the patients without leucocytospermia were positive for ETs. Samples with leucocytospermia have a higher number of ETs and would be related to the amount of leucocytes in the SF. The morphological predominant ETs were diffuse (diffETs) and spread (sprETs). The formation of ETs indicates leucocyte activation in semen, and it was observed that ETosis does not depend exclusively on the presence of bacterial contamination.

Background Acute ruminal acidosis (ARA) is a metabolic disease of cattle characterized by an aseptic synovitis. ARA is the result of an increased intake of highly fermentable carbohydrates that frequently occurs in dairy cattle subjected to high production requirements. In human joint diseases such as rheumatoid arthritis and gout, several pro-inflammatory molecules are increased in the synovial fluid, including cytokines, prostaglandin E2 (PGE2), metalloproteinases, and neutrophil extracellular traps (NETs). The aim of this study was to identify the presence of proinflammatory mediators and neutrophils in the synovial fluid of heifers with ARA, induced by an oligofructose overload. Five heifers were challenged with an oligofructose overload (13 g/kg BW) dissolved in water. As a control, a similar vehicle volume was used in four heifers. Synovial fluid samples were collected from the tarso-crural joint and PGE2, IL-6, IL-1β, ATP, lactate dehydrogenase (LDH), albumin, glucose, matrix metalloproteinase-9 (MMP-9), cellular free DNA, NETs, and serpin B1 were analyzed at 0, 9, and 24 h post treatment. Results At 9 h post oligofructose overload, an increase of IL-1β, IL-6, PGE2, serpin B1 and LDH was detected in the joints when compared to the control group. At 24 h, the synovial fluid was yellowish, viscous, turbid, and contained abundant neutrophils. An increase of DNA-backbone-like traps, histone 3 (H3cit), aggregated neutrophil extracellular traps (aggNETs), and serpin B1 were observed 24 h post treatment. Furthermore, albumins, LDH, ATP, MMP-9, IL-6, and IL-1β were increased after 24 h. Conclusions The overall results indicate that IL-1β, IL-6 and PGE2, were the earliest proinflammatory parameters that increased in the synovial fluid of animals with ARA. Furthermore, the most sever inflammatory response in the joint was observed after 24 h and could be associated with a massive presence of neutrophils and release of aggNETs. Electronic supplementary material The online version of this article (10.1186/s12917-019-1974-x) contains supplementary material, which is available to authorized users.

So far, neither the feline lungworms Aelurostrongylus abstrusus and Troglostrongylus brevior nor the canine lungworm Angiostrongylus vasorum was reported in wildlife or intermediate hosts from Austria. The slug Arion vulgaris represents an invasive species in Europe and serves as intermediate host for several lungworm species. This study aimed to analyse the occurrence of metastrongyloid lungworm larvae in slugs in Vienna, Austria. Therefore, 193 A. vulgaris were collected in the central Prater park in summer 2016. Specimens were artificially digested, analysed microscopically for lungworm larvae, and species were confirmed via PCR and sequencing. Out of 193, five slugs were positive to lungworms (2.6%), one for A. vasorum, two for A. abstrusus (genotypes A and B) and one for T. brevior, and one slug had a mixed infection of A. abstrusus and T. brevior larvae. The current study is the first evidence on the endemicity of these metastrongyloid lungworm species in Austria.

Simple Summary Within innate immunity, polymorphonuclear neutrophils (PMN) are the most abundant leukocyte population. Alongside PMN, monocytes, eosinophils, and basophils are also known to exist. All of them can release extracellular traps (ETs), a complex web-like structure composed of chromatin decorated with nuclear histones, granular enzymes, peptides, and proteins, to firmly entrap invasive pathogens, thereby slowing dissemination and helping to develop proper immune responses against bacteria, fungi, viruses, and parasites. Here, we showed for the first time that Atlantic salmon-derived PMN released ETs-like structures in vitro, in response to highly pathogenic facultative intracellular rickettsial bacteria Piscirickettsia salmonis. The release of ET-like structures from PMN could be a new alternative to improve farmed salmon’s defense against pathogens. Abstract Extracellular traps (ETs) are webs of DNA, citrullinated histones, anti-microbial peptides, and proteins that were not previously reported in Atlantic salmon (Salmo salar). ETs are mainly released from polymorphonuclear neutrophils (PMN) and are considered a novel PMN-derived effector mechanism against different invasive pathogens. Here, we showed that Atlantic salmon-derived PMN released ETs-like structures in vitro in response to highly pathogenic facultative intracellular rickettsial bacteria Piscirickettsia salmonis. PMN were isolated from pre-smolt Atlantic salmon and stimulated in vitro with oleic acid and P. salmonis. Extracellular DNA was measured using the PicoGreen™ dye, while immunofluorescence image analysis was used to confirm the classical components of salmonid-extruded ETs. Future studies are required to better understand the role of Atlantic salmon-derived ETs orchestrating innate/adaptive immunity and the knowledge on regulation pathways involved in this cell death process. Thus, comprehension of salmonid-derived ETs against P. salmonis might represent novel alternative strategies to improve host innate defense mechanisms of farmed salmon against closely related rickettsial bacteria, as a complement to disease prevention and control strategies.

