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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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draft genome sequence and annotation of the apicomplexan parasite besnoitia besnoiti
Genome Announcements, 2017Co-Authors: Gereon Schares, Pratap Venepally, Hernan LorenziAbstract:ABSTRACT The apicomplexan parasite besnoitia besnoiti is the causative agent of bovine besnoitiosis that affects livestock, particularly cattle. The definitive host of B. besnoiti is unknown and its transmission only partially understood. Here, we report the first draft genome sequence, assembly, and annotation of this parasite.
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importance of serological cross reactivity among toxoplasma gondii hammondia spp neospora spp sarcocystis spp and besnoitia besnoiti
Parasitology, 2017Co-Authors: Luis Fernando Pita Gondim, José Roberto Mineo, Gereon ScharesAbstract:Toxoplasma gondii, Neospora spp., Sarcocystis spp., Hammondia spp. and besnoitia besnoiti are genetically related cyst-forming coccidia. Serology is frequently used for the identification of T. gondii, Neospora spp. and B. besnoiti-exposed individuals. Serologic cross-reactions occur in different tests among animals infected with T. gondii and H. hammondi, as well as among animals infected by T. gondii and N. caninum. Infections caused by N. caninum and N. hughesi are almost indistinguishable by serology. Neospora caninum, B. besnoiti and Sarcocystis spp. infections in cattle show some degree of serologic cross-reactivity. Antibody cross-reactivity between Neospora spp. and H. heydorni-infected animals is suspected, but not proven to occur. We review serologic cross-reactivity among animals and/or humans infected with T. gondii, Neospora spp., Sarcocystis spp., Hammondia spp. and B. besnoiti. Emphasis is laid upon antigens and serological methods for N. caninum diagnosis which were tested for cross-reactivity with related protozoa. Species-specific antigens, as well as stage-specific proteins have been identified in some of these parasites and have promising use for diagnosis and epidemiological surveys.
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importance of serological cross reactivity among toxoplasma gondii hammondia spp neospora spp sarcocystis spp and besnoitia besnoiti
Parasitology, 2017Co-Authors: Luis Fernando Pita Gondim, José Roberto Mineo, Gereon ScharesAbstract:Toxoplasma gondii, Neospora spp., Sarcocystis spp., Hammondia spp. and besnoitia besnoiti are genetically related cyst-forming coccidia. Serology is frequently used for the identification of T. gondii, Neospora spp. and B. besnoiti-exposed individuals. Serologic cross-reactions occur in different tests among animals infected with T. gondii and H. hammondi, as well as among animals infected by T. gondii and N. caninum. Infections caused by N. caninum and N. hughesi are almost indistinguishable by serology. Neospora caninum, B. besnoiti and Sarcocystis spp. infections in cattle show some degree of serologic cross-reactivity. Antibody cross-reactivity between Neospora spp. and H. heydorni-infected animals is suspected, but not proven to occur. We review serologic cross-reactivity among animals and/or humans infected with T. gondii, Neospora spp., Sarcocystis spp., Hammondia spp. and B. besnoiti. Emphasis is laid upon antigens and serological methods for N. caninum diagnosis which were tested for cross-reactivity with related protozoa. Species-specific antigens, as well as stage-specific proteins have been identified in some of these parasites and have promising use for diagnosis and epidemiological surveys.
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besnoitia besnoiti lytic cycle in vitro and differences in invasion and intracellular proliferation among isolates
Parasites & Vectors, 2016Co-Authors: Javier Regidorcerrillo, Paula Garcialunar, D Gutierrezexposito, Nelson Marreros, Arcangelo Gentile, Gereon Schares, J P Dubey, Philippe JacquietAbstract:Background Bovine besnoitiosis, caused by the protozoan besnoitia besnoiti, reduces productivity and fertility of affected herds. besnoitiosis continues to expand in Europe and no effective control tools are currently available. Experimental models are urgently needed. Herein, we describe for the first time the kinetics of standardised in vitro models for the B. besnoiti lytic cycle. This will aid to study the pathogenesis of the disease, in the screening for vaccine targets and drugs potentially useful for the treatment of besnoitiosis.
