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Gema Alvarezgarcia - One of the best experts on this subject based on the ideXlab platform.
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rna seq analyses reveal that endothelial activation and fibrosis are induced early and progressively by Besnoitia besnoiti host cell invasion and proliferation
Frontiers in Cellular and Infection Microbiology, 2020Co-Authors: Alejandro Jimenezmelendez, Chandra Ramakrishnan, Adrian B Hehl, Giancarlo Russo, Gema AlvarezgarciaAbstract:The pathogenesis of bovine besnoitiosis and the molecular bases that govern disease progression remain to be elucidated. Thus, we have employed an in vitro model of infection based on primary bovine aortic endothelial cells (BAEC), target cells during the acute infection. Host-parasite interactions were investigated by RNA-Seq at two post-infection (pi) time points: 12 hpi, when tachyzoites have already invaded host cells, and 32 hpi, when tachyzoites have replicated for at least two generations. Additionally, the gene expression profile of B. besnoiti tachyzoites was studied at both pi time points. Up to 446 differentially expressed B. taurus genes (DEGs) were found in BAEC between both pi time points: 249 DEGs were up-regulated and 197 DEGs were down-regulated at 32 hpi. Upregulation of different genes encoding cytokines, chemokines, leukocyte adhesion molecules predominantly at 12 hpi implies an activation of endothelial cells, whilst upregulation of genes involved in angiogenesis and extracellular matrix organization was detected at at both time points. NF-κB and TNF-α signalling pathways appeared to be mainly modulated upon infection, coordinating the expression of several effector proteins with proinflammatory and pro-fibrotic phenotypes. These mediators are thought to be responsible for macrophage recruitment setting the basis for chronic inflammation and fibrosis characteristic of chronic besnoitiosis. Angiogenesis regulation also predominated, and this multistep process was evidenced by the upregulation of markers involved in both early (eg. growth factors and matrix metalloproteinases) and late steps (eg. integrins and vasohibin). Besnoitia besnoiti orthologue genes present in other Toxoplasmatinae members and involved in the lytic cycle have shown to be differentially expressed among the two time points studied, with a higher expression at 32 hpi (eg. ROP40, ROP5B, MIC1, MIC10). This study gives molecular clues on B. besnoiti- BAECs interaction and shows the progression of type II endothelial cell activation upon parasite invasion and proliferation.
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lytic cycle of Besnoitia besnoiti tachyzoites displays similar features in primary bovine endothelial cells and fibroblasts
Parasites & Vectors, 2019Co-Authors: Alejandro Jimenezmelendez, Luis Miguel Ortegamora, Maria Fernandezalvarez, Alexandra Calle, Miguel Angel Ramirez, Carlos Diezmadiaz, Patricia Vazquezarbaizar, Gema AlvarezgarciaAbstract:Bovine besnoitiosis, caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti, is a chronic and debilitating cattle disease that continues to spread in Europe in the absence of control tools. In this scenario, in vitro culture systems are valuable tools to carry out drug screenings and to unravel host-parasite interactions. However, studies performed in bovine target cells are scarce. The objective of the present study was to obtain primary bovine aortic endothelial cells (BAECs) and fibroblast cell cultures, target cells during the acute and the chronic stage of the disease, respectively, from healthy bovine donors. Afterwards, expression of surface (CD31, CD34 and CD44) and intracellular markers (vimentin and cytokeratin) was studied to characterize cell populations by flow cytometry. Next, the lytic cycle of B. besnoiti tachyzoites was studied in both target cells. Invasion rates (IRs) were determined by immunofluorescence at several time points post-infection, and proliferation kinetics were studied by quantitative PCR (qPCR). Finally, the influence of bovine viral diarrhea virus (BVDV) co-infection on the host cell machinery, and consequently on B. besnoiti invasion and proliferation, was investigated in BAECs. Morphology and cytometry results confirmed the endothelial and fibroblast origins. CD31 was the surface marker that best discriminated between BAECs and fibroblasts, since fibroblasts lacked CD31 labelling. Expression of CD34 was weak in low-passage BAECs and absent in high-passage BAECs and fibroblasts. Positive labelling for CD44, vimentin and cytokeratin was observed in both BAECs and fibroblasts. Regarding the lytic cycle of the parasite, although low invasion rates (approximately 3–4%) were found in both cell culture systems, more invasion was observed in BAECs at 24 and 72 hpi. The proliferation kinetics did not differ between BAECs and fibroblasts. BVDV infection favoured early Besnoitia invasion but there was no difference in tachyzoite yields observed in BVDV-BAECs compared to BAECs. We have generated and characterized two novel standardized in vitro models for Besnoitia besnoiti infection based on bovine primary target BAECs and fibroblasts, and have shown the relevance of BVDV coinfections, which should be considered in further studies with other cattle pathogens.
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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, Luis Miguel Ortegamora, Andrew Hemphill, Vreni Balmer, Laura Ricosan Roman, 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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exposure to neospora spp and Besnoitia spp in wildlife from israel
International journal for parasitology. Parasites and wildlife, 2018Co-Authors: Monica L Mazuz, Gema Alvarezgarcia, Luis Miguel Ortegamora, Varda Shkap, Roni King, Igor Savisky, D GutierrezexpositoAbstract:Abstract Neosporosis and besnoitiosis, caused by cyst-forming protozoa Neospora caninum and Besnoitia besnoiti, respectively, are parasitic infestations of livestock in Israel. These parasites cause significant economic losses in cattle due to reproductive and productive disorders. Both parasites have been detected in several wild ruminant species throughout other regions of the world, while the existence of a sylvatic life cycle in Israel remains uncertain. Thus, a wide panel of 871 sera from two wild carnivores and nine wild ruminant species were tested. All sera were first analysed by MAT for an initial screening and positive samples were confirmed a posteriori by Western blot. Additionally, a complementary IFAT was used for the detection of antibodies against N. caninum. Neospora antibodies were present in six out of the 11 species investigated, whereas Besnoitia antibodies were undetected. Golden jackal, red fox, addax, Arabian oryx, Persian fallow deer, mouflon, mountain gazelle, Nubian ibex, scimitar horned oryx and water buffalo were seropositive against N. caninum infection by IFAT and/or MAT. Moreover, the presence of Neospora spp.-specific antibodies was confirmed by Western blot in golden jackal (6/189; 3.2%), red fox (1/75; 1.3%), Persian fallow deer (13/232; 5.6%), mouflon (1/15; 16.7%), Nubian ibex (22/55; 40%) and water buffalo (12/18; 66.7%). Addax (1/49) and water buffalo (1/18) were MAT-seropositive against B. besnoiti but were seronegative by Western blot. Hence, Neospora sylvatic cycle is present in Israel and may cross over to a domestic life cycle. In contrast, wildlife species investigated are unlikely to present a risk of transmitting Besnoitia to livestock in Israel.
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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, Gereon Schares, L M Ortegamora, Carlos Diezmadiaz, 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.
Gereon Schares - One of the best experts on this subject based on the ideXlab platform.
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Serological survey of Neospora spp. and Besnoitia spp. in horses in Portugal.
Veterinary Parasitology: Regional Studies and Reports, 2020Co-Authors: Helga Waap, Uillians Volkart De Oliveira, Telmo Nunes, Jacinto Gomes, Tiago Gomes, Andrea Bärwald, Alexandre Dias Munhoz, Gereon ScharesAbstract:Abstract Equine neosporosis is regarded to be caused either by Neospora hughesi or Neospora caninum and equine besnoitiosis is caused by Besnoitia bennetti, both of which are apicomplexan parasites. N. caninum is the only known Neospora species in Europe, where equine N. caninum infections have been reported as being associated to abortion and reproductive failure. N. hughesi is prevalent in North America and was predominantly linked to neurological disorders. B. bennetti is considered an emergent disease in donkeys in North America and evidence for B. bennetti infection was recently reported in Europe. Though N. caninum and Besnoitia besnoiti are prevalent in cattle in Portugal, little is known about neosporosis in horses and, to the best of our knowledge, no information was hitherto available for Besnoitia spp. The aim of this study was thus to carry out a serological survey to determine the seroprevalence of these parasites in naturally exposed horses in Portugal. A total of 385 animals were screened by the Indirect Fluorescent Antibody Test at the cut-off value 1:50 and positive results were confirmed by Western blot. Exposure to Neospora spp. and Besnoitia spp. was confirmed in 9.1% (95% Confidence Interval [CI]: 6.6–12.4%) and 0.3% (95% CI: 0.0–1.5%) of horses, respectively. Considering the putative economic and animal health impact of neosporosis in horses and the consequences of a possible spread of equine besnoitiosis in Europe and elsewhere, more comprehensive studies are needed to characterize the species detected in serological surveys, evaluate the geographical distribution and assess possible risk factors that could favor transmission.
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sensitive quantitative detection of Besnoitia darlingi and related parasites in intermediate hosts and to assess felids as definitive hosts for known and as yet undescribed related parasite species
International journal for parasitology. Parasites and wildlife, 2020Co-Authors: Gereon Schares, Andrea Bärwald, J P Dubey, Benjamin M Rosenthal, Mareen Tuschy, Franz Josef ConrathsAbstract:Abstract Besnoitia darlingi, B. neotomofelis and B. oryctofelisi are closely related coccidian parasites with cats as definitive hosts. While B. darlingi uses opossums as intermediate hosts, B. neotomofelis and B. oryctofelisi have been described in Southern Plains woodrats (Neotoma micropus) from the USA and in domestic rabbits from Argentina, respectively. A comparison of the Internal Transcribed Spacer-1 (ITS-1) region of the ribosomal DNA (rDNA) of these Besnoitia spp. showed only a few differences. The present study aimed at developing a real-time PCR to detect B. darlingi, B. neotomofelis and B. oryctofelisi in tissues of intermediate and in faeces of definitive hosts in order to support studies of these organisms’ epidemiology and pathogenesis. The established PCR was based on primer regions distinct from the ITS-1 sequences of ungulate Besnoitia spp. and made use of a Besnoitia universal probe. To monitor inhibition, a heterologous internal control was established based on the enhanced green fluorescent protein gene. The real-time PCR reacted with B. darlingi, B. neotomofelis and B. oryctofelisi, while the novel PCR did not recognize ungulate Besnoitia spp. (B. besnoiti, B. bennetti, B. tarandi). DNA of Apicomplexa ascribed to other Besnoitia-related genera, including other gut parasites of cats (Cryptosporidium parvum, Giardia duodenalis, Tritrichomonas foetus), was not recognized. The real-time PCR had an analytic sensitivity of less than 1 tachyzoite per reaction. In feline faeces spiked with B. darlingi oocysts, the limit of detection was a DNA amount equivalent to 1 oocyst per PCR reaction. In B. darlingi infected ɣ-interferon knock-out mice, the lung was identified as the predilection organ. In conclusion, this real-time PCR should advance further studies on these parasites and may inspire research on related species, not only in the Americas, but also in other parts of the world.
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development and characterization of monoclonal antibodies against Besnoitia besnoiti tachyzoites
Parasitology, 2019Co-Authors: Paula Garcialunar, Gereon Schares, Javier Regidorcerrillo, Alejandro Jimenezmelendez, Andrew Hemphill, A Sanzfernandez, I Garciasoto, Ivan Pastorfernandez, M Fernandezalvarez, L M 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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a new lyophilized tachyzoite based elisa to diagnose Besnoitia spp infection in bovids and wild ruminants improves specificity
Veterinary Parasitology, 2017Co-Authors: Paula Garcialunar, Gereon Schares, L M Ortegamora, Carlos Diezmadiaz, 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.
Helder Cortes - One of the best experts on this subject based on the ideXlab platform.
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Besnoitia besnoiti impact on fertility of cattle exploited in Mediterranean pastures (Alentejo)
2020Co-Authors: Helder Cortes, Alexandre Leitao, J. Chagas E Silva, M.c. Baptista, R.m. Pereira, A.e.m. Horta, M.i. Vasques, J. P. Barbas, C.c. MarquesAbstract:Besnoitia besnoiti is a bovine parasite endemic in many tropical and subtropical areas whose prevalence in the Mediterranean countries such as Portugal seems to be increasing. Most infections are mild or subclinical, characterized by the formation of numerous cutaneous and sub-cutaneous microcysts, lowering the quality of skins for the leather industry. Male sterility or impaired fertility is a common sequela in breeding bulls, and is one of the most negative aspects of the disease in animals that survive infection. Our objective was to investigate if asymptomatic Besnoitiosis leads to bovine infertility, by comparing seminal parameters pre and post-thawing, in vitro fertilization (IVF) and embryo rates between asymptomatic infected (n=3) and uninfected (n=5) bulls, exploited in an extensive production system in Alentejo-Portugal. Skin biopsies were submitted to histopathological analyses to identify B. besnoiti cysts in sires. Semen was collected by electroejaculation and sperm quality parameters before cryopreservation and after thawing were analyzed using ANOVA. The quality of semen collected from asymptomatic infected and uninfected bulls presented no differences before cryopreservation. From all the post-thawed sperm quality parameters (motility and hypoosmotic swelling test; post-swim-up motility, activity, concentration and agglutination; fertilization and embryo rates) evaluated, only post-thawed (51.0±36.3 vs. 42.3±10.6%, P≤0.05) and post-swim-up (36.3±18.8 vs 25.1±12.0 %, P≤0.009) motility were significantly different between asymptomatic infected and uninfected bulls, respectively. Semen from asymptomatic Besnoitia besnoiti infected bulls may maintain fertilization ability. However the presence of these animals in herds represents a risk of spreading the disease leading to further economic losses.
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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 and toxoplasma gondii two apicomplexan strategies to manipulate the host cell centrosome and golgi apparatus
Parasitology, 2014Co-Authors: Rita M Cardoso, Helder Cortes, Alexandre Leitao, Sofia Nolasco, Joao Goncalves, 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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prevalence and geographic distribution of Besnoitia besnoiti infection in cattle herds in portugal
Parasitology Research, 2014Co-Authors: Helga Waap, Telmo Nunes, Helder Cortes, 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, Liliana M R Silva, Carlos Hermosilla, Helder Cortes, 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.
D Gutierrezexposito - One of the best experts on this subject based on the ideXlab platform.
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exposure to neospora spp and Besnoitia spp in wildlife from israel
International journal for parasitology. Parasites and wildlife, 2018Co-Authors: Monica L Mazuz, Gema Alvarezgarcia, Luis Miguel Ortegamora, Varda Shkap, Roni King, Igor Savisky, D GutierrezexpositoAbstract:Abstract Neosporosis and besnoitiosis, caused by cyst-forming protozoa Neospora caninum and Besnoitia besnoiti, respectively, are parasitic infestations of livestock in Israel. These parasites cause significant economic losses in cattle due to reproductive and productive disorders. Both parasites have been detected in several wild ruminant species throughout other regions of the world, while the existence of a sylvatic life cycle in Israel remains uncertain. Thus, a wide panel of 871 sera from two wild carnivores and nine wild ruminant species were tested. All sera were first analysed by MAT for an initial screening and positive samples were confirmed a posteriori by Western blot. Additionally, a complementary IFAT was used for the detection of antibodies against N. caninum. Neospora antibodies were present in six out of the 11 species investigated, whereas Besnoitia antibodies were undetected. Golden jackal, red fox, addax, Arabian oryx, Persian fallow deer, mouflon, mountain gazelle, Nubian ibex, scimitar horned oryx and water buffalo were seropositive against N. caninum infection by IFAT and/or MAT. Moreover, the presence of Neospora spp.-specific antibodies was confirmed by Western blot in golden jackal (6/189; 3.2%), red fox (1/75; 1.3%), Persian fallow deer (13/232; 5.6%), mouflon (1/15; 16.7%), Nubian ibex (22/55; 40%) and water buffalo (12/18; 66.7%). Addax (1/49) and water buffalo (1/18) were MAT-seropositive against B. besnoiti but were seronegative by Western blot. Hence, Neospora sylvatic cycle is present in Israel and may cross over to a domestic life cycle. In contrast, wildlife species investigated are unlikely to present a risk of transmitting Besnoitia to livestock in Israel.
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absence of antibodies specific to Besnoitia spp in european sheep and goats from areas in spain where bovine besnoitiosis is endemic
Parasitology Research, 2017Co-Authors: D Gutierrezexposito, Luis Miguel Ortegamora, Ignasi Marco, Santiago Lavin, Javier Carvajalvalilla, Angel Morales, Gema AlvarezgarciaAbstract:Besnoitia besnoiti and B. caprae, which infect bovids (cattle and antelopes) and goats, respectively, are responsible for besnoitiosis, a chronic and debilitating disease. Bovine besnoitiosis is considered to be a reemerging disease in Central and Western Europe. In addition, infection by Besnoitia spp. has been reported in reindeer from Sweden and Finland. Recently, the parasite was also detected in roe deer and red deer from Spain, where an interconnection between the domestic and sylvatic cycles of B. besnoiti has been presumed. In contrast, caprine besnoitiosis seems to be enzootic to Kenya and Iran. The presence of Besnoitia spp. in small domestic ruminants has never been explored in Europe, and the role that these species might play in the epidemiology of bovine besnoitiosis, as intermediate hosts or reservoirs of B. besnoiti, remains unknown. Herein, the first serosurvey conducted in European sheep and goats from areas in Spain where bovine besnoitiosis is endemic is described. Convenience sampling was conducted of 1943 sheep and 342 goats close to cattle from the Pyrenees and Central Spain that were infected with endemic Besnoitia spp. Serum samples were first analyzed by ELISA and then by confirmatory Western blot. Specific antibodies were not found in any sampled animal. Thus, sheep are unlikely to play a role in the epidemiology of bovine besnoitiosis, at least in the sampled areas. A larger serosurvey is necessary to determine whether goats might be a putative reservoir. To confirm the results of this study, sheep and goats should be further studied in other European countries and regions where their numbers are high and where bovine besnoitiosis is spreading.
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the role of wild ruminants as reservoirs of Besnoitia besnoiti infection in cattle
Veterinary Parasitology, 2016Co-Authors: D Gutierrezexposito, Javier Regidorcerrillo, Maria C Arnal, David Martinezduran, Miguel Revilla, Daniel Fernandez De Luco, Alejandro Jimenezmelendez, R Calerobernal, M Habela, Ignacio GarciabocanegraAbstract:Abstract Bovine besnoitiosis, a parasitic disease caused by Besnoitia besnoiti, has been reported mainly in beef cattle raised under extensive pastoral systems and is considered to be re-emerging in Western Europe. Horizontal transmission probably occurs either by means of blood sucking arthropods or as a consequence of direct contact between infected and non-infected cattle. However, the role that wild ruminants (e.g., red deer (Cervus elaphus) and roe deer (Capreolus capreolus)) may play in the parasite life cycle as putative reservoirs remains elusive. Thus, we investigated the presence of Besnoitia spp. infection in 2608 wild ruminants located in areas where bovine besnoitiosis is present and identified the Besnoitia species detected. First, a serosurvey was conducted in red deer (n = 309), roe deer (n = 417), Pyrenean chamois (Rupicapra p. pyrenaica, n = 383) and Iberian wild goat (Capra pyrenaica hispanica, n = 288) from two areas of Aragon, northeastern Spain, where bovine besnoitiosis is endemic. Second, red deer (n = 820), roe deer (n = 37), fallow deer (Dama dama, n = 166), Iberian wild goat (n = 86) and European mouflon (Ovis orientalis musimon, n = 102) from southwestern Spain, where new outbreaks have recently been reported, were also sampled. The presence of Besnoitia spp.-specific antibodies was confirmed by western blot in one red deer and one roe deer from the Pyrenees, and Besnoitia spp. DNA was detected by ITS1-PCR in the seropositive red deer. Besnoitia genotyping based on 6 microsatellite (MS) analyses was carried out in red deer samples and compared with B. besnoiti genotypes from 7 in vitro isolates and 3 infected bovines, B. tarandi (1 isolate) and B. bennetti (from tissues of an infected donkey) for Besnoitia spp. assignation. Multilocus MS analysis of B. besnoiti, B. tarandi and B. bennetti showed specific genotypes for each species. A restricted genetic diversity with two genotypes by variation in a unique MS marker was revealed among the 7 B. besnoiti isolates. Incomplete Besnoitia spp. genotype of 3 MS markers from red deer samples entirely matched the B. besnoiti genotypes. Accordingly, this work gives clues for the presence of B. besnoiti infection in red deer from Western Europe. Further molecular genotyping is needed to confirm that red deer may act as an intermediate host of B. besnoiti, although the low prevalences that were found indicate that wild ruminant species do not pose a significant risk of transmitting the infection to cattle.
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Besnoitia besnoiti lytic cycle in vitro and differences in invasion and intracellular proliferation among isolates
Parasites & Vectors, 2016Co-Authors: Gereon Schares, Paula Garcialunar, Javier Regidorcerrillo, D Gutierrezexposito, J P Dubey, Caroline F Frey, Nelson Marreros, Arcangelo Gentile, 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.
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proteomics reveals differences in protein abundance and highly similar antigenic profiles between Besnoitia besnoiti and Besnoitia tarandi
Veterinary Parasitology, 2014Co-Authors: Paula Garcialunar, Javier Regidorcerrillo, L M Ortegamora, D Gutierrezexposito, Gema AlvarezgarciaAbstract:Abstract Besnoitia besnoiti and Besnoitia tarandi are two cyst-forming apicomplexan parasites of the genus Besnoitia . B. besnoiti uses cattle as an intermediate host, in which it causes a disease that progresses in two sequential phases: the acute anasarca stage and the chronic scleroderma stage. Reindeer and caribou act as intermediate hosts for B. tarandi , which causes clinical signs similar to those caused by B. besnoiti . Previous studies demonstrated high molecular similarity, as determined by 18S and ITS-1 RNA sequences, between these Besnoitia spp., and strong serological cross-reactivity between these species has recently been demonstrated. Thus, a difference gel electrophoresis approach and mass spectrometry analysis were used to describe the proteomes and explore differences in protein abundance between B. besnoiti and B. tarandi in tachyzoite extracts. Immunoproteomes were also compared using 2-DE immunoblotting with polyclonal sera from experimentally infected rabbits. From approximately 1400 spots detected in DIGE-gels, 28 and 29 spots were differentially abundant in B. besnoiti and B. tarandi tachyzoites, respectively (±1.5-fold, p B. besnoiti and B. tarandi , respectively. Of the 32 differentially abundant spots analyzed by MALDI-TOF/MS, 6 up-regulated B. besnoiti proteins (LDH; HSP90; purine nucleoside phosphorylase and 3 hypothetical proteins) and 6 up-regulated B. tarandi proteins (G3PDH; LDH; PDI; mRNA decapping protein and 2 hypothetical proteins) were identified. Interestingly, no specific antigen spots were recognized by sera on any of the Besnoitia species studied and a similar antigen profile has been observed for B. tarandi and B. besnoiti sera when cross reactions were studied. This fact corroborates the difficulty in discerning Besnoitia infections using current serological assays. The present study underscores the importance of sequencing the B. besnoiti genome for species diversity studies of the genus Besnoitia .
J P Dubey - One of the best experts on this subject based on the ideXlab platform.
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sensitive quantitative detection of Besnoitia darlingi and related parasites in intermediate hosts and to assess felids as definitive hosts for known and as yet undescribed related parasite species
International journal for parasitology. Parasites and wildlife, 2020Co-Authors: Gereon Schares, Andrea Bärwald, J P Dubey, Benjamin M Rosenthal, Mareen Tuschy, Franz Josef ConrathsAbstract:Abstract Besnoitia darlingi, B. neotomofelis and B. oryctofelisi are closely related coccidian parasites with cats as definitive hosts. While B. darlingi uses opossums as intermediate hosts, B. neotomofelis and B. oryctofelisi have been described in Southern Plains woodrats (Neotoma micropus) from the USA and in domestic rabbits from Argentina, respectively. A comparison of the Internal Transcribed Spacer-1 (ITS-1) region of the ribosomal DNA (rDNA) of these Besnoitia spp. showed only a few differences. The present study aimed at developing a real-time PCR to detect B. darlingi, B. neotomofelis and B. oryctofelisi in tissues of intermediate and in faeces of definitive hosts in order to support studies of these organisms’ epidemiology and pathogenesis. The established PCR was based on primer regions distinct from the ITS-1 sequences of ungulate Besnoitia spp. and made use of a Besnoitia universal probe. To monitor inhibition, a heterologous internal control was established based on the enhanced green fluorescent protein gene. The real-time PCR reacted with B. darlingi, B. neotomofelis and B. oryctofelisi, while the novel PCR did not recognize ungulate Besnoitia spp. (B. besnoiti, B. bennetti, B. tarandi). DNA of Apicomplexa ascribed to other Besnoitia-related genera, including other gut parasites of cats (Cryptosporidium parvum, Giardia duodenalis, Tritrichomonas foetus), was not recognized. The real-time PCR had an analytic sensitivity of less than 1 tachyzoite per reaction. In feline faeces spiked with B. darlingi oocysts, the limit of detection was a DNA amount equivalent to 1 oocyst per PCR reaction. In B. darlingi infected ɣ-interferon knock-out mice, the lung was identified as the predilection organ. In conclusion, this real-time PCR should advance further studies on these parasites and may inspire research on related species, not only in the Americas, but also in other parts of the world.
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bobcats lynx rufus are natural definitive host of Besnoitia darlingi
Veterinary Parasitology, 2017Co-Authors: S K Verma, Benjamin M Rosenthal, Camila K Cerqueiracezar, F H A Murata, Matthew J Lovallo, J P DubeyAbstract:Abstract Bovine besnoitiosis, caused by Besnoitia besnoiti, is an economically important disease of cattle in many countries but its transmission remains a mystery. Wild felids are suspected to be its definitive hosts. The domestic cat (Felis catus) is known experimental definitive host for Besnoitia species of rodents. Here, we report for Besnoitia darlingi the first identification of a natural definitive host, the bobcat (Lynx rufus). Oocysts resembling Toxoplasma gondii (unsporulated; 10.9 ± 0.8 × 12.1 ± 0.2 μm; n = 5) were detected microscopically in the feces of two of 25 free ranging wild bobcats from Mississippi, USA. After detailed investigation, we identified these oocysts as B. darlingi and not T. gondii. The IFN-γ gene knockout (KO) mice fed oocysts from bobcats died of acute besnoitiosis and tachyzoites were found in their tissues. Oocysts were also mildly pathogenic to outbred Swiss Webster mice (SW) (Mus musculus). The SW mice fed oocysts became ill but generally survived and developed characteristic thick-walled Besnoitia tissue cysts in their tongue and heart muscles and brains. Two laboratory-raised domestic cats (Felis catus) excreted B. darlingi oocysts after ingesting murine tissues infected with bobcat-derived oocysts. The parasite was successfully cultivated in African green monkey kidney fibroblast cells (CV-1 cell line) seeded with infected murine tissue homogenate. The multilocus PCR-DNA sequencing (18S rDNA, 28S rDNA, and ITS-1) from culture-derived tachyzoites confirmed the parasite as B. darlingi. Our results suggest that bobcats may be an important link in the sylvatic cycle of Besnoitia species and bioassay or molecular tests are needed to differentiate Toxoplasma gondii-like oocysts in feces of felids, both domestic and wild cats.
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Besnoitia besnoiti lytic cycle in vitro and differences in invasion and intracellular proliferation among isolates
Parasites & Vectors, 2016Co-Authors: Gereon Schares, Paula Garcialunar, Javier Regidorcerrillo, D Gutierrezexposito, J P Dubey, Caroline F Frey, Nelson Marreros, Arcangelo Gentile, 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.
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development of early tissue cysts and associated pathology of Besnoitia besnoiti in a naturally infected bull bos taurus from south africa
Journal of Parasitology, 2013Co-Authors: J P Dubey, Gereon Schares, E Van Wilpe, D J C Blignaut, J H WilliamsAbstract:Abstract: Besnoitia besnoiti is an apicomplexan that causes serious economic loss in cattle in many countries and the disease is now spreading in Europe. At least 2 phases of bovine besnoitiosis are recognized clinically. An acute febrile phase characterized by anasarca and necrosis of skin is associated with multiplication of tachyzoites in vascular endothelium; this phase is short-lived and rarely diagnosed. Chronic besnoitiosis characterized by dermal lesions is associated with the presence of macroscopic tissue cysts and is easily diagnosed. Here we report the development of early B. besnoiti tissue cysts in a naturally infected Hugenoot bull from South Africa. Tissue cysts were 10–70 μm in diameter, contained 1–12 bradyzoites, and were most numerous in the dermis, testicles, and pampiniform venous plexus. Amylopectin granules in bradyzoites stained red with periodic acid Schiff (PAS) reaction. Bradyzoites varied in size and in the intensity of PAS reaction (some were PAS-negative), some were plump, a...
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serological evidence of Besnoitia spp infection in canadian wild ruminants and strong cross reaction between Besnoitia besnoiti and Besnoitia tarandi