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.

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

Increased short-chain fatty acid (SCFA) production is associated with subacute ruminal acidosis (SARA) and activation of inflammatory processes. In humans and rodents, SCFAs modulate inflammatory responses in the gut via free fatty acid receptor 2 (FFA2). In bovines, butyric acid is one of the most potent FFA2 agonists. Its expression in bovine neutrophils has recently been demonstrated, suggesting a role in innate immune response in cattle. This study aimed to evaluate if butyric acid modulates oxidative and non-oxidative functions or if it can potentiate other inflammatory mediators in bovine neutrophils. Our results showed that butyric acid can activate bovine neutrophils, inducing calcium (Ca(2+)) influx and mitogen-activated protein kinase (MAPK) phosphorylation, two second messengers involved in FFA2 activation. Ca(2+) influx induced by butyric acid was dependent on the extracellular and intracellular Ca(2+) source and phospholipase C (PLC) activation. Butyric acid alone had no significant effect on reactive oxygen species (ROS) production and chemotaxis; however, a priming effect on platelet-activating factor (PAF), a potent inflammatory mediator, was observed. Butyric acid increased CD63 expression and induced the release of neutrophil granule markers matrix metalloproteinase-9 (MMP-9) and lactoferrin. Finally, we observed that butyric acid induced neutrophil extracellular trap (NET) formation without affecting cellular viability. These findings suggest that butyric acid, a component of the ruminal fermentative process, can modulate the innate immune response of ruminants.

In total, 2 pet guinea pigs ( Cavia porcellus ) that were 5 years old were presented with pruritus, erythema, alopecia, and crusting of the ventral abdomen. Skin scrapings revealed multiple nematode larvae, identified as Pelodera strongyloides based on morphologic characteristics. Treatment consisted of systemic ivermectin and fenbendazole in addition to topical benzoyl peroxide application. P. strongyloides is a free-living saprophytic nematode, which is ubiquitously present in any decaying organic material, such as soil or feed. The nematode is capable of invading mammalian skin, resulting in pruritic dermatitis. Diagnosis of the nematode induced skin disease is based on history, including husbandry, presence of a ventrally distributed dermatitis, and demonstration of larvae in skin scrapings. Dermal peloderosis cases have been reported in different animal species, including dogs, cattle, horses, sheep, and a laboratory housed guinea pig. This is the first description of P. strongyloides dermatitis in pet guinea pigs. P. strongyloides dermatitis should be considered as a differential diagnosis for alopecic and crusty skin lesions in guinea pigs.

Objective To investigate the presence of neutrophil extracellular traps (NETs) in vivo by analysing intestinal sections from experimentally Eimeria bovis - and naturally Eimeria arloingi -infected animals. Methods Intestinal samples of Eimeria arloingi - and Eimeria bovis -infected animals were analysed by using immunohistochemical and fluorescence approach by using monoclonal antibodies. Results Classical NET components were confirmed by co-localization of extracellular DNA being decorated with neutrophil elastase and histones in Eimeria -infected tissue samples. Here, extrusion of NETs was exclusively detected in intestinal polymorphonuclear neutrophils infiltrating Eimeria -infected sites. In vivo NETs were either found in close proximity or in direct contact to different Eimeria stages suggesting a stage-independent process. NETs were also found within the gut lumen driven by polymorphonuclear neutrophils that were contacting released oocysts. Conclusions We postulate that NETs might play an important role in innate defence reactions in coccidiosis therefore significantly altering the outcome of infection.