Helder Cortes - One of the best experts on this subject based on the ideXlab platform.
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besnoitia besnoiti and toxoplasma gondii two apicomplexan strategies to manipulate the host cell centrosome and golgi apparatus
Parasitology, 2014Co-Authors: Rita M Cardoso, Alexandre Leitao, Sofia Nolasco, Joao Goncalves, Helder Cortes, Helena SoaresAbstract:: besnoitia besnoiti and Toxoplasma gondii are two closely related parasites that interact with the host cell microtubule cytoskeleton during host cell invasion. Here we studied the relationship between the ability of these parasites to invade and to recruit the host cell centrosome and the Golgi apparatus. We observed that T. gondii recruits the host cell centrosome towards the parasitophorous vacuole (PV), whereas B. besnoiti does not. Notably, both parasites recruit the host Golgi apparatus to the PV but its organization is affected in different ways. We also investigated the impact of depleting and over-expressing the host centrosomal protein TBCCD1, involved in centrosome positioning and Golgi apparatus integrity, on the ability of these parasites to invade and replicate. Toxoplasma gondii replication rate decreases in cells over-expressing TBCCD1 but not in TBCCD1-depleted cells; while for B. besnoiti no differences were found. However, B. besnoiti promotes a reorganization of the Golgi ribbon previously fragmented by TBCCD1 depletion. These results suggest that successful establishment of PVs in the host cell requires modulation of the Golgi apparatus which probably involves modifications in microtubule cytoskeleton organization and dynamics. These differences in how T. gondii and B. besnoiti interact with their host cells may indicate different evolutionary paths.
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a review on bovine besnoitiosis a disease with economic impact in herd health management caused by besnoitia besnoiti franco and borges
Parasitology, 2014Co-Authors: Helder Cortes, Alexandre Leitao, Bruno Gottstein, Andrew HemphillAbstract:Bovine besnoitiosis is caused by the largely unexplored apicomplexan parasite besnoitia besnoiti. In cows, infection during pregnancy often results in abortion, and chronically infected bulls become infertile. Similar to other apicomplexans B. besnoiti has acquired a largely intracellular lifestyle, but its complete life cycle is still unknown, modes of transmission have not been entirely resolved and the definitive host has not been identified. Outbreaks of bovine besnoitiosis in cattle were described in the 1990s in Portugal and Spain, and later several cases were also detected in France. More cases have been reported recently in hitherto unaffected countries, including Italy, Germany, Switzerland, Hungary and Croatia. To date, there is still no effective pharmaceutical compound available for the treatment of besnoitiosis in cattle, and progress in the identification of novel targets for intervention through pharmacological or immunological means is hampered by the lack of molecular data on the genomic and transcriptomic level. In addition, the lack of an appropriate small animal laboratory model, and wide gaps in our knowledge on the host-parasite interplay during the life cycle of this parasite, renders vaccine and drug development a cost- and labour-intensive undertaking.
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prevalence and geographic distribution of besnoitia besnoiti infection in cattle herds in portugal
Parasitology Research, 2014Co-Authors: Helga Waap, Helder Cortes, Telmo Nunes, Alexandre LeitaoAbstract:Bovine besnoitiosis, caused by the apicomplexan parasite besnoitia besnoiti is considered an emergent disease in Europe. This study aimed to determine the prevalence and geographic distribution of B. besnoiti in cattle herds in continental Portugal and to identify potential spatial clustering of infection. A stratified two-stage cross-sectional serological survey was carried out between March 2012 and May 2013 with the five administrative NUTS II regions, Norte, Centro, Lisboa, Alentejo, and Algarve, as the stratification level. Sera from 391 herds in 220 parishes and 83 municipalities were analyzed by a serial testing strategy, with the modified agglutination test (B-MAT) as the first screening assay and the immunofluorescent antibody test (IFAT) as the confirmatory test. Within-herd prevalence of positive herds varied between 0.7 and 72.4 % and was ≥10.3 % in half of the infected herds. Using a Bayesian approach, the true prevalence of B. besnoiti in cattle herds was determined to be 5.1 % (confidence interval (CI), 3.1–7.8 %) and the mean within-herd prevalence of positive herds was 33.0 % (CI, 20.3–46.0 %). The sensitivity and specificity of the B-MAT were estimated to be 96.9 % (CI, 93.7–98.8 %) and 99.7 % (CI, 99.6–99.8 %), whereas those of the IFAT were 89.6 % (CI, 86.0–92.5 %) and 99.7 % (CI, 98.5–99.9 %), respectively. Spatial scan statistics analysis identified one spatial cluster covering the majority of the Alentejo region. Seropositive herds were detected for the first time outside Alentejo, in the region Centro and in the northeast of Portugal. Further epidemiological research is needed to identify eco-biological factors, which could explain the geographic clustering of B. besnoiti in Portugal.
