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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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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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monoclonal antibody directed against neospora caninum Tachyzoite carbohydrate epitope reacts specifically with apical complex associated sialylated beta tubulin
Journal of Parasitology, 2006Co-Authors: Sangeetha Srinivasan, Timothy V Baszler, Angela Leepin, Nathalie Vonlaufen, Sanya J Sanderson, Jonathan M Wastling, Andrew HemphillAbstract:Monoclonal antibodies (mabs) were generated against whole sonicated Neospora caninum Tachyzoites as immunogen. Initial ELISA screening of the reactivity of hybridoma culture supernatants using the same antigen and antigen treated with sodium periodate prior to antibody binding resulted in the identification of 8 supernatants with reactivity against putative carbohydrate epitopes. Following immunoblotting, mab6D12 (IgG1), binding a 52/48-kDa doublet, and mab6C6 (IgM), binding a 190/180-kDa doublet, were selected for further studies. Immunofluorescence of Tachyzoite-infected cultures localized the corresponding epitopes not to the surface, but to interior epitopes at the apical part of N. caninum Tachyzoites. During in vitro Tachyzoite to bradyzoite stage conversion, mab6C6 labeling translocated toward the cyst periphery, while for mab6D12 no changes in localization were noted. Upon extraction of Tachyzoites with the nonionic detergent Triton-X-100, the 52-kDa band recognized by mab6D12 was present exclusively in the insoluble, cytoskeletal fraction of both N. caninum and Toxoplasma gondii Tachyzoites. Tandem mass spectrometry analysis identified this protein as N. caninum beta tubulin. The 48-kDa band labeled by mab6D12 was a Vero cell protein contamination. The protein(s) reacting with mab6C6 could not be conclusively identified by mass spectrometry. Immunofluorescence consistently failed to label T. gondii Tachyzoites, indicating that beta tubulin in T. gondii and N. caninum could be differentially modified or that the reactive epitope in T. gondii is masked. Immunogold TEM of isolated apical cytoskeletal preparations and dual immunofluorescence with antibody to tubulin confirmed that mab6D12 binds to the anterior part of apical complex-associated microtubules. The sodium periodate sensitivity of the beta tubulin associated epitope was confirmed by immunoblotting and ELISA, and treatment of N. caninum cytoskeletal proteins with sialidase prior to mab6D12 labeling resulted in a profound loss of antibody binding, suggesting that mab6D12 reacts with sialylated beta tubulin.
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neospora caninum protein disulfide isomerase is involved in Tachyzoite host cell interaction
International Journal for Parasitology, 2005Co-Authors: Arunasalam Naguleswaran, Nathalie Vonlaufen, Ferial Alaeddine, Christophe Guionaud, Sabrina Sonda, Paul Jenoe, Meike Mevissen, Andrew HemphillAbstract:We have previously shown that treatment of Neospora caninum Tachyzoites with the aspartyl protease inhibitor pepstatin A reduces host cell invasion [Naguleswaran, A., Muller, N., Hemphill, A., 2003. Neospora caninum and Toxoplasma gondii: a novel adhesion/invasion assay reveals distinct differences in Tachyzoite-host cell interactions. Exp. Parasitol. 104, 149-158]. Pepstatin A-affinity-chromatography led to the isolation of a major band of approximately 52 kDa which was identified as a homologue of a previously described Toxoplasma gondii putative protein disulfide isomerase (TgPDI) through tandem mass spectrometry. A BLAST search against N. caninum expressed sequence tags (ESTs) on the ApiDots server using TgPDI cDNA as query sequence revealed a 2251 bp PDI-like consensus (NcPDI), which shows 94% identity to the T. gondii homologue. In N. caninum Tachyzoites, NcPDI was found mainly in the soluble hydrophilic fraction. Immunofluorescence showed that expression of NcPDI was dramatically down-regulated in the bradyzoite stage, and immunogold-EM on Tachyzoites localised the protein to the cytoplasm, mostly in close vicinity to the nuclear membrane, to the micronemes, and to the parasite cell surface. However, NcPDI was absent in rhoptries and dense granules. Preincubation of Tachyzoites with the sulfhydryl blocker 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoic acid (pCMBA), and with the PDI inhibitor bacitracin reduced adhesion of parasites to host cells. In addition, incubation of N. caninum Tachyzoites with affinity-purified anti-NcPDI antibodies reduced host cell adhesion. PDIs catalyse the formation, reduction or isomerisation of disulfide bonds. Many major components of the adhesion and invasion machinery of apicomplexan parasites are cysteine-rich and dependent on correct folding via disulfide bond formation. Thus, our data points towards an important role for surface-associated NcPDI in Neospora-host cell interaction.