In constructed wetlands, organic pollutants are mainly degraded via microbial processes. Helophytes, plants that are commonly used in these systems, provide oxygen and root exudates to the rhizosphere, stimulating microbial degradation. While the treatment performance of constructed wetlands can be remarkable, a mechanistic understanding of microbial degradation processes in the rhizosphere is still limited. We investigated microbial toluene removal in a constructed wetland model system combining 16S rRNA gene sequencing, metaproteomics and (13) C-toluene in situ protein-based stable isotope probing (protein-SIP). The rhizospheric bacterial community was dominated by Burkholderiales and Rhizobiales, each contributing about 20% to total taxon abundance. Protein-SIP data revealed that the members of Burkholderiaceae, the proteins of which showed about 73% of (13) C-incorporation, were the main degraders of toluene in the planted system, while the members of Comamonadaceae were involved to a lesser extent in degradation (about 64% (13) C-incorporation). Among the Burkholderiaceae, one of the key players of toluene degradation could be assigned to Ralstonia pickettii. We observed that the main pathway of toluene degradation occurred via two subsequent monooxygenations of the aromatic ring. Our study provides a suitable approach to assess the key processes and microbes that are involved in the degradation of organic pollutants in complex rhizospheric ecosystems.

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.

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.

Toxoplasma gondii is a cosmopolitan zoonotic parasite and nowadays considered as an emerging neozoan pathogen in the marine environment. Cetacean innate immune reactions against T. gondii stages have not yet been investigated. Thus, T. gondii tachyzoites were utilized to trigger neutrophil extracellular traps (NETs) in bottlenose dolphin (Tursiops truncatus) polymorphonuclear neutrophils (PMN). Scanning electron microscopy unveiled T. gondii tachyzoites as potent and rapid inducers of cetacean-derived NETosis. Co-localization of extracellular chromatin with global histones, granulocytic myeloperoxidase and neutrophil elastase confirmed classical characteristics of NETosis. Interestingly, different phenotypes of NETs were induced by tachyzoites resulting in spread, diffuse and aggregated NET formation and moreover, 'anchored' and 'cell free' NETosis was also detected. Current data indicate that cetacean-derived NETosis might represent an early, ancient and well-conserved host innate defense mechanism that not only acts against T. gondii but might also occur in response to other closely related emerging apicomplexan parasites affecting marine cetaceans.

Reptiles are becoming popular pets in many parts of the world. They are also known to harbor numerous gastrointestinal parasites. We used faecal smears to examine 748 stool samples from 14 different agamid lizard species. In addition, we used coproantigen ELISA tests (11 samples) and immunofluorescence assays (IFA) (19 samples) to detect reptile Cryptosporidium infections. In 28 cases, veterinarians requested therapy to treat oxyurid- and/or Isospora amphiboluri-infections and resent fecal samples after proposed therapy and anti-parasitic treatments had been applied. We also performed complete dissections of 24 deceased agamas in order to specify protozoan and helminth parasite infections. Overall, the examined fecal samples contained 6 different taxa. Oxyurids (Pharyngodonidae) were the most prevalent nematodes (41.2%), followed by I. amphiboluri (17.0%), Entamoeba spp. (0.8%), Choleoeimeria spp. (0.5%), Trichomonas spp. (0.3%), Cryptosporidium spp. (0.3%) and Strongyloides-like nematodes (0.1%). I. amphiboluri infections were significantly more prevalent (Chi-square test: χ2 = 21,5, df = 1, P