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neutrophil extracellular traps as innate immune reaction against the emerging apicomplexan parasite besnoitia besnoiti
PLOS ONE, 2014Co-Authors: Tamara Munoz Caro, Helder Cortes, Liliana M. R. Silva, Carlos Hermosilla, Anja TaubertAbstract:besnoitia besnoiti infection in cattle is an important emerging protozoan disease in Europe causing economic losses and severe clinical signs, such as generalized dermatitis, orchitis, and vulvitis in affected animals. Neutrophil extracellular trap (NET) formation was recently demonstrated as an important effector mechanism of PMN acting against several invading pathogens. In the present study, interactions of bovine PMN with tachyzoites of B. besnoiti were investigated in this respect in vitro. For the demonstration and quantification of NETs, extracellular DNA was stained by Sytox Orange or Pico Green. Fluorescent illustrations as well as scanning electron microscopy analyses (SEM) showed PMN-promoted NET formation rapidly being induced upon contact with B. besnoiti tachyzoites. Co-localization of extracellular DNA with histones, neutrophil elastase (NE) and myeloperoxidase (MPO) in parasite entrapping structures confirmed the classical characteristics of NET. Exposure of PMN to viable, UV attenuated and dead tachyzoites showed a significant induction of NET formation, but even tachyzoite homogenates significantly promoted NETs when compared to negative controls. NETs were abolished by DNase treatment and were reduced after PMN preincubation with NADPH oxidase-, NE- and MPO-inhibitors. Tachyzoite-triggered NET formation led to parasite entrapment as quantitative assays indicated that about one third of tachyzoites were immobilized in NETs. In consequence, tachyzoites were hampered from active invasion of host cells. Thus, transfer of tachyzoites, previously being confronted with PMN, to adequate host cells resulted in significantly reduced infection rates when compared to PMN-free infection controls. To our knowledge, we here report for the first time B. besnoiti-induced NET formation. Our results indicate that PMN-triggered extracellular traps may represent an important effector mechanism of the host early innate immune response against B. besnoiti which may lead to diminishment of initial parasite infection rates during the acute infection phase.
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first cross sectional serological survey on besnoitia besnoiti in cattle in italy
Parasitology Research, 2013Co-Authors: Laura Rinaldi, Antonio Bosco, Vincenzo Musella, Maria Paola Maurelli, Helder Cortes, Giuseppe CringoliAbstract:A cross-sectional serological survey was conducted to evaluate the prevalence of besnoitiosis in cattle farms located in a region of southern Italy. A geographical information system (GIS) was used in order to uniformly sample the bovine farms (n = 88) throughout the entire region. Blood samples were collected from 528 autochthonous cattle and sera were tested for antibodies to besnoitia besnoiti using an enzyme-linked immunosorbent assay test. The farm prevalence was 83.0 % (73/88), and the individual animal prevalence was 44.1 % (233/528). The availability of geo-referenced point or areal data on bovine besnoitiosis and the construction of prevalence maps by GIS are suggested for dissemination of information to veterinarians on this emerging infection in cattle.
Luis Fernando Pita Gondim - One of the best experts on this subject based on the ideXlab platform.
