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.

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Leukocytes play a major role in combating infections either by phagocytosis, release of antimicrobial granules, or extracellular trap (ET) formation. ET formation is preceded by a certain leukocyte cell death form, known as ETosis, an evolutionarily conserved mechanism of the innate immune system also observed in marine mammals. Besides several biomolecules and microbial stimuli, marine mammal ETosis is also trigged by various terrestrial protozoa and metazoa, considered nowadays as neozoan parasites, which are circulating in oceans worldwide and causing critical emerging marine diseases. Recent studies demonstrated that pinniped- and cetacean-derived polymorphonuclear neutrophils (PMNs) and monocytes are able to form different phenotypes of ET structures composed of nuclear DNA, histones, and cytoplasmic peptides/proteases against terrestrial apicomplexan parasites, e.g., Toxoplasma gondii and Neospora caninum. Detailed molecular analyses and functional studies proved that marine mammal PMNs and monocytes cast ETs in a similar way as terrestrial mammals, entrapping and immobilizing T. gondii and N. caninum tachyzoites. Pinniped- and cetacean leukocytes induce vital and suicidal ETosis, with highly reliant actions of nicotinamide adenine dinucleotide phosphate oxidase (NOX), generation of reactive oxygen species (ROS), and combined mechanisms of myeloperoxidase (MPO), neutrophil elastase (NE), and DNA citrullination via peptidylarginine deiminase IV (PAD4).This scoping review intends to summarize the knowledge on emerging protozoans in the marine environment and secondly to review limited data about ETosis mechanisms in marine mammalian species.

Neutrophil extracellular traps (NETs) are web-like structures composed of nuclear DNA decorated with histones and cytoplasmic peptides which antiparasitic properties have not previously been investigated in cetaceans. Polymorphonuclear neutrophils (PMN) were isolated from healthy bottlenose dolphins (Tursiops truncatus), and stimulated with Neospora caninum tachyzoites and the NETs-agonist zymosan. In vitro interactions of PMN with the tachyzoites resulted in rapid extrusion of NETs. For the demonstration and quantification of cetacean NETs, extracellular DNA was stained by using either Sytox Orange® or Pico Green®. Scanning electron microscopy (SEM) and fluorescence analyses demonstrated PMN-derived release of NETs upon exposure to tachyzoites of N. caninum. Co-localization studies of N. caninum induced cetacean NETs proved the presence of DNA adorned with histones (H1, H2A/H2B, H3, H4), neutrophil elastase (NE), myeloperoxidase (MPO) and pentraxin (PTX) confirming the molecular properties of mammalian NETosis. Dolphin-derived N. caninum-NETosis were efficiently suppressed by DNase I and diphenyleneiodonium (DPI) treatments. Our results indicate that cetacean-derived NETs represent an ancient, conserved and relevant defense effector mechanism of the host innate immune system against N. caninum and probably other related neozoan parasites circulating in the marine environment.

BackgroundNeospora caninum is an intracellular obligate apicomplexan parasite responsible for multisystemic lesions in dogs. Being definitive hosts and reservoirs, dogs excrete environmentally resistant oocysts. Breeding bitches represent a susceptible dog group and infected bitches may spread this parasite through transplacental transmission.ResultsA total of 218 serum samples of German breeding bitches were collected to determine the presence of N. caninum. Antibodies were detected in 16 (7.33%) bitches using a commercial indirect enzyme-linked immunosorbent assay (ELISA). Immunoblotting analysis confirmed all seropositive samples detected by ELISA, proving that the animals were infected with N. caninum. The owners were interviewed regarding breed, age, environment, type, vaccine status, feeding habits and the presence of reproductive disorders. Seropositive animals were between the ages of two to seven years; three of them were kept in kennels while the others were household dogs, one of which was additionally a hunting dog. Owners of four seropositive bitches reported one gestation, while multiple pregnancies had been recorded for the other twelve bitches. Fourteen bitches were regularly vaccinated and six were fed with fresh raw meat.ConclusionsAlthough the results confirmed a low incidence of N. caninum seropositive German breeding bitches, further epidemiological and surveillance studies are required to complement our findings regarding the current situation of neosporosis in this specific canine population of Germany.

a b s t r a c tA total of 359 sheep serum samples from 15 farms were analyzed for the presence of antibodies against Maedi Visna Virus (MVV) by Enzyme-linked Immunosorbent Assay (ELISA). Additionally, a survey was applied to the sheep owners in order to determine management measures and presence of clinical symptoms of Maedi-Visna in the flocks. Logistic regression with random effects was performed to assess risk factors to seropositivity. Seven serum samples were positive to MVV belonging to six flocks, determining low flock seropositivity (0 to 7.1%). Only one of the five regions studied was negative, determining low overall prevalence (1.95%) and low positivity by region (0.0% to 0.84%). One seropositive animal with symptoms related to MVV was sacrificed for necropsy. An enlargement of mesenteric and celiac lymph nodes was determined, histopathology of lungs revealed acute interstitial pneumonia. Analysis of organs, fluids and blood of this sheep by Polymerase Chain Reaction (PCR) to detect MVV yielded negative results. In order to confirm the serological results, serum and blood samples from two remaining seropositive animals were analyzed six months later, one resulted seronegative and the other seropositive in ELISA, but both negative in PCR. No risk factors were associated to seropositivity. Since MVV produces latency, we conclude, that the positive serological results were due to false positive reactions. Even though, 52.0% of the participating farms had introduced animals, embryos, or semen from other farms or from abroad without any sanitary certification, MVV seems to be present in Costa Rica probably in a very low prevalence. Further studies are required to implement control measures and prevent the spread of the disease.

The presence of antibodies against Toxoplasma gondii and Neospora caninum were analyzed in 392 sheep sera from ten Costa Rican ovine flocks using indirect immuno-enzymatic assays. Additionally, general information about sheep management, environment, and clinical reproductive disorders was assessed through a questionnaire to inquire factors related to these apicomplexan parasites. A total of 161 (41.1%) serum samples reacted positive to T. gondii, 43 (10.9%) to N. caninum and 26 (6.63%) to both parasites. Toxoplasma gondii serorreactors were detected in all the analyzed flocks (100.0%), meanwhile N. caninum antibodies were found in nine flocks (90%), from the six Costa Rican regions. Factors associated with T. gondii were the co-presence of cattle (OR = 5.06; C.I.95%; 2.08–12.30; p: <0.001), grey foxes (Urocyon cinereoargenteus) and opossums (Didelphis marsupialis) (OR = 2.44; C.I.95%; 1.50–3.95; p: <0.001) inside or around the farms, and the presence of peccaries (Tayassu sp.) (OR = 0.35; C.I.95%; 0.16–0.74; p: 0.0058) was a variable associated with N. caninum seropositivity. The obtained results of T. gondii and N. caninum infections in sheep flocks from Costa Rica should be considered for the proper prevention and control strategies against these apicomplexan abortive parasites.

A total of 391 goats from 13 dairy flocks from all Costa Rican regions were analyzed for Toxoplasma gondii-, Neospora caninum-and Coxiella burnetii-related seroprevalence by enzyme-linked immunosorbent assays (ELISA). Additionally, a risk factor analysis for these parasitic infections was performed based on a questionnaire considering several environmental and housing/management factors. A total of 62.1% (243/391) of individual serum samples revealed seropositive for T. gondii, 7.9% (31/391) for N. caninum, and 1.8% (7/391) for C. burnetii. At herd level, the overall seroprevalence for T. gondii was 100%, for N. caninum 69.2% and for C. burnetii 7.7%. However, no clinical signs related to toxoplasmosis, neosporosis or Q fever were apparent in these flocks. T. gondii-related risk factors were the contact with cats (OR = 3.44; CI 95%; 2.0-5.91), dogs (OR = 5.75; CI 95%; 2.84-11.66), and white-tailed deer (Odocoileus virginianus) (OR = 0.15; CI 95%; 0.08-0.26) within or around the farms. The presence of reproductive males in each flock (OR = 0.32; CI 95%; 0.14-0.74) and the coexistence of sheep (OR = 0.46; CI 95%; 0.2-1.08) and cattle (OR = 5.94; CI 95%; 1.70-20.78) revealed as protective and risk factors respectively for N. caninum infections. This study determined for the first time the seroprevalences of N. caninum, T. gondii and C. burnetii in Costa Rican goat flocks. Particularly, the high withinherd seroprevalences determined for T. gondii requires further surveillance to complement these findings.