Background Several metastrongyloid lungworms are unreported pathogens in Colombia. Angiostrongylus vasorum and Crenosoma vulpis target the cardiopulmonary system of domestic and wild canids. Aelurostrongylus abstrusus and Troglostrongylus brevior infect felids and considering that six wild felid species exist in Colombia, knowledge of feline lungworm infections is important for their conservation. The zoonotic metastrongyloids Angiostrongylus costaricensis and Angiostrongylus cantonensis can cause severe gastrointestinal and neurological diseases. Angiostrongylus costaricensis has been reported in Colombia, while Ang. cantonensis is present in neighbouring countries. Research on the epidemiology of metastrongyloids in Colombia and South America more broadly requires evaluating the role that gastropods play as intermediate hosts in their life cycles. This study assessed the prevalence of metastrongyloid larvae in populations of the invasive giant African snail, Lissachatina fulica, in Colombia. Methodology/Principal findings A total of 609 Lissachantina fulica were collected from 6 Colombian municipalities. The snails were then cryo-euthanized, artificially digested and the sediments examined microscopically for the presence of metastrongyloid larvae. Based on morphological characteristics 53.3% (56/107) of the snails from Puerto Leguízamo (Department of Putumayo) were infected with Ael. abstrusus larvae, 8.4% (9/107) with Ang. vasorum larvae, 6.5% (7/107) with T. brevior larvae and 5.6% (6/107) with C. vulpis larvae, being the region with highest prevalences of the four species. Snails from Andes (Department of Antioquia) and Tulúa (Department of Valle del Cauca) were positive for Ang. vasorum larvae with a prevalence of 4.6 (11/238) and 6.3% (4/64), respectively. Species identifications were confirmed by PCR and sequencing. Conclusions/Significance This epidemiological survey reports for first time the presence of Ael. abstrusus, T. brevior, C. vulpis and Ang. vasorum in L. fulica in a number of regions of Colombia., Author summary Several lungworm species are neglected pathogens in Colombia. Angiostrongylus vasorum and Crenosoma vulpis target the cardiopulmonary system of domestic and wild canids. Aelurostrongylus abstrusus and Troglostrongylus brevior infect domestic cats as well as wild felids. Angiostrongylus costaricensis and Angiostrongylus cantonensis may cause severe gastrointestinal or neurological diseases in humans, respectively. Snails/slugs are necessary intermediate hosts in the life cycles of these parasites. We assessed the prevalence of metastrongyloid larvae in 609 specimens of the giant African snail, Lissachatina fulica, from 6 Colombian municipalities. In Puerto Leguízamo, 53.3% of the snails were infected with Ael. abstrusus larvae, 8.4% with Ang. vasorum larvae, 6.5% with T. brevior larvae and 5.6% with C. vulpis larvae. Snails from Andes and Tulúa were positive for Ang. vasorum larvae with a prevalence of 4.6 and 6.3%, respectively. This epidemiological study reports for first time the presence of Ael. abstrusus, T. brevior, C. vulpis and Ang. vasorum in the invasive giant African snail in various parts of Colombia.

Heartworm disease is a zoonotic vector-borne disease caused by Dirofilaria immitis mainly affecting canids. Infectious third-stage larvae (L3) are transmitted to the definitive hosts via culicid mosquitoes; adult nematodes reside in the pulmonary arteries and in the right heart releasing unsheathed first-stage larvae (microfilariae) into the bloodstream leading to chronic and sometimes fatal disease. So far, early innate immune reactions triggered by these different D. immitis stages in the canine host have scarcely been investigated. Therefore, D. immitis microfilariae and L3 were analyzed for their capacity to induce neutrophil extracellular traps (NETs) in canine polymorphonuclear neutrophils (PMN). Overall, scanning electron microscopy analysis revealed both larval stages as strong inducers of canine NETosis. Co-localization of PMN-derived extracellular DNA with granulocytic histones, neutrophil elastase, or myeloperoxidase in parasite-entrapping structures confirmed the classical characteristics of NETosis. Quantitative analyses showed that both larval stages triggered canine NETs in a time-dependent but dose-independent manner. Moreover, parasite-induced NET formation was not influenced by the parasites viability since heat-inactivated microfilariae and L3 also induced NETs. In addition, parasite/PMN confrontation promoted significant entrapment but not killing of microfilariae and L3. Both, NETosis and larval entrapment was significantly reversed via DNase I treatments while treatments with the NADPH oxidase inhibitor diphenyleneiodonium failed to significantly influence these reactions. Interestingly, different types of NETs were induced by microfilariae and L3 since microfilarial stages merely induced spread and diffuse NETs while the larger L3 additionally triggered aggregated NET formation.

Eimeria infections are common in sheep industry worldwide nonetheless very little is known on infection dynamics and associated risk factors influencing the outcome of clinical ovine coccidiosis. The aim of the present work was to analyse intrinsic (e. g. host age) and extrinsic (flock size, farm size and animal density) risk factors influencing dynamics of Eimeria spp. infections in sheep farms of the semi-arid region of Murcia in southern Spain. Overall, 64 ovine flocks in Murcia were surveyed and a total of 396 faecal samples were collected between May and July 2015. Flotation and McMaster techniques were performed for parasitological investigation. Excreted oocysts were identified on the basis of morphological as well as morphometric characteristics. In all analysed samples Eimeria spp. oocysts (100%) were found and 11 different Eimeria species were identified. Most prevalent species was E. ovinoidalis (97%) followed by E. parva/E.pallida (97%), E. weybridgensis (90.9%) and E. crandallis (89.4%), respectively. Concomitant infections with more than one Eimeria species were observed in all analysed samples. Analysis of intrinsic/extrinsic risk factor revealed age, herd/farm size and animal density to be the main factors significantly influencing Eimeria oocysts per gram of faeces counts (OPG) and overall prevalence of Eimeria spp. infections in sheep.