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importance of serological cross reactivity among toxoplasma gondii hammondia spp neospora spp sarcocystis spp and besnoitia besnoiti
Parasitology, 2017Co-Authors: Luis Fernando Pita Gondim, José Roberto Mineo, Gereon ScharesAbstract:Toxoplasma gondii, Neospora spp., Sarcocystis spp., Hammondia spp. and besnoitia besnoiti are genetically related cyst-forming coccidia. Serology is frequently used for the identification of T. gondii, Neospora spp. and B. besnoiti-exposed individuals. Serologic cross-reactions occur in different tests among animals infected with T. gondii and H. hammondi, as well as among animals infected by T. gondii and N. caninum. Infections caused by N. caninum and N. hughesi are almost indistinguishable by serology. Neospora caninum, B. besnoiti and Sarcocystis spp. infections in cattle show some degree of serologic cross-reactivity. Antibody cross-reactivity between Neospora spp. and H. heydorni-infected animals is suspected, but not proven to occur. We review serologic cross-reactivity among animals and/or humans infected with T. gondii, Neospora spp., Sarcocystis spp., Hammondia spp. and B. besnoiti. Emphasis is laid upon antigens and serological methods for N. caninum diagnosis which were tested for cross-reactivity with related protozoa. Species-specific antigens, as well as stage-specific proteins have been identified in some of these parasites and have promising use for diagnosis and epidemiological surveys.
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importance of serological cross reactivity among toxoplasma gondii hammondia spp neospora spp sarcocystis spp and besnoitia besnoiti
Parasitology, 2017Co-Authors: Luis Fernando Pita Gondim, José Roberto Mineo, Gereon ScharesAbstract:Toxoplasma gondii, Neospora spp., Sarcocystis spp., Hammondia spp. and besnoitia besnoiti are genetically related cyst-forming coccidia. Serology is frequently used for the identification of T. gondii, Neospora spp. and B. besnoiti-exposed individuals. Serologic cross-reactions occur in different tests among animals infected with T. gondii and H. hammondi, as well as among animals infected by T. gondii and N. caninum. Infections caused by N. caninum and N. hughesi are almost indistinguishable by serology. Neospora caninum, B. besnoiti and Sarcocystis spp. infections in cattle show some degree of serologic cross-reactivity. Antibody cross-reactivity between Neospora spp. and H. heydorni-infected animals is suspected, but not proven to occur. We review serologic cross-reactivity among animals and/or humans infected with T. gondii, Neospora spp., Sarcocystis spp., Hammondia spp. and B. besnoiti. Emphasis is laid upon antigens and serological methods for N. caninum diagnosis which were tested for cross-reactivity with related protozoa. Species-specific antigens, as well as stage-specific proteins have been identified in some of these parasites and have promising use for diagnosis and epidemiological surveys.
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frequency of antibodies against besnoitia besnoiti in brazilian cattle
Veterinary Parasitology, 2014Co-Authors: Rosângela Soares Uzeda, Muller Ribeiro Andrade, Ana Maria Antonello, F S Vogel, Luis Gustavo Corbellini, Luis Fernando Pita GondimAbstract:Abstract besnoitia besnoiti is a cyst-forming parasite that has been associated with economic losses in Africa and Europe. besnoitiosis is considered as a re-emergent disease in the European continent. It is unknown whether cattle are exposed to B. besnoiti in the Americas, thus the aim of this study was to serologically investigate antibodies against B. besnoiti in a total of 2014 cattle serum samples from two states from Brazil. All samples were evaluated by IFAT and part of the positive sera was tested by Western blot (WB) using tachyzoites extracts under non-reducing condition. A total of 3.48% (70/2014) of the tested sera reacted positively by IFAT with titers of 200 (85.7%), 400 (10%) and 800 (4.3%). When 47 positive samples were assessed by WB a range of antigens from 7 to 206 kDa was recognized by the IFAT-positive sera. The results are suggestive of exposure of Brazilian cattle to B. besnoiti due to the titers (≥200) observed for some sera using IFAT. However, the antigens recognized by the IFAT-positive animals did not completely match with the WB patterns previously described by other working groups. It is possible that Brazilian cattle are exposed to B. besnoiti strains with different antigenic composition of those described in the European and African continent. Further studies are needed to confirm the presence of B. besnoiti or other besnoitia species in Brazilian cattle.
Alexandre Leitao - One of the best experts on this subject based on the ideXlab platform.
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Serological survey of Toxoplasma gondii and besnoitia besnoiti in a wildlife conservation area in southern Portugal
Veterinary Parasitology: Regional Studies and Reports, 2016Co-Authors: Helga Waap, Telmo Nunes, Alexandre LeitaoAbstract:Abstract Toxoplasma gondii and besnoitia besnoiti are closely related apicomplexan protozoa. T. gondii is a zoonotic pathogen which may cause serious disease in man and warm-blooded animals, including wild species. B. besnoiti causes bovine besnoitiosis, an emergent disease in Europe, which is linked to important production losses. Unlike T. gondii , the life cycle of B. besnoiti remains a mystery, since the definitive host has not yet been identified. The aim of this work was to determine the seroprevalence of T. gondii in wildlife and feral cats from a hitherto unsampled area in southern Portugal and to identify likely candidates for definitive and/or other intermediate hosts of B. besnoiti . A total of 260 animals were screened for T. gondii and B. besnoiti by the modified agglutination tests, using the cut-off value of 1:20 and 1:80, respectively. The prevalence of T. gondii was 85.3% in Egyptian mongoose ( Herpestes ichneumon ; n = 34), 83.3% in wildcats ( Felis silvestris ; n = 6), 66.7% in stone martens ( Martes foina ; n = 6), 47.1% in genets ( Genetta genetta ; n = 17), 40% in foxes ( Vulpes vulpes ; n = 25), 39.2% in cats ( Felis catus ; n = 79), 33.3% in European polecats ( Mustela putorius ; n = 3), 21.4% in red deer ( Cervus elaphus ; n = 14), 7.7% in wild boars ( Sus scrofa ; n = 26), 2.8% in rabbits ( Oryctolagus cuniculus ; n = 36) and 0% in European otters ( Lutra lutra ; n = 2), European badgers ( Meles meles ; n = 6) and rodents (n = 5). None of the species tested was positive for B. besnoiti . Based on the present results, the monitoring of T. gondii in native animal populations may be of major importance for wildlife conservation strategies and human health protection, while the search for other hosts of B. besnoiti requires further investigations in wild and domestic species.
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besnoitia besnoiti and toxoplasma gondii two apicomplexan strategies to manipulate the host cell centrosome and golgi apparatus
Parasitology, 2014Co-Authors: Rita M Cardoso, Alexandre Leitao, Sofia Nolasco, Joao Goncalves, Helder Cortes, Helena SoaresAbstract:: besnoitia besnoiti and Toxoplasma gondii are two closely related parasites that interact with the host cell microtubule cytoskeleton during host cell invasion. Here we studied the relationship between the ability of these parasites to invade and to recruit the host cell centrosome and the Golgi apparatus. We observed that T. gondii recruits the host cell centrosome towards the parasitophorous vacuole (PV), whereas B. besnoiti does not. Notably, both parasites recruit the host Golgi apparatus to the PV but its organization is affected in different ways. We also investigated the impact of depleting and over-expressing the host centrosomal protein TBCCD1, involved in centrosome positioning and Golgi apparatus integrity, on the ability of these parasites to invade and replicate. Toxoplasma gondii replication rate decreases in cells over-expressing TBCCD1 but not in TBCCD1-depleted cells; while for B. besnoiti no differences were found. However, B. besnoiti promotes a reorganization of the Golgi ribbon previously fragmented by TBCCD1 depletion. These results suggest that successful establishment of PVs in the host cell requires modulation of the Golgi apparatus which probably involves modifications in microtubule cytoskeleton organization and dynamics. These differences in how T. gondii and B. besnoiti interact with their host cells may indicate different evolutionary paths.