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.
Luis Miguel Ortegamora - One of the best experts on this subject based on the ideXlab platform.
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the impact of bki 1294 therapy in mice infected with the apicomplexan parasite neospora caninum and re infected during pregnancy
Frontiers in Veterinary Science, 2020Co-Authors: Pablo Winzer, Luis Miguel Ortegamora, Adriana Aguadomartinez, Nicoleta Anghel, Dennis Imhof, Dominic Ritler, Joachim Muller, Ghalia Boubaker, Kayode K Ojo, Wesley C VanvoorhisAbstract:Exposure of Neospora caninum Tachyzoites to BKI-1294 in vitro results in the formation of long-lived multinucleated complexes (MNCs). However, in vivo treatment of BALB/c mice with BKI-1294 shortly after N. caninum infection during pregnancy was safe and profoundly reduced pup mortality and vertical transmission. We hypothesized that the formation of MNCs could trigger immune responses that contribute to BKI efficacy in vivo. In this study, mice were first vaccinated with a sublethal dose of N. caninum Tachyzoites and were treated with BKI-1294. We then investigated the effects of these treatments after mating and re-infection during pregnancy. Effects on fertility, pup survival, vertical transmission, and parasite load in dams were evaluated. Cytokines in sera or splenocyte culture supernatants were assessed by either ELISA or the Luminex™ 200 system, and humoral immune responses against Tachyzoite and MNC antigens were compared by ELISA, Western blotting and immunoproteomics. Our results showed that BKI-1294 treatment of live-vaccinated mice reduced the cerebral parasite load in the dams, but resulted in higher neonatal pup mortality and vertical transmission. In live-vaccinated mice, cytokine levels, most notably IFN-y, IL-10, and IL-12, were consistently lower in BKI-1294 treated animals compared to non-treated mice. In addition, comparative Western blotting identified two protein bands in MNC extracts that were only recognized by sera of live-vaccinated mice treated with BKI-1294, and were not found in Tachyzoite extracts. We conclude that treatment of live-vaccinated mice with BKI-1294 influenced the cellular and humoral immune responses against infection, affected the safety of the live-vaccine, and decreased protection against re-infection and vertical transmission during pregnancy.
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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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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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identification of besnoitia besnoiti proteins that showed differences in abundance between Tachyzoite and bradyzoite stages by difference gel electrophoresis
Parasitology, 2013Co-Authors: Aranzazu Fernandezgarcia, Paula Garcialunar, Gema Alvarezgarcia, Virginia Maruganhernandez, V Riscocastillo, Adriana Aguadomartinez, Luis Miguel OrtegamoraAbstract:: Bovine besnoitiosis is a chronic and debilitating disease, caused by the apicomplexan parasite Besnoitia besnoiti. Infection of cattle by B. besnoiti is governed by the Tachyzoite stage, which is related to acute infection, and the bradyzoite stage gathered into macroscopic cysts located in subcutaneous tissue in the skin, mucosal membranes and sclera conjunctiva and related to persistence and chronic infection. However, the entire life cycle of this parasite and the molecular mechanisms underlying Tachyzoite-to-bradyzoite conversion remain unknown. In this context, a different antigenic pattern has been observed between Tachyzoite and bradyzoite extracts. Thus, to identify stage-specific proteins, a difference gel electrophoresis (DIGE) approach was used on Tachyzoite and bradyzoite extracts followed by mass spectrometry (MS) analysis. A total of 130 and 132 spots were differentially expressed in bradyzoites and Tachyzoites, respectively (average ratio ± 1.5, P<0.05 in t-test). Furthermore, 25 differentially expressed spots were selected and analysed by MALDI-TOF/MS. As a result, 5 up-regulated bradyzoite proteins (GAPDH, ENO1, LDH, SOD and RNA polymerase) and 5 up-regulated Tachyzoite proteins (ENO2; LDH; ATP synthase; HSP70 and PDI) were identified. The present results set the basis for the identification of new proteins as drug targets. Moreover, the role of these proteins in Tachyzoite-to-bradyzoite conversion and the role of the host cell environment should be a subject of further research.