Aedes aegypti represents the principal vector of many arthropod-borne diseases in tropical areas worldwide. Since mosquito control strategies are mainly based on use of insecticides, resistance development can be expected to occur in frequently exposed Ae. aegypti populations. Surveillance on resistance development as well as testing of insecticide susceptibility is therefore mandatory and needs further attention by national/international public health authorities. In accordance, we here conducted a study on Ae. aegypti resistance development towards several often used insecticides, i.e., malathion, deltamethrin, permethrin, λ-cyhalothrin, bendiocarb, and cyfluthrin, in the periurban area of Banjarmasin city, Kalimantan, Indonesia. Our results clearly showed resistance development of Ae. aegypti populations against tested insecticides. Mortalities of Ae. aegypti were less than 90% with the highest resistance observed against 0.75% permethrin. Collected mosquitoes from Banjarmasin also presented high level of resistance development to 0.1% bendiocarb. Molecular analysis of voltage-gated sodium channel (Vgsc) gene showed significant association of V1016G gene point mutation in resistance Ae. aegypti phenotypes against 0.75% permethrin. However, F1534C gene point mutation did not correlate to Ae. aegypti insecticide resistance to 0.75% permethrin. Irrespective of periurban areas in Kalimantan considered as less densed island of Indonesia, Ae. aegypti-derived resistance to different routinely applied insecticides occurred. Our findings evidence that Ae. aegypti insecticide resistance is most likely spreading into less populated areas and thus needs further surveillance in order to delay Ae. aegypti resistance development.

Metastrongyloid parasites represent sparsely studied parasites of dogs and cats in Germany. Recent European surveys indicate that these parasites are spreading in Europe. Actual data on prevalence of Angiostrongylus vasorwn in dogs and foxes reveal several endemic foci in Germany. However, actual data on the prevalence of A. vasorum and other metastrongyloid lungworm larvae in a wide range of slug and snail intermediate hosts, such as Anion lusitanicus, are missing for Germany. To fill this gap, we conducted an epidemiological survey on native German slugs in selected regions of Hesse and Rhineland-Palatinate. The focus was on slugs, because in study areas slugs appear to be more abundant than snails. Slugs were collected throughout different seasons of the year in areas that were previously proven to be hyperendemic for A. vasorum fox infections. Overall, a total of 2701 slugs were collected and examined for lungworm larvae via artificial digestion. The number of A. vasorum larvae per slug varied considerably (1-546 larvae per specimen). Some hotspot areas with high A. vasorum prevalence in slugs (up to 19.4%) were identified. The overall A. vasorum prevalence varied with season with largest number of slugs infected in summer (9.1%) and lowest number in winter (0.8%). The current study revealed a total A. vasorum prevalence of 4.7% in slugs based on microscopic analyses. Confirmation of lungworm species was made by specific duplex-real-time PCRs. Hence, these data demonstrate that final hosts are at a permanent risk for A. vasorum infections during all seasons when living in investigated areas. Besides A. vasorum, other lungworm larvae were also detected, such as Crenosoma vulpis (the fox lungworm, 2.3%) and Aelurostrongylus abstrusus (feline lungworm, 0.2%).

Extracellular traps (ETs) are composed of nuclear DNA as backbone adorned with histones, cytoplasmic antimicrobial peptides/proteins which are released from a range of vertebrate and invertebrate host immune cells in response to several invading pathogens. Until now this ancient novel innate defence mechanism has not been demonstrated in any marine mammal. Interactions of harbour seal (Phoca vitulina)-PMN and -monocytes with viable tachyzoites of Toxoplasma gondii were investigated in this respect in vitro. For the demonstration and quantification of harbour seal PMN- and monocyte-derived ETs, extracellular DNA was stained with Sytox Orange. Fluorescence assays as well as scanning electron microscopy (SEM) analyses demonstrated PMN- and monocyte-promoted ET formation rapidly being induced upon contact with T. gondii-tachyzoites. The co-localisation of extracellular DNA decorated with histones (H3), neutrophil elastase (NE) and myeloperoxidase (MPO) in parasite entrapping structures confirmed the classical characteristics of PMN- and monocyte-promoted ETs. Exposure of harbour seal PMN and monocytes to viable tachyzoites resulted in a significant induction of ETs when compared to negative controls. Harbour seal-ETs were efficiently abolished by DNase I treatment and were reduced after PMN and monocytes pre-incubation with the NADPH oxidase inhibitor diphenilane iodondium. Tachyzoites of T. gondii were firmly entrapped and immobilised within harbour seal-ET structures. To our best knowledge, we here report for the first time on T. gondii-induced ET formation in harbour seal-PMN and -monocytes. Our results strongly indicate that PMN- and monocyte-triggered ETs represent a relevant and ancient conserved effector mechanism of the pinniped innate immune system as reaction against the pathogenic protozoon T. gondii and probably against other foreign pathogens occurring in the ocean environment.