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a review on bovine besnoitiosis a disease with economic impact in herd health management caused by besnoitia besnoiti franco and borges
Parasitology, 2014Co-Authors: Helder Cortes, Alexandre Leitao, Bruno Gottstein, Andrew HemphillAbstract:Bovine besnoitiosis is caused by the largely unexplored apicomplexan parasite besnoitia besnoiti. In cows, infection during pregnancy often results in abortion, and chronically infected bulls become infertile. Similar to other apicomplexans B. besnoiti has acquired a largely intracellular lifestyle, but its complete life cycle is still unknown, modes of transmission have not been entirely resolved and the definitive host has not been identified. Outbreaks of bovine besnoitiosis in cattle were described in the 1990s in Portugal and Spain, and later several cases were also detected in France. More cases have been reported recently in hitherto unaffected countries, including Italy, Germany, Switzerland, Hungary and Croatia. To date, there is still no effective pharmaceutical compound available for the treatment of besnoitiosis in cattle, and progress in the identification of novel targets for intervention through pharmacological or immunological means is hampered by the lack of molecular data on the genomic and transcriptomic level. In addition, the lack of an appropriate small animal laboratory model, and wide gaps in our knowledge on the host-parasite interplay during the life cycle of this parasite, renders vaccine and drug development a cost- and labour-intensive undertaking.
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prevalence and geographic distribution of besnoitia besnoiti infection in cattle herds in portugal
Parasitology Research, 2014Co-Authors: Helga Waap, Helder Cortes, Telmo Nunes, Alexandre LeitaoAbstract:Bovine besnoitiosis, caused by the apicomplexan parasite besnoitia besnoiti is considered an emergent disease in Europe. This study aimed to determine the prevalence and geographic distribution of B. besnoiti in cattle herds in continental Portugal and to identify potential spatial clustering of infection. A stratified two-stage cross-sectional serological survey was carried out between March 2012 and May 2013 with the five administrative NUTS II regions, Norte, Centro, Lisboa, Alentejo, and Algarve, as the stratification level. Sera from 391 herds in 220 parishes and 83 municipalities were analyzed by a serial testing strategy, with the modified agglutination test (B-MAT) as the first screening assay and the immunofluorescent antibody test (IFAT) as the confirmatory test. Within-herd prevalence of positive herds varied between 0.7 and 72.4 % and was ≥10.3 % in half of the infected herds. Using a Bayesian approach, the true prevalence of B. besnoiti in cattle herds was determined to be 5.1 % (confidence interval (CI), 3.1–7.8 %) and the mean within-herd prevalence of positive herds was 33.0 % (CI, 20.3–46.0 %). The sensitivity and specificity of the B-MAT were estimated to be 96.9 % (CI, 93.7–98.8 %) and 99.7 % (CI, 99.6–99.8 %), whereas those of the IFAT were 89.6 % (CI, 86.0–92.5 %) and 99.7 % (CI, 98.5–99.9 %), respectively. Spatial scan statistics analysis identified one spatial cluster covering the majority of the Alentejo region. Seropositive herds were detected for the first time outside Alentejo, in the region Centro and in the northeast of Portugal. Further epidemiological research is needed to identify eco-biological factors, which could explain the geographic clustering of B. besnoiti in Portugal.
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besnoitia besnoiti protein disulfide isomerase bbpdi molecular characterization expression and in silico modelling
Experimental Parasitology, 2011Co-Authors: Eduardo Marcelino, Alexandre Leitao, Sofia Nolasco, Joana Morais, Andrew Hemphill, Helder Cortes, Tiago M. Martins, Carlos NovoAbstract:besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor an efficacious vaccine is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neosporacaninum and Toxoplasmagondii, which are closely related to B. besnoiti, PDI play an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we present the nucleotide sequence of the B. besnoiti PDI gene. BbPDI belongs to the thioredoxin-like superfamily (cluster 00388) and is included in the PDI_a family (cluster defined cd02961) and the PDI_a_PDI_a′_c subfamily (cd02995). A 3D theoretical model was built by comparative homology using Swiss-Model server, using as a template the crystallographic deduced model of Tapasin-ERp57 (PDB code 3F8U chain C). Analysis of the phylogenetic tree for PDI within the phylum apicomplexa reinforces the close relationship among B. besnoiti, N. caninum and T. gondii. When subjected to a PDI-assay based on the polymerisation of reduced insulin, recombinant BbPDI expressed in E. coli exhibited enzymatic activity, which was inhibited by bacitracin. Antiserum directed against recombinant BbPDI reacted with PDI in Western blots and by immunofluorescence with B. besnoiti tachyzoites and bradyzoites.
Andrew Hemphill - 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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repurposing of commercially available anti coccidials identifies diclazuril and decoquinate as potential therapeutic candidates against besnoitia besnoiti infection
Veterinary Parasitology, 2018Co-Authors: Alejandro Jimenezmelendez, Vreni Balmer, Laura Ricosan Roman, Luis Miguel Ortegamora, Andrew Hemphill, Gema AlvarezgarciaAbstract:Abstract Repurposing of currently marketed compounds with proven efficacy against apicomplexan parasites was used as an approach to define novel candidate therapeutics for bovine besnoitiosis. besnoitia besnoiti tachyzoites grown in MARC-145 cells were exposed to different concentrations of toltrazuril, diclazuril, imidocarb, decoquinate, sulfadiazine and trimethoprim alone or in combination with sulfadiazine. Drugs were added either just prior to infection of MARC-145 cells (0 h post infection, hpi) or at 6 hpi. A primary evaluation of drug effects was done by direct immunofluorescence staining and counting. Potential effects on the host cells were assessed using a XTT kit for cell proliferation. Compounds displaying promising efficacy were selected for IC50 and IC99 determination by qPCR. In addition, the impact of drugs on the tachyzoite ultrastructure was assessed by TEM and long-term treatment assays were performed. Cytotoxicity assays confirmed that none of the compounds affected the host cells. Decoquinate and diclazuril displayed invasion inhibition rates of 90 and 83% at 0 h pi and 73 and 72% at 6 h pi, respectively. The remaining drugs showed lower efficacy and were not further studied. Decoquinate and diclazuril exhibited IC99 values of 100 nM and 29.9 μM, respectively. TEM showed that decoquinate primarily affected the parasite mitochondrium, whilst diclazuril interfered in cytokinesis of daughter zoites. The present study demonstrates the efficacy of diclazuril and decoquinate against B. besnoiti in vitro and further assessments of safety and efficacy of both drugs should be performed in the target species.
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in vitro efficacy of bumped kinase inhibitors against besnoitia besnoiti tachyzoites
International Journal for Parasitology, 2017Co-Authors: Alejandro Jimenezmelendez, Javier Regidorcerrillo, Vreni Balmer, Adrian B Hehl, Alexandra M Wallace, Tess R Smith, Dustin J Maly, Luis Miguel Ortegamora, Andrew Hemphill, Wesley C Van VoorhisAbstract:Abstract besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a chronic and debilitating disease that causes systemic and skin manifestations and sterility in bulls. Neither treatments nor vaccines are currently available. In the search for therapeutic candidates, calcium-dependent protein kinases have arisen as promising drug targets in other apicomplexans (e.g. Neospora caninum , Toxoplasma gondii , Plasmodium spp. and Eimeria spp.) and are effectively targeted by bumped kinase inhibitors. In this study, we identified and cloned the gene coding for Bb CDPK1. The impact of a library of nine bumped kinase inhibitor analogues on the activity of recombinant Bb CDPK1 was assessed by luciferase assay. Afterwards, those were further screened for efficacy against besnoitiabesnoiti tachyzoites grown in Marc-145 cells. Primary tests at 5 µM revealed that eight compounds exhibited more than 90% inhibition of invasion and proliferation. The compounds BKI 1294, 1517, 1553 and 1571 were further characterised, and EC 99 (1294: 2.38 µM; 1517: 2.20 µM; 1553: 3.34 µM; 1571: 2.78 µM) were determined by quantitative real-time polymerase chain reaction in 3-day proliferation assays. Exposure of infected cultures with EC 99 concentrations of these drugs for up to 48 h was not parasiticidal. The lack of parasiticidal action was confirmed by transmission electron microscopy, which showed that bumped kinase inhibitor treatment interfered with cell cycle regulation and non-disjunction of tachyzoites, resulting in the formation of large multi-nucleated complexes which co-existed with viable parasites within the parasitophorous vacuole. However, it is possible that, in the face of an active immune response, parasite clearance may occur. In summary, bumped kinase inhibitors may be effective drug candidates to control besnoitiabesnoiti infection. Further in vivo experiments should be planned, as attainment and maintenance of therapeutic blood plasma levels in calves, without toxicity, has been demonstrated for BKIs 1294, 1517 and 1553.
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a review on bovine besnoitiosis a disease with economic impact in herd health management caused by besnoitia besnoiti franco and borges
Parasitology, 2014Co-Authors: Helder Cortes, Alexandre Leitao, Bruno Gottstein, Andrew HemphillAbstract:Bovine besnoitiosis is caused by the largely unexplored apicomplexan parasite besnoitia besnoiti. In cows, infection during pregnancy often results in abortion, and chronically infected bulls become infertile. Similar to other apicomplexans B. besnoiti has acquired a largely intracellular lifestyle, but its complete life cycle is still unknown, modes of transmission have not been entirely resolved and the definitive host has not been identified. Outbreaks of bovine besnoitiosis in cattle were described in the 1990s in Portugal and Spain, and later several cases were also detected in France. More cases have been reported recently in hitherto unaffected countries, including Italy, Germany, Switzerland, Hungary and Croatia. To date, there is still no effective pharmaceutical compound available for the treatment of besnoitiosis in cattle, and progress in the identification of novel targets for intervention through pharmacological or immunological means is hampered by the lack of molecular data on the genomic and transcriptomic level. In addition, the lack of an appropriate small animal laboratory model, and wide gaps in our knowledge on the host-parasite interplay during the life cycle of this parasite, renders vaccine and drug development a cost- and labour-intensive undertaking.
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besnoitia besnoiti protein disulfide isomerase bbpdi molecular characterization expression and in silico modelling
Experimental Parasitology, 2011Co-Authors: Eduardo Marcelino, Alexandre Leitao, Sofia Nolasco, Joana Morais, Andrew Hemphill, Helder Cortes, Tiago M. Martins, Carlos NovoAbstract:besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a disease with a high prevalence in tropical and subtropical regions and re-emerging in Europe. Despite the great economical losses associated with besnoitiosis, this disease has been underestimated and poorly studied, and neither an effective therapy nor an efficacious vaccine is available. Protein disulfide isomerase (PDI) is an essential enzyme for the acquisition of the correct three-dimensional structure of proteins. Current evidence suggests that in Neosporacaninum and Toxoplasmagondii, which are closely related to B. besnoiti, PDI play an important role in host cell invasion, is a relevant target for the host immune response, and represents a promising drug target and/or vaccine candidate. In this work, we present the nucleotide sequence of the B. besnoiti PDI gene. BbPDI belongs to the thioredoxin-like superfamily (cluster 00388) and is included in the PDI_a family (cluster defined cd02961) and the PDI_a_PDI_a′_c subfamily (cd02995). A 3D theoretical model was built by comparative homology using Swiss-Model server, using as a template the crystallographic deduced model of Tapasin-ERp57 (PDB code 3F8U chain C). Analysis of the phylogenetic tree for PDI within the phylum apicomplexa reinforces the close relationship among B. besnoiti, N. caninum and T. gondii. When subjected to a PDI-assay based on the polymerisation of reduced insulin, recombinant BbPDI expressed in E. coli exhibited enzymatic activity, which was inhibited by bacitracin. Antiserum directed against recombinant BbPDI reacted with PDI in Western blots and by immunofluorescence with B. besnoiti tachyzoites and bradyzoites.