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Andrew Hemphill - One of the best experts on this subject based on the ideXlab platform.
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neospora caninum structure and fate of multinucleated complexes induced by the bumped kinase inhibitor bki 1294
Pathogenetics, 2020Co-Authors: Pablo Winzer, Luis Miguel Ortegamora, J. Muller, Nicoleta Anghel, Dennis Imhof, Vreni Balmer, Wesley C Van Voorhis, Andrew HemphillAbstract:Background: Bumped kinase inhibitors (BKIs) are potential drugs for neosporosis treatment in farm animals. BKI-1294 exposure results in the formation of multinucleated complexes (MNCs), which remain viable in vitro under constant drug pressure. We investigated the formation of BKI-1294 induced MNCs, the re-emergence of viable Tachyzoites following drug removal, and the localization of CDPK1, the molecular target of BKIs. Methods: N. caninum Tachyzoites and MNCs were studied by TEM and immunofluorescence using antibodies directed against CDPK1, and against NcSAG1 and IMC1 as markers for Tachyzoites and newly formed zoites, respectively. Results: After six days of drug exposure, MNCs lacked SAG1 surface expression but remained intracellular, and formed numerous zoites incapable of disjoining from each other. Following drug removal, proliferation continued, and zoites lacking NcSAG1 emerged from the periphery of these complexes, forming infective Tachyzoites after 10 days. In intracellular Tachyzoites, CDPK1 was evenly distributed but shifted towards the apical part once parasites were extracellular. This shift was not affected by BKI-1294. Conclusions: CDPK1 has a dynamic distribution depending on whether parasites are located within a host cell or outside. During MNC-to-tachyzoite reconversion newly formed Tachyzoites are generated directly from MNCs through zoites of unknown surface antigen composition. Further in vivo studies are needed to determine if MNCs could lead to a persistent reservoir of infection after BKI treatment.
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development and characterization of monoclonal antibodies against besnoitia besnoiti Tachyzoites
Parasitology, 2019Co-Authors: Paula Garcialunar, Javier Regidorcerrillo, Alejandro Jimenezmelendez, A Sanzfernandez, I Garciasoto, Ivan Pastorfernandez, Gereon Schares, Andrew Hemphill, Maria Fernandezalvarez, Luis Miguel OrtegamoraAbstract:This is the first report on the development and characterization of eight monoclonal antibodies (MABs) generated against whole- and membrane-enriched tachyzoite extracts of the apicomplexan parasite Besnoitia besnoiti. Confocal laser scanning immunofluorescence microscopy was used to localize respective epitopes in B. besnoiti Tachyzoites along the lytic cycle. A pattern compatible with dense granule staining was observed with MABs 2.A.12, 2.F.3 and 2.G.4, which could be confirmed by immunogold electron microscopy for MABs 2.A.12 and 2.F.3. In particular, MABs 2.F.3 and 2.G.4 were secreted during early invasion, proliferation and egress phases. MABs 3.10.8 and 5.5.11 labelled the tachyzoite surface, whilst MABs 1.17.8, 8.9.2 and 2.G.A recognized the apical tip, which is reminiscent for microneme localization. Besides, the epitopes recognized by the latter two (MABs 8.9.2 and 2.G.A) exhibited a redistribution from the anterior part across the parasite surface towards the posterior end during invasion. Most MABs developed were genus-specific. Indeed, the MABs cross-reacted neither with T. gondii nor with N. caninum Tachyzoites. In summary, we have generated MABs that will be useful to study the key processes in the lytic cycle of the parasite and with additional promising diagnostic value. However, the molecular identity of the antigens recognized remains to be elucidated.
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in vitro treatment of besnoitia besnoiti with the naphto quinone buparvaquone results in marked inhibition of tachyzoite proliferation mitochondrial alterations and rapid adaptation of Tachyzoites to increased drug concentrations
Parasitology, 2019Co-Authors: J. Muller, Vera Manser, Andrew HemphillAbstract:: We here assessed the in vitro efficacy of the naptho-quinone buparvaquone (BPQ) against Besnoitia besnoiti Tachyzoites in vitro. BPQ is currently licensed for the treatment of theileriosis in cattle in many countries, but not in the EU. In 4-day treatment assays, BPQ massively impaired tachyzoite proliferation with an IC50 of 10 ± 3 nm, and virtually complete inhibition was obtained in the presence of nm BPQ. Exposure to 1 µm BPQ leads to ultrastructural changes affecting initially the mitochondrial matrix and the cristae. After 96 h, most parasites were largely distorted, filled with cytoplasmic amylopectin granules and vacuoles containing components of unknown composition. Host cell mitochondria did not appear to be notably affected by the drug. However, upon prolonged exposure (14-16 days) to increased BPQ concentrations, B. besnoiti Tachyzoites exhibited the capacity to adapt, and they resumed proliferation at dosages of up to 10 µm BPQ, albeit at a lower rate. These BPQ-adapted parasites maintained this lower susceptibility to BPQ treatment after freeze-thawing, and inspection by the transmission electron microscopy revealed that they underwent proliferation in the absence of structurally intact mitochondria.