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.

Neospora caninum represents a relevant apicomplexan parasite causing severe reproductive disorders in cattle worldwide. Neutrophil extracellular trap (NET) generation was recently described as an efficient defense mechanism of polymorphonuclear neutrophils (PMN) acting against different parasites. In vitro interactions of bovine PMN with N. caninum were analyzed at different ratios and time spans. Extracellular DNA staining was used to illustrate the typical molecules of NETs [i.e., histones (H3), neutrophil elastase (NE), myeloperoxidase (MPO), pentraxin] via antibody-based immunofluorescence analyses. Functional inhibitor treatments were applied to reveal the role of several enzymes [NADPH oxidase (NOX), NE, MPO, PAD4], ATP-dependent P2Y2 receptor, store-operated Ca++entry (SOCE), CD11b receptor, ERK1/2- and p38 MAPK-mediated signaling pathway in tachyzoite-triggered NETosis. N. caninum tachyzoites triggered NETosis in a time- and dose-dependent manner. Scanning electron microscopy analyses revealed NET structures being released by bovine PMN and entrapping tachyzoites. N. caninum-induced NET formation was found not to be NOX-, NE-, MPO-, PAD4-, ERK1/2-, and p38 MAP kinase-dependent process since inhibition of these enzymes led to a slight decrease of NET formation. CD11b was also identified as a neutrophil receptor being involved in NETosis. Furthermore, N. caninum-triggered NETosis depends on Ca++ influx as well as neutrophil metabolism since both the inhibition of SOCE and of P2Y2-mediated ATP uptake diminished NET formation. Host cell invasion assays indicated that PMN-derived NETosis hampered tachyzoites from active host cell invasion, thereby inhibiting further intracellular replication. NET formation represents an early and effective mechanism of response of the innate immune system, which might reduce initial infection rates during the acute phase of cattle neosporosis.

Gurltia paralysans is a poorly documented metastrongyloid nematode of cats, which mainly parasitizes the veins in the spinal cord subarachnoid space and parenchyma. Parasitic paraparesis caused by G. paralysans is a lesser-known spinal cord disease affecting domestic and wild felids of South America. Regions where feline gurltiosis is endemic include the southern parts of Chile and Argentina. Intra vitam diagnosis of feline gurltiosis remains challenging and is based primarily on neurological signs and the exclusion of other ethiologies for feline myelopathies. In view of the lack of information in the literature for this neglected feline neurological parasitosis, we have undertaken a detailed redescription and molecular characterization to expand on the previously available details in the original descriptions by Wolffhugel in 1993. The specimens used in this study were collected from spinal cord lesions of gurltiosis-affected domestic cats. Female and male specimens were morphologically and morphometrically examined using light and scanning electron microscopy. Molecular characterization was performed by sequencing a partial region of the nuclear ribosomal DNA and cytochrome oxidase gene of this parasite, and phylogenetic trees were constructed from the 28S D2-D3 and ITS2 regions using the Maximum Likelihood method. Sequence matching and phylogenetic analysis with these new sequences were consistent with the morphological classification of G. paralysans being within the Metastrongyloidea superfamily, but no consistent relation to a specific metastrongyloid family. The newly developed G. paralysans-specific PCR described here not only provides a useful diagnostic tool for feline gurltiosis in domestic cats living in endemic areas, but could also be used in large-scale epidemiological surveys on the intermediate mollusk host and the final host. By combining the morphology, molecular, and phylogenetic data we have reliably identified G. paralysans and confirmed its taxonomic status within the Metastrongyloidea.

Eimeria arloingi infections can cause severe haemorrhagic enteritis in young goat kids, thereby leading to high economic losses in goat industry worldwide. We aimed to isolate a new E. arloingi strain and establish a suitable in vitro culture system for the first merogony. E. arloingi oocysts were collected from naturally infected goat kids in the province of Alentejo, Portugal. For the maintenance of E. arloingi (strain A), kids kept under strict parasite-free conditions were orally infected with 10(3) sporulated oocysts each. Further, a new excystation protocol was successfully established to obtain viable sporozoites for further in vitro development in primary bovine umbilical vein endothelial cells (BUVEC). Overall, E. arloingi first merogony was successfully accomplished in BUVEC leading to macromeront formation (up to 150 μm) and the release of fully developed merozoites I stages. Moreover, host endothelial cell-parasite interactions were investigated in order to determine the extent of modulation carried out by E. arloingi in BUVEC during the first merogony. Gene transcription of adhesion molecules (E-selectin, P-selectin, VCAM-1, ICAM-1) was enhanced in the first hours post-infection (p.i.) in E. arloingi-infected BUVEC. BUVEC activation due to invasion was also shown by increased chemokine (CXCL8, CCL2, CCL5), cytokine (GM-CSF) and COX-2 gene transcription. The new E. arloingi (strain A) will be useful for better comprehension of early host innate immune reactions against this parasite in vitro/in vivo as well as to further our investigations in the complex Eimeria-host endothelial cell interactions.

Coccidiosis caused by the apicomplexan protozoa Eimeria ninakohlyakimovae has a high impact on animal health and profitability of the goat industry worldwide. Primary E. ninakohlyakimovae infections induce clinical disease mainly in goat kids, as homologous re-infections are commonly under immunological control. Nevertheless, there is evidence of fatal acute E. ninakohlyakimovae challenge-infections to occur in goat kids. So far, the nature of the immune response promoting protection or failure in E. ninakohlyakimovae challenge-infected goat kids has not been investigated. Therefore, we here analyzed cellular immune responses and histopathological alterations in the gut mucosa of fatal E. ninakohlyakimovae challenge-infected goat kids. Overall, a severe eosinophilic enteritis was observed in affected animals. Hyperplasia of epithelial cells, hypertrophy of globet cells and marked hyperplasia of Peyer's patches and mesenteric lymph nodes draining affected areas were also relevant findings. The majority of goat kids suffering from acute E. ninakohlyakimovae challenge-infections showed severe diarrhoea and some of them even died within 11–16 days post challenge, indicating that the mechanisms of pathogenicity might be related to the first generation macromeronts. Interestingly, infiltration of eosinophils and, in less extent, of intraepithelial lymphocytes and neutrophils was observed even within degraded E. ninakohlyakimovae first generation macromeronts. The excessive immune response mounted against intracellular E. ninakohlyakimovae stages (sporozoites/meronts I) as measured by intestinal leucocyte infiltration might result in severe pro-inflammatory reactions and contribute to the fatal outcome of the challenge infection.

The current study was conducted to isolate a field strain of Eimeria ninakohlyakimovae, characterize its infectivity and the response to challenge under experimental conditions. The isolated strain (GC) induced a prepatent period of 14-15 days p.i., a patency of 7±2 days and a noticeable pathogenicity in infected goat kids. Challenge trials resulting in a decrease of oocysts per gram counts as well as a milder intensity of clinical signs in re-infected animals indicated the capacity of this strain to induce protective immune response. Altogether, the data reported in the present study suggest that the strain E. ninakohlyakimovae GC is a useful tool for the investigation of mechanisms of pathogenicity as well as host protective immune response in caprine coccidiosis, representing a valuable prerequisite for the development of future strategies in prophylaxis and control of this important parasitic disease in goat.

Constructed wetlands (CWs) are successfully applied for the treatment of waters contaminated with aromatic compounds. In these systems, plants provide oxygen and root exudates to the rhizosphere and thereby stimulate microbial degradation processes. Root exudation of oxygen and organic compounds depends on photosynthetic activity and thus may show day-night fluctuations. While diurnal changes in CW effluent composition have been observed, information on respective fluctuations of bacterial activity are scarce. We investigated microbial processes in a CW model system treating toluene-contaminated water which showed diurnal oscillations of oxygen concentrations using metaproteomics. Quantitative real-time PCR was applied to assess diurnal expression patterns of genes involved in aerobic and anaerobic toluene degradation. We observed stable aerobic toluene turnover by Burkholderiales during the day and night. Polyhydroxyalkanoate synthesis was upregulated in these bacteria during the day, suggesting that they additionally feed on organic root exudates while reutilizing the stored carbon compounds during the night via the glyoxylate cycle. Although mRNA copies encoding the anaerobic enzyme benzylsuccinate synthase ( bssA ) were relatively abundant and increased slightly at night, the corresponding protein could not be detected in the CW model system. Our study provides insights into diurnal patterns of microbial processes occurring in the rhizosphere of an aquatic ecosystem. IMPORTANCE Constructed wetlands are a well-established and cost-efficient option for the bioremediation of contaminated waters. While it is commonly accepted knowledge that the function of CWs is determined by the interplay of plants and microorganisms, the detailed molecular processes are considered a black box. Here, we used a well-characterized CW model system treating toluene-contaminated water to investigate the microbial processes influenced by diurnal plant root exudation. Our results indicated stable aerobic toluene degradation by members of the Burkholderiales during the day and night. Polyhydroxyalkanoate synthesis in these bacteria was higher during the day, suggesting that they additionally fed on organic root exudates and reutilized the stored carbon compounds during the night. Our study illuminates microbial processes occurring in the rhizosphere of an aquatic ecosystem.

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.

The proteome of Eimeria bovis meront I-carrying host cells was analyzed by two-dimensional gel electrophoresis (2DE) at 14 days p.i. and compared to non-infected control cells. A total of 221 protein spots were modulated in their abundance in E. bovis-infected host cells and were subsequently analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectometry (MALDI-TOF-MS). These analyses identified 104 proteins in total with 25 host cell proteins being up-regulated and 79 proteins being down-regulated in E. bovis-infected host cells. Moreover, 20 newly expressed proteins were identified exclusively in E. bovis-infected host cells and were most likely of parasite origin. Parasite-induced differences in protein abundance concerned distinct functional categories, with most proteins being involved in host cell metabolism, cell structure, protein fate and gene transcription. Some of the modulated molecules also indicated regulatory processes on the level of host cell stress response (HSP70, HSP90), host cell apoptosis (caspase 8) and actin elongation/depolymerization (α-actinin-1, gelsonin, tropomodulin-3, transgelin). Since merozoites I were already released shortly after cell sampling, the current data reflect the situation at the end of first merogony. This is the first proteomic approach on E. bovis-infected host cells that was undertaken to gain a rather broad insight into Eimeria-induced host cell modulation. The data processed in this investigation should provide a useful basis for more detailed analyses concerning Eimeria-host cell interactions.

Neutrophils are recruited into the female reproductive tract following insemination to eliminate excess spermatozoa and bacteria introduced by the breeding process. In natural breeding, bovine semen is deposited in the vagina and sperm migrate into the uterus, leaving the bulk of seminal plasma (SP) behind. Current artificial insemination protocols introduce variable amounts of SP and semen extender into the uterus. Beside phagozytosis and secretion of immune modulators, polymorphonuclear neutrophils (PMN) are able to form neutrophil extracellular traps (NETs) extruding their DNA into the extracellular environment and ensnare pathogens including sperm cells. Recently, a time-dependent increase in bovine NETs formation has been reported. In the absence of SP and extender only a low degree of NETs formation by the sperm cells alone was detected (Fichtner et al. 2017 Reprod. Domest. Anim. 52(S1), 16). The aim of the present study was to investigate the effect of different semen extenders with various supplements or varying SP concentrations on NETs formation. Bovine PMN were isolated via Ficoll gradient centrifugation from peripheral blood. Semen extenders, purchased from 2 companies, were supplemented either with no animal protein or fresh egg yolk or “egg yolk-like substances” (gamma-irradiated sterile egg yolk or phospholipids as liposomes). The SP collected from ejaculates of eight 5-year-old bulls was added to the incubation medium in concentrations of 1, 3, 5, 10, 15, and 20%. For NETs induction, PMN and semen extenders or SP concentrations were co-cultured for 60 min; PMN and incubation medium alone served as negative controls. After incubation, the samples were treated with micrococcal nuclease and stained with PicoGreen. Quantification was performed by spectrofluorometric analyses using an automated plate monochrome reader (Varioscan Flash; Thermo Scientific, Waltham, MA, USA). Relative fluorescence intensities (FI in arbitrary units, AU) were calculated by subtracting the values of the negative controls from the ones obtained from the different samples. For statistical analyses, a one-way ANOVA followed by Tukey’s HSD test was used. Experiments were repeated at least 9 times. No significant differences in the relative FI were detected between the 2 animal protein-free extenders. A significantly higher FI was observed in one of the extenders supplemented with egg yolk compared with the other. The same held true for one extender completed with an egg yolk-like substance. Relative FI significantly increased from 1 to 5% SP, stayed constant up to 10%, followed by a slight decrease up to a concentration of 20%. These data indicate that formation of NETs in vitro depends on the composition of the extender itself and the protein source used by different companies. Furthermore, formation of NETs is also dependent on the dose of SP. From these results, it could be speculated that semen extenders plus additives and SP may affect fertility. The financial support of the Förderverein Bioökonomieforschung e.V. (FBF) is gratefully acknowledged.