J P Dubey - One of the best experts on this subject based on the ideXlab platform.
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cxcl9 is important for recruiting immune t cells into the brain and inducing an accumulation of the t cells to the areas of Tachyzoite proliferation to prevent reactivation of chronic cerebral infection with toxoplasma gondii
American Journal of Pathology, 2015Co-Authors: Eri Ochiai, J P Dubey, Yasuhiro Suzuki, Morgan Brogli, Tomoya Kudo, Xisheng WangAbstract:T cells are required to maintain the latency of chronic infection with Toxoplasma gondii in the brain. Here, we examined the role of non–glutamic acid-leucine-arginine CXC chemokine CXCL9 for T-cell recruitment to prevent reactivation of infection with T. gondii. Severe combined immunodeficient (SCID) mice were infected and treated with sulfadiazine to establish a chronic infection. Immune T cells from infected wild-type mice were transferred into the SCID mice in combination with treatment with anti-CXCL9 or control sera. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Numbers of CD4+ and CD8+ T cells isolated from the brains were markedly less in mice treated with anti-CXCL9 serum than in mice treated with control serum at 3 days after sulfadiazine discontinuation. Amounts of Tachyzoite (acute stage form of T. gondii)-specific SAG1 mRNA and numbers of foci associated with Tachyzoites were significantly greater in the former than the latter at 5 days after sulfadiazine discontinuation. An accumulation of CD3+ T cells into the areas of Tachyzoite growth was significantly less frequent in the SCID mice treated with anti-CXCL9 serum than in mice treated with control serum. These results indicate that CXCL9 is crucial for recruiting immune T cells into the brain and inducing an accumulation of the T cells into the areas where Tachyzoites proliferate to prevent reactivation of chronic T. gondii infection.
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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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unexpected oocyst shedding by cats fed toxoplasma gondii Tachyzoites in vivo stage conversion and strain variation
Veterinary Parasitology, 2005Co-Authors: J P DubeyAbstract:Tachyzoites, bradyzoites (in tissue cysts), and sporozoites (in oocysts) are the three infectious stages of Toxoplasma gondii. The prepatent period (time to shedding of oocysts after primary infection) varies with the stage of T. gondii ingested by the cat. The prepatent period (pp) after ingesting bradyzoites is short (3-10 days) while it is long (18 days or longer) after ingesting oocysts or Tachyzoites, irrespective of the dose. The conversion of bradyzoites to Tachyzoites and Tachyzoites to bradyzoites is biologically important in the life cycle of T. gondii. In the present paper, the pp was used to study in vivo conversion of Tachyzoites to bradyzoites using two isolates, VEG and TgCkAr23. T. gondii organisms were obtained from the peritoneal exudates (pex) of mice inoculated intraperitoneally (i.p.) with these isolates and administered to cats orally by pouring in the mouth or by a stomach tube. In total, 94 of 151 cats shed oocysts after ingesting pex. The pp after ingesting pex was short (5-10 days) in 50 cats, intermediate (11-17) in 30 cats, and long (18 or higher) in 14 cats. The strain of T. gondii (VEG, TgCKAr23) or the stage (bradyzoite, Tachyzoite, and sporozoite) used to initiate infection in mice did not affect the results. In addition, six of eight cats fed mice infected 1-4 days earlier shed oocysts with a short pp; the mice had been inoculated i.p. with bradyzoites of the VEG strain and their whole carcasses were fed to cats 1, 2, 3, or 4 days post-infection. Results indicate that bradyzoites may be formed in the peritoneal cavities of mice inoculated intraperitoneally with T. gondii and some bradyzoites might give rise directly to bradyzoites without converting to Tachyzoites.
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neospora caninum identification of 19 38 and 40 kda surface antigens and a 33 kda dense granule antigen using monoclonal antibodies
Experimental Parasitology, 1999Co-Authors: Gereon Schares, J P Dubey, Jean-françois Dubremetz, Andrea Barwald, A Loyens, Franz Josef ConrathsAbstract:Neospora caninum, a coccidian parasite closely related to Toxoplasma gondii, can infect a broad host range and is regarded as an important cause of bovine abortion worldwide. In the present study, four antigens of N. caninum were partially characterized using monoclonal antibodies. Immunofluorescence of viable Tachyzoites as well as the immunoprecipitation of antigens extracted from Tachyzoites previously labeled by surface biotinylation revealed that three of these antigens with apparent molecular weights of 40, 38, and 19 kDa are located in the outer surface membrane of this parasite stage. Further evidence for the surface localization of the 38-kDa antigen was obtained by immunoelectron microscopy. In addition to the surface molecules, an antigen located in dense granules and in the tubular network of the parasitophorous vacuole was detected by another monoclonal antibody. When Tachyzoite antigens separated under nonreducing conditions were probed on Western blots, this antibody reacted mainly with a 33-kDa antigen. Immunohistochemical analysis of infected tissue sections indicated that the 33-kDa dense granule antigen is present in both Tachyzoites and bradyzoites, while the 38-kDa surface antigen from Tachyzoites seems to be absent in bradyzoites.
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serological diagnosis of bovine neosporosis by neospora caninum monoclonal antibody based competitive inhibition enzyme linked immunosorbent assay
Journal of Clinical Microbiology, 1996Co-Authors: Timothy V Baszler, J P Dubey, Donald P Knowles, Bruce A Mathison, Terry F McelwainAbstract:Neospora caninum, a protozoan parasite closely related to Toxoplasma gondii, causes abortion and congenital infection in cattle. To investigate specific methods of antemortem diagnosis, the antibody responses of infected cows were evaluated by immunoblot assay and competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) by using a monoclonal antibody (MAb), MAb 4A4-2, against N. caninum Tachyzoites. MAb 4A4-2 bound diffusely to the exterior surface of N. caninum Tachyzoites and recognized a single 65-kDa band in immunoblots. MAb 4A4-2 was unreactive to antigens of two closely related apicomplexan protozoa, Toxoplasma gondii and Sarcocystis cruzi. Binding of MAb 4A4-2 was inhibited by mild periodate treatment of N. caninum antigen, demonstrating the carbohydrate nature of the epitope. Immunoblot analysis of N. caninum Tachyzoite antigens with sera from cows with confirmed Neospora-induced abortion revealed at minimum 14 major antigens ranging from 11 to 175 kDa. Although the recognized antigens varied from cow to cow, antigens of 116, 65, and 25 kDa were detected in all cows with abortion confirmed to be caused by N. caninum. The binding of MAb 4A4-2 to N. caninum Tachyzoite antigen was consistently inhibited by sera from Neospora-infected cows in a CI-ELISA format and was not inhibited by sera from Neospora antibody-negative cows. Furthermore, sera from cattle experimentally infected with T. gondii, S. cruzi, Sarcocystis hominis, or Sarcocystis hirsuta, which had cross-reactive antibodies recognizing multiple N. caninum antigens by immunoblot assay, did not inhibit binding of MAb 4A4-2 in the CI-ELISA. Thus, MAb 4A4-2 binds a carbohydrate epitope on a single N. caninum Tachyzoite surface antigen that is recognized consistently and specifically by Neospora-infected cattle.
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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.