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host cells participate in the in vitro effects of novel diamidine analogues against Tachyzoites of the intracellular apicomplexan parasites neospora caninum and toxoplasma gondii
Antimicrobial Agents and Chemotherapy, 2008Co-Authors: Angela Leepin, Angela Studli, Reto Brun, Chad E Stephens, David W Boykin, Andrew HemphillAbstract:The in vitro effects of 19 dicationic diamidine derivatives against the proliferative tachyzoite stages of the apicomplexan parasites Neospora caninum and Toxoplasma gondii were investigated. Four compounds (DB811, DB786, DB750, and DB766) with similar structural properties exhibited profound inhibition of tachyzoite proliferation. The lowest 50% inhibitory concentrations were found for DB786 (0.21 microM against Neospora and 0.22 microM against Toxoplasma) and DB750 (0.23 microM against Neospora and 0.16 microM against Toxoplasma), with complete proliferation inhibition at 1.7 microM for both drugs against both species. DB750 and DB786 were chosen for further studies. Electron microscopy of N. caninum-infected human foreskin fibroblast (HFF) cultures revealed distinct alterations and damage of parasite ultrastructure upon drug treatment, while host cells remained unaffected. For true parasiticidal efficacy against N. caninum, a treatment duration of 3 h at 1.7 microM was sufficient for DB750, while a longer treatment period (24 h) was necessary for DB786. Pretreatment of Tachyzoites for 1 h prior to host cell exposure had no effect on infectivity. However, pretreatment of uninfected host cells had a significant adverse effect on N. caninum proliferation: exposure of HFFs to 1.7 microM DB750 for 6, 12, or 24 h, followed by infection with N. caninum Tachyzoites and subsequent culture in the absence of DB750, resulted in significantly delayed parasite proliferation. This suggests that either (i) these compounds or their respective active metabolites were still present after the removal of the drugs or (ii) the drug treatments reversibly impaired some functional activities in HFFs that were essential for parasite proliferation and/or survival.
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in vitro efficacy of nitro and bromo thiazolyl salicylamide compounds thiazolides against besnoitia besnoiti infection in vero cells
Parasitology, 2007Co-Authors: Helder Cortes, Alexandre Leitao, N Mueller, Arunasalam Naguleswaran, Marco Esposito, Andrew HemphillAbstract:Nitazoxanide (NTZ) and its deacetylated metabolite tizoxanide (TIZ) exhibit considerable in vitro activity against Besnoitia besnoiti Tachyzoites grown in Vero cells. Real-time-PCR was used to assess B. besnoiti tachyzoite adhesion, invasion, and intracellular proliferation in vitro. A number of NTZ-derivatives, including Rm4822 and Rm4803, were generated, in which the thiazole-ring-associated nitro-group was replaced by a bromo-moiety. We here show that replacement of the nitro-group on the thiazole ring with a bromo (as it occurs in Rm4822) does not impair the efficacy of the drug, but methylation of the salicylate ring at the ortho-position in a bromo-derivative (Rm4803) results in complete abrogation of the antiparasitic activity. Treatment of extracellular B. besnoiti Tachyzoites with NTZ has an inhibitory effect on host cell invasion, while treatments with TIZ, Rm4822 do not. TEM demonstrates that the effects of Rm4822 treatment upon the parasites are similar to the damage induced by NTZ. This includes increased vacuolization of the parasite cytoplasm, and loss of the structural integrity of the parasitophorous vacuole and its membrane. Thus, Rm4822, due to the absence of a potentially mutagenic nitro-group, may represent an important potential addition to the anti-parasitic arsenal for food animal production, especially in cattle.
Anja Taubert - One of the best experts on this subject based on the ideXlab platform.
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metabolic requirements of besnoitia besnoiti tachyzoite triggered netosis
Parasitology Research, 2020Co-Authors: Ershun Zhou, Ivan Conejeros, Sybille Mazurek, Ulrich Gärtner, Carlos Hermosilla, Anja TaubertAbstract:Besnoitia besnoiti is the causative agent of bovine besnoitiosis, a disease affecting both, animal welfare and cattle productivity. NETosis represents an important and early host innate effector mechanism of polymorphonuclear neutrophils (PMN) that also acts against B. besnoiti Tachyzoites. So far, no data are available on metabolic requirements of B. besnoiti tachyzoite-triggered NETosis. Therefore, here we analyzed metabolic signatures of tachyzoite-exposed PMN and determined the relevance of distinct PMN-derived metabolic pathways via pharmacological inhibition experiments. Overall, tachyzoite exposure induced a significant increase in glucose and serine consumption as well as glutamate production in PMN. Moreover, tachyzoite-induced cell-free NETs were significantly diminished via PMN pre-treatments with oxamate and dichloroacetate which both induce an inhibition of lactate release as well as oxythiamine, which inhibits pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and transketolase, thereby indicating a key role of pyruvate- and lactate-mediated metabolic pathways for proper tachyzoite-mediated NETosis. Furthermore, NETosis was increased by enhanced pH conditions; however, inhibitors of MCT-lactate transporters (AR-C141900, AR-C151858) failed to influence NET formation. Moreover, a significant reduction of tachyzoite-induced NET formation was also achieved by treatments with oligomycin A (inhibitor of ATP synthase) and NF449 (purinergic receptor P2X1 antagonist) thereby suggesting a pivotal role of ATP availability for tachyzoite-mediated NETosis. In summary, the current data provide first evidence on carbohydrate-related metabolic pathways and energy supply to be involved in B. besnoiti tachyzoite-induced NETosis.
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histone h2a and bovine neutrophil extracellular traps induce damage of besnoitia besnoiti infected host endothelial cells but fail to affect total parasite proliferation
Biology, 2019Co-Authors: Ivan Conejeros, Zahady D Velasquez, Daniela Grob, Hannah Salecker, Ershun Zhou, Carlos Hermosilla, Anja TaubertAbstract: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.
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simultaneous and positively correlated net formation and autophagy in besnoitia besnoiti tachyzoite exposed bovine polymorphonuclear neutrophils
Frontiers in Immunology, 2019Co-Authors: Ershun Zhou, Ivan Conejeros, Zahady D Velasquez, Ulrich Gärtner, Carlos Hermosilla, Tamara Munozcaro, Anja TaubertAbstract:: Given that B. besnoiti Tachyzoites infect host endothelial cells of vessels in vivo, they become potential targets for professional phagocytes [e.g., polymorphonuclear neutrophils (PMN)] when in search for adequate host cells or in case of host cell lysis. It was recently reported that B. besnoiti-Tachyzoites can efficiently be trapped by neutrophil extracellular traps (NETs) released by bovine PMN. So far, the potential role of autophagy in parasite-triggered NET formation is unclear. Thus, we here analyzed autophagosome formation and activation of AMP-activated protein kinase α (AMPKα) in potentially NET-forming innate leukocytes being exposed to B. besnoiti Tachyzoites. Blood was collected from healthy adult dairy cows, and bovine PMN were isolated via density gradient centrifugation. Scanning electron microscopy confirmed PMN to undergo NET formation upon contact with B. besnoiti Tachyzoites. Nuclear area expansion (NAE) analysis and cell-free and anchored NETs quantification were performed in B. besnoiti-induced NET formation. Interestingly, Tachyzoites of B. besnoiti additionally induced LC3B-related autophagosome formation in parallel to NET formation in bovine PMN. Notably, both rapamycin- and wortmannin-treatments failed to influence B. besnoiti-triggered NET formation and autophagosome formation. Also, isolated NETs fail to induce autophagy suggesting independence between both cellular processes. Finally, enhanced phosphorylation of AMP activated kinase α (AMPKα), a key regulator molecule of autophagy, was observed within the first minutes of interaction in tachyzoite-exposed PMN thereby emphasizing that B. besnoiti-triggered NET formation indeed occurs in parallel to autophagy.
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antiparasitic efficacy of curcumin against besnoitia besnoiti Tachyzoites in vitro
Frontiers in Veterinary Science, 2019Co-Authors: Maria Eugenia Cervantesvalencia, Yazmin Alcalacanto, Graciela Tapia, Carlos Hermosilla, Anja Taubert, Liliana M. R. SilvaAbstract:: Besnoitia besnoiti is the causative agent of bovine besnoitiosis. B. besnoiti infections lead to reduced fertility and productivity in cattle causing high economic losses, not only in Europe, but also in Asia and Africa. Mild to severe clinical signs, such as anasarca, oedema, orchitis, hyperkeratosis, and characteristic skin and mucosal cysts, are due to B. besnoiti tachyzoite and bradyzoite replication in intermediate host tissues. So far, there are no commercially available effective drugs against this parasite. Curcumin, a polyphenolic compound from Curcuma longa rhizome is well-known for its antioxidant, anti-inflammatory, immunomodulatory and also anti-protozoan effects. Hence, the objective of this study was to evaluate the effects of curcumin on viability, motility, invasive capacity, and proliferation of B. besnoiti Tachyzoites replicating in primary bovine umbilical vein endothelial cells (BUVEC) in vitro. Functional inhibition assays revealed that curcumin treatments reduce tachyzoite viability and induce lethal effects in up to 57% of Tachyzoites (IC50 in 5.93 μM). Referring to general motility, significant dose-dependent effects of curcumin treatments were observed. Interestingly, curcumin treatments only dampened helical gliding and twirling activities whilst longitudinal gliding motility was not significantly affected. In addition, curcumin pretreatments of Tachyzoites resulted in a dose-dependent reduction of host cell invasion as detected by infections rates at 1 day p. i. These findings demonstrate feeding cattle with Curcuma longa rhizomes may represent a new strategy for besnoitiosis treatment.
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Image_1_Antiparasitic Efficacy of Curcumin Against Besnoitia besnoiti Tachyzoites in vitro.TIF
2019Co-Authors: María Eugenia Cervantes-valencia, Graciela Tapia, Carlos Hermosilla, Anja Taubert, Yazmín Alcalá-canto, Liliana M. R. SilvaAbstract:Besnoitia besnoiti is the causative agent of bovine besnoitiosis. B. besnoiti infections lead to reduced fertility and productivity in cattle causing high economic losses, not only in Europe, but also in Asia and Africa. Mild to severe clinical signs, such as anasarca, oedema, orchitis, hyperkeratosis, and characteristic skin and mucosal cysts, are due to B. besnoiti tachyzoite and bradyzoite replication in intermediate host tissues. So far, there are no commercially available effective drugs against this parasite. Curcumin, a polyphenolic compound from Curcuma longa rhizome is well-known for its antioxidant, anti-inflammatory, immunomodulatory and also anti-protozoan effects. Hence, the objective of this study was to evaluate the effects of curcumin on viability, motility, invasive capacity, and proliferation of B. besnoiti Tachyzoites replicating in primary bovine umbilical vein endothelial cells (BUVEC) in vitro. Functional inhibition assays revealed that curcumin treatments reduce tachyzoite viability and induce lethal effects in up to 57% of Tachyzoites (IC50 in 5.93 μM). Referring to general motility, significant dose-dependent effects of curcumin treatments were observed. Interestingly, curcumin treatments only dampened helical gliding and twirling activities whilst longitudinal gliding motility was not significantly affected. In addition, curcumin pretreatments of Tachyzoites resulted in a dose-dependent reduction of host cell invasion as detected by infections rates at 1 day p. i. These findings demonstrate feeding cattle with Curcuma longa rhizomes may represent a new strategy for besnoitiosis treatment.
Carlos Hermosilla - One of the best experts on this subject based on the ideXlab platform.
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metabolic requirements of besnoitia besnoiti tachyzoite triggered netosis
Parasitology Research, 2020Co-Authors: Ershun Zhou, Ivan Conejeros, Sybille Mazurek, Ulrich Gärtner, Carlos Hermosilla, Anja TaubertAbstract:Besnoitia besnoiti is the causative agent of bovine besnoitiosis, a disease affecting both, animal welfare and cattle productivity. NETosis represents an important and early host innate effector mechanism of polymorphonuclear neutrophils (PMN) that also acts against B. besnoiti Tachyzoites. So far, no data are available on metabolic requirements of B. besnoiti tachyzoite-triggered NETosis. Therefore, here we analyzed metabolic signatures of tachyzoite-exposed PMN and determined the relevance of distinct PMN-derived metabolic pathways via pharmacological inhibition experiments. Overall, tachyzoite exposure induced a significant increase in glucose and serine consumption as well as glutamate production in PMN. Moreover, tachyzoite-induced cell-free NETs were significantly diminished via PMN pre-treatments with oxamate and dichloroacetate which both induce an inhibition of lactate release as well as oxythiamine, which inhibits pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and transketolase, thereby indicating a key role of pyruvate- and lactate-mediated metabolic pathways for proper tachyzoite-mediated NETosis. Furthermore, NETosis was increased by enhanced pH conditions; however, inhibitors of MCT-lactate transporters (AR-C141900, AR-C151858) failed to influence NET formation. Moreover, a significant reduction of tachyzoite-induced NET formation was also achieved by treatments with oligomycin A (inhibitor of ATP synthase) and NF449 (purinergic receptor P2X1 antagonist) thereby suggesting a pivotal role of ATP availability for tachyzoite-mediated NETosis. In summary, the current data provide first evidence on carbohydrate-related metabolic pathways and energy supply to be involved in B. besnoiti tachyzoite-induced NETosis.
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histone h2a and bovine neutrophil extracellular traps induce damage of besnoitia besnoiti infected host endothelial cells but fail to affect total parasite proliferation
Biology, 2019Co-Authors: Ivan Conejeros, Zahady D Velasquez, Daniela Grob, Hannah Salecker, Ershun Zhou, Carlos Hermosilla, Anja TaubertAbstract: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.
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simultaneous and positively correlated net formation and autophagy in besnoitia besnoiti tachyzoite exposed bovine polymorphonuclear neutrophils
Frontiers in Immunology, 2019Co-Authors: Ershun Zhou, Ivan Conejeros, Zahady D Velasquez, Ulrich Gärtner, Carlos Hermosilla, Tamara Munozcaro, Anja TaubertAbstract:: Given that B. besnoiti Tachyzoites infect host endothelial cells of vessels in vivo, they become potential targets for professional phagocytes [e.g., polymorphonuclear neutrophils (PMN)] when in search for adequate host cells or in case of host cell lysis. It was recently reported that B. besnoiti-Tachyzoites can efficiently be trapped by neutrophil extracellular traps (NETs) released by bovine PMN. So far, the potential role of autophagy in parasite-triggered NET formation is unclear. Thus, we here analyzed autophagosome formation and activation of AMP-activated protein kinase α (AMPKα) in potentially NET-forming innate leukocytes being exposed to B. besnoiti Tachyzoites. Blood was collected from healthy adult dairy cows, and bovine PMN were isolated via density gradient centrifugation. Scanning electron microscopy confirmed PMN to undergo NET formation upon contact with B. besnoiti Tachyzoites. Nuclear area expansion (NAE) analysis and cell-free and anchored NETs quantification were performed in B. besnoiti-induced NET formation. Interestingly, Tachyzoites of B. besnoiti additionally induced LC3B-related autophagosome formation in parallel to NET formation in bovine PMN. Notably, both rapamycin- and wortmannin-treatments failed to influence B. besnoiti-triggered NET formation and autophagosome formation. Also, isolated NETs fail to induce autophagy suggesting independence between both cellular processes. Finally, enhanced phosphorylation of AMP activated kinase α (AMPKα), a key regulator molecule of autophagy, was observed within the first minutes of interaction in tachyzoite-exposed PMN thereby emphasizing that B. besnoiti-triggered NET formation indeed occurs in parallel to autophagy.
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antiparasitic efficacy of curcumin against besnoitia besnoiti Tachyzoites in vitro
Frontiers in Veterinary Science, 2019Co-Authors: Maria Eugenia Cervantesvalencia, Yazmin Alcalacanto, Graciela Tapia, Carlos Hermosilla, Anja Taubert, Liliana M. R. SilvaAbstract:: Besnoitia besnoiti is the causative agent of bovine besnoitiosis. B. besnoiti infections lead to reduced fertility and productivity in cattle causing high economic losses, not only in Europe, but also in Asia and Africa. Mild to severe clinical signs, such as anasarca, oedema, orchitis, hyperkeratosis, and characteristic skin and mucosal cysts, are due to B. besnoiti tachyzoite and bradyzoite replication in intermediate host tissues. So far, there are no commercially available effective drugs against this parasite. Curcumin, a polyphenolic compound from Curcuma longa rhizome is well-known for its antioxidant, anti-inflammatory, immunomodulatory and also anti-protozoan effects. Hence, the objective of this study was to evaluate the effects of curcumin on viability, motility, invasive capacity, and proliferation of B. besnoiti Tachyzoites replicating in primary bovine umbilical vein endothelial cells (BUVEC) in vitro. Functional inhibition assays revealed that curcumin treatments reduce tachyzoite viability and induce lethal effects in up to 57% of Tachyzoites (IC50 in 5.93 μM). Referring to general motility, significant dose-dependent effects of curcumin treatments were observed. Interestingly, curcumin treatments only dampened helical gliding and twirling activities whilst longitudinal gliding motility was not significantly affected. In addition, curcumin pretreatments of Tachyzoites resulted in a dose-dependent reduction of host cell invasion as detected by infections rates at 1 day p. i. These findings demonstrate feeding cattle with Curcuma longa rhizomes may represent a new strategy for besnoitiosis treatment.
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Image_1_Antiparasitic Efficacy of Curcumin Against Besnoitia besnoiti Tachyzoites in vitro.TIF
2019Co-Authors: María Eugenia Cervantes-valencia, Graciela Tapia, Carlos Hermosilla, Anja Taubert, Yazmín Alcalá-canto, Liliana M. R. SilvaAbstract:Besnoitia besnoiti is the causative agent of bovine besnoitiosis. B. besnoiti infections lead to reduced fertility and productivity in cattle causing high economic losses, not only in Europe, but also in Asia and Africa. Mild to severe clinical signs, such as anasarca, oedema, orchitis, hyperkeratosis, and characteristic skin and mucosal cysts, are due to B. besnoiti tachyzoite and bradyzoite replication in intermediate host tissues. So far, there are no commercially available effective drugs against this parasite. Curcumin, a polyphenolic compound from Curcuma longa rhizome is well-known for its antioxidant, anti-inflammatory, immunomodulatory and also anti-protozoan effects. Hence, the objective of this study was to evaluate the effects of curcumin on viability, motility, invasive capacity, and proliferation of B. besnoiti Tachyzoites replicating in primary bovine umbilical vein endothelial cells (BUVEC) in vitro. Functional inhibition assays revealed that curcumin treatments reduce tachyzoite viability and induce lethal effects in up to 57% of Tachyzoites (IC50 in 5.93 μM). Referring to general motility, significant dose-dependent effects of curcumin treatments were observed. Interestingly, curcumin treatments only dampened helical gliding and twirling activities whilst longitudinal gliding motility was not significantly affected. In addition, curcumin pretreatments of Tachyzoites resulted in a dose-dependent reduction of host cell invasion as detected by infections rates at 1 day p. i. These findings demonstrate feeding cattle with Curcuma longa rhizomes may represent a new strategy for besnoitiosis treatment.
Gereon Schares - One of the best experts on this subject based on the ideXlab platform.
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development and characterization of monoclonal antibodies against besnoitia besnoiti Tachyzoites
Parasitology, 2019Co-Authors: Paula Garcialunar, Javier Regidorcerrillo, Alejandro Jimenezmelendez, A Sanzfernandez, I Garciasoto, Ivan Pastorfernandez, Gereon Schares, Andrew Hemphill, Maria Fernandezalvarez, Luis Miguel OrtegamoraAbstract:This is the first report on the development and characterization of eight monoclonal antibodies (MABs) generated against whole- and membrane-enriched tachyzoite extracts of the apicomplexan parasite Besnoitia besnoiti. Confocal laser scanning immunofluorescence microscopy was used to localize respective epitopes in B. besnoiti Tachyzoites along the lytic cycle. A pattern compatible with dense granule staining was observed with MABs 2.A.12, 2.F.3 and 2.G.4, which could be confirmed by immunogold electron microscopy for MABs 2.A.12 and 2.F.3. In particular, MABs 2.F.3 and 2.G.4 were secreted during early invasion, proliferation and egress phases. MABs 3.10.8 and 5.5.11 labelled the tachyzoite surface, whilst MABs 1.17.8, 8.9.2 and 2.G.A recognized the apical tip, which is reminiscent for microneme localization. Besides, the epitopes recognized by the latter two (MABs 8.9.2 and 2.G.A) exhibited a redistribution from the anterior part across the parasite surface towards the posterior end during invasion. Most MABs developed were genus-specific. Indeed, the MABs cross-reacted neither with T. gondii nor with N. caninum Tachyzoites. In summary, we have generated MABs that will be useful to study the key processes in the lytic cycle of the parasite and with additional promising diagnostic value. However, the molecular identity of the antigens recognized remains to be elucidated.
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a new lyophilized tachyzoite based elisa to diagnose besnoitia spp infection in bovids and wild ruminants improves specificity
Veterinary Parasitology, 2017Co-Authors: Paula Garcialunar, Luis Miguel Ortegamora, Carlos Diezmadiaz, Gereon Schares, Gema AlvarezgarciaAbstract:Abstract Recent studies have reported that routinely used whole or soluble Besnoitia besnoiti tachyzoite (TZ) extract-based ELISAs potentially give rise to a high number of false-positive results, which may compromise control and the epidemiological studies of bovine besnoitiosis. Thus, western blot (WB) has been recommended as a confirmatory test. In the present study, a new ELISA test that employs lyophilized Tachyzoites for the first time (BbSALUVET ELISA 2.0) was developed and validated with cattle sera (n = 606) under a worst-case scenario. False positive and false negative, soluble TZ extract-based BbSALUVET ELISA 1.0 reactors were overrepresented, and WB was used as the reference test. One commercial test (PrioCHECK Besnoitia Ab 2.0, which employs whole TZ extract) and a recently developed membrane-enriched ELISA (APure-BbELISA) were also tested. The three ELISAs showed high AUC values (>0.9). However, the best diagnostic performance corresponded to the BbSALUVET ELISA 2.0 and the APure-BbELISA [(92% sensitivity (Se) and 98% specificity (Sp)] followed by PrioCHECK Besnoitia Ab 2.0 (88% Se, 98% Sp, and 4.5% doubtful results). In addition, the BbSALUVET ELISA 2.0 was validated with wild ruminant sera, and excellent performance (96% Se, 97% Sp, and 4% doubtful results) was obtained again. A different antigenic composition of the lyophilized Tachyzoites, compared with whole or soluble tachyzoite extracts, may be responsible for the improved diagnostic performance. This study proposes the use of the BbSALUVET ELISA 2.0 in cattle prior to entry to herds free of the disease and in valuable samples prior to a selective culling without the need of a confirmatory Western Blot test in positive samples due to its excellent specificity.
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exploring the life cycle of besnoitia besnoiti experimental infection of putative definitive and intermediate host species
Veterinary Parasitology, 2011Co-Authors: Walter Basso, Maja Rutten, Gereon Schares, N S Gollnick, Peter DeplazesAbstract:The biology of Besnoitia besnoiti, the cause of bovine besnoitiosis, is poorly understood. Its definitive host is unknown, and information on potential intermediate hosts is scarce. In order to investigate potential definitive and intermediate hosts for European isolates of B. besnoiti, domestic dogs, cats, rabbits, guinea pigs (Cavia orcellus), gerbils (Meriones unguiculatus), common voles (Microtus arvalis) and NMRI-mice were inoculated with B. besnoiti isolated from naturally infected German cattle. Dogs and cats were fed 5×106 B. besnoiti Tachyzoites (isolate Bb-GER1), or tissue cysts containing at least 2×107 B. besnoiti bradyzoites obtained from the skin of a naturally infected Limousin cow from the same herd where strain Bb-GER1 was isolated. Rodents and rabbits were subcutaneously inoculated with either 5×105 Bb-GER1 Tachyzoites or 5×105 bradyzoites. Groups of 2–4 non-inoculated animals of each species were monitored as negative controls. Feces from all dogs and cats were daily examined by a sedimentation–flotation technique for at least 11 weeks after inoculation but no B. besnoiti oocysts were identified. Cats fed Tachyzoites and dogs did not seroconvert, but specific antibodies to B. besnoiti Tachyzoites were detected by IFAT (titer≥100) in 2 out of 3 cats fed tissue cysts since 5–7 weeks post infection. By immunoblot, these two cats exhibited a reaction pattern against tachyzoite antigens similar to that observed in naturally infected cattle. Antibodies against B. besnoiti Tachyzoites were detected in all inoculated rodent species and rabbits by both, IFAT and immunoblot since 3 weeks post-inoculation. Rabbits and rodents, subcutaneously inoculated with same doses of inactivated bradyzoites remained serologically negative (IFAT titer < 50). Clinical signs observed in the inoculated rabbits included fever, serous conjunctivitis and transient swelling of the testes. No clinical abnormalities were noticed in the other tested animal species. Voles developed pneumonia as observed by histological examination. B. besnoiti-DNA was detected by PCR in blood from rabbits, gerbils and voles at 9 days post-infection, and in skin, heart, lung, striated muscle and kidney tissues from voles at 19–21 weeks post-infection. Domestic dogs and cats could not be shown to be definitive hosts of B. besnoiti, but cats seroconverted after feeding on B. besnoiti tissue cysts indicating that B. besnoiti stages had invaded the cats’ tissues. The molecular and serological results from this study indicate that European B. besnoiti isolates may infect cats, rabbits, guinea pigs, gerbils, mice
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Characterisation of NcGRA7 and NcSAG4 proteins: Immunolocalisation and their role in the host cell invasion by Neospora caninum Tachyzoites
Acta Parasitologica, 2010Co-Authors: Adriana Aguado-martínez, Gereon Schares, Gema Álvarez-garcía, Verónica Risco-castillo, Aurora Fernández-garcía, Virginia Marugán-hernández, Luis M. Ortega-moraAbstract:Neospora caninum negatively impacts bovine reproductive performance around the world. Addressing this problem requires a greater understanding of the parasite’s molecular biology. In this study, monoclonal antibodies against recombinant proteins were successfully developed and employed to characterise two different proteins of N. caninum : the acute phase-associated NcGRA7 and the chronic phase-associated NcSAG4. Immunofluorescence with the anti-rNcGRA7 monoclonal antibody suggested that NcGRA7 trafficks from tachyzoite dense granules to the matrix of the parasitophorous vacuole and parasite’s surroundings. Furthermore, NcGRA7 is also expressed in the bradyzoite stage and localised on the matrix of bradyzoite-positive vacuoles. NcGRA7 appears to be partially involved in the tachyzoite-invasion mechanisms, as an anti-rNcGRA7 monoclonal antibody partially inhibited in vitro tachyzoite-invasion. A monoclonal antibody specific for NcSAG4 confirmed this protein’s bradyzoitespecific expression both by western blot and immunofluorescence. However, some bradyzoite-positive vacuoles only weakly expressed NcSAG4, if it was expressed at all. The specificity of the anti-rNcSAG4 monoclonal antibody was confirmed by the recognition of the NcSAG4 in the membrane surface of Nc-1SAG4^c transgenic Tachyzoites, which constitutively expresses NcSAG4. Blocking NcSAG4 of Nc-1SAG4^c Tachyzoites with the monoclonal antibody did not affect host cell invasion. However, its implication on the host cell adhesion or host immune evasion should not be discarded.
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comparative evaluation of immunofluorescent antibody and new immunoblot tests for the specific detection of antibodies against besnoitia besnoiti Tachyzoites and bradyzoites in bovine sera
Veterinary Parasitology, 2010Co-Authors: Gereon Schares, Josef Selmair, Ana Rostaher, J C Scharr, M Majzoub, Walter Basso, J P Dubey, Martin C. Langenmayer, Helder CortesAbstract:Besnoitia besnoiti, an apicomplexan parasite causes economically important disease in cattle in many countries of Africa and Asia is re-emerging in Europe. Serological identification of infected cattle is important because introduction of these animals into naive herds seems to play a major role in the transmission of the parasite. We report new, simplified immunoblot-based serological tests for the detection of B. besnoiti-specific antibodies. Antigens were used under non-reducing conditions in the immunoblots, because reduction of the antigen with beta-mercaptoethanol diminished the antigenicity in both, Tachyzoites and bradyzoites. Ten B. besnoiti tachyzoite and ten bradyzoite antigens of 15-45 kDa molecular weight were recognized by B. besnoiti infected cattle, but not or only weakly detected by cattle infected with related protozoan parasites, Neospora caninum, Toxoplasma gondii, Sarcocystis cruzi, Sarcocystis hominis, or Sarcocystis hirsuta. The sensitivity and specificity of B. besnoiti immunoblots were determined with sera from 62 German cattle with clinically confirmed besnoitiosis and 404 sera from unexposed German cattle including 214 sera from animals with a N. caninum-specific antibody response. Using a new scoring system, the highest specificity (100%) and sensitivity (90%) of the immunoblots were observed when reactivity to at least four of the ten selected tachyzoite or bradyzoite antigens was considered as positive. When a cut-off based on this scoring system was applied to both the tachyzoite- and the bradyzoite-based immunoblots, there was an almost perfect agreement with the indirect fluorescent antibody test with a titre of 200 as the positive cut-off. We identified and partially characterized 10 tachyzoite and 10 bradyzoite B. besnoiti antigens which may help to develop new specific and sensitive serological tests based on individual antigens and in the identification of possible vaccine candidates. (C) 2010 Elsevier B.V. All rights reserved
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immunogenic multistage recombinant protein vaccine confers partial protection against experimental toxoplasmosis mimicking natural infection in murine model
Trials in Vaccinology, 2016Co-Authors: Yaprak Gedik, Y Gürüz, Sultan Gulce Iz, Aysu Degirmenci Doskaya, Ismet Deliloglu S Gurhan, M DöskayaAbstract:Toxoplasma gondii is a protozoan parasite that can infect warm-blooded animals including humans. Vaccination studies mostly use tachyzoite specific proteins however in natural route of infection, toxoplasmosis initiates with tissue cysts (bradyzoites) or oocysts (sporozoites) and thereafter stage conversion takes place where the Tachyzoites take action and cause acute infection continues with Tachyzoites. Despite this knowledge, challenging models used in the vaccination studies prefer administration of tissue cyst forming strains intraperitoneally or subcutaneously instead of oral administration which is the natural route of infection. In the present study, a multivalent adjuvanted recombinant protein vaccine that contains bradyzoite specific BAG1 and tachyzoite specific GRA1 protein and controls were administered to female Swiss Webster outbred mice. Humoral and cellular immune responses were analyzed by Rec-ELISA, Western blot, and flow cytometry. Mice were infected orally with T. gondii PRU strain tissue cysts using feeding needle to mimic the natural route of infection. 40 days after challenging microscopy and Real Time PCR were performed to determine the protection level. Analysis of sera obtained from vaccinated mice showed strong anti-BAG1 and anti-GRA1 IgG responses. The IgG2a response was significantly higher (P < 0.0001) and the ratio of CD8 + T lymphocytes secreting IFN-γ almost doubled compared to PBS control which are indicative of protection against toxoplasmosis. The amount of tissue cysts in vaccinated group was reduced 10.5% compared to control group. To generate a protective vaccine against toxoplasmosis, multistage vaccines and usage of challenging models mimicking natural route of infection are critical cornerstones. In this study, we generated a BAG1 and GRA1 multistage vaccine that induced strong immune response in which the protection was not at anticipated level. In addition, the murine model was orally challenged with tissue cysts to mimic natural route of infection.
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toxoplasma gondii rh ankara production of evolving Tachyzoites using a novel cell culture method
Experimental Parasitology, 2011Co-Authors: A Degirmenci, M Döskaya, C Ciçek, M Korkmaz, Y Gürüz, Ayse Caner, A UnerAbstract:Abstract Toxoplasma gondii is one of the most researched parasite due to its easy growth both in vitro and in vivo. Tachyzoites, derived from mouse or rat peritoneum encounters ethical and economical problems when used for research or diagnostic purposes. Currently, research has focused on determining the most suitable cell culture environment to reach highest amount of viable Tachyzoites with least host cell contamination. However, gene expression changes that take place throughout the adaptation of evolving T. gondii strains to continuous cell cultures appear as a problem. The present study aimed to determine a novel cell culture strategy for T. gondii RH Ankara strain Tachyzoites to harvest abundant Tachyzoites with least host cell contamination and minimal antigenic variation at predetermined dates to use as an antigen source in serological assays that will facilitate reduction in animal use. To achieve this purpose, T. gondii RH Ankara strain Tachyzoites were incubated with HeLa cell at different ratios for two or three days. In all flasks incubated for two days, viability rate reached to 100% and HeLa cell contamination decreased to levels between 0.12–0.5 × 106/ml. In the flasks with HeLa-tachyzoite ratio 1/8, the tachyzoite yield and viability ratio were 3 × 106/ml and 100%, respectively, with accompanying 10 fold decrease (0.12 × 106/ml) in HeLa contamination. During continuous production, highest tachyzoite yield was obtained from the first passage (3.55 × 106/ml) and until the end of third subculture viability rates and HeLa cell contaminations were between 98.2–99.4% and 0.31–0.37 × 106/ml, respectively. ELISA, IFA and Western blot analyses showed that the quality, specificity and sensitivity of the antigen harvested from the first passage of cell culture performed at two days intervals were comparable to the antigen harvested from mice and decreased in the following subcultures. Overall, these results demonstrated that T. gondii RH Ankara strain is still evolving to adapt to cell culture environment and therefore such strains continuously produced in cell cultures should be avoided for serological assays. However, the two day short interval cell culture method described herein offers a chance to reduce the animal use intended for the preparation of serological assays’ antigen from local evolving strains.
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Behaviour of Toxoplasma gondii RH Ankara strain Tachyzoites during continuous production in various cell lines.
Parasitology, 2005Co-Authors: M Döskaya, A Degirmenci, A Deirmenc, C Ciçek, M Korkmaz, Y Gürüz, A UnerAbstract:Toxoplasma gondii is an obligate intracellular protozoan parasite. The objective of the present study was to examine the behaviour of Toxoplasma gondii RH Ankara strain Tachyzoites in a cell culture environment. The study represents the first step in determining whether T. gondii RH Ankara strain Tachyzoites, grown in cell culture, are of sufficient quality to allow cessation of in vivo tachyzoite production for diagnostic assays. In the present study, T. gondii RH Ankara strain Tachyzoites were continuously produced in myeloma X63.Ag8.653, HeLa, Hep-2, and Vero cell cultures for 2 months. The average size of the Tachyzoites was 3 x 5.7 microm prior to the first inoculation but after continuous production, a marked decrease was noted in average tachyzoite size. The smallest tachyzoite size, was 1 x 2.1 microm after 2 months, in myeloma cell cultures even though the yield of Tachyzoites increased. With other cell cultures, tachyzoite yields were not as high as myeloma cell culture although decrease in size was less. The smallest decrease in tachyzoite size, averaging 2 x 3.8 microm after 2 months, was observed in Tachyzoites produced in HeLa cell cultures. A virulence assay in small groups of BALB/c mice, using Tachyzoites derived from cell cultures, was also conducted. The preliminary results of the virulence assay suggest that as the size of the Tachyzoites decreased, the virulence in mice decreased. Future research will focus on the effect of the size of cell culture-derived T. gondii RH Ankara strain Tachyzoites on the virulence, protein expression, and the reliability of diagnostic assays. Ultimately, the behaviour of Tachyzoites from various T. gondii strains will be observed in cell culture to determine if size is altered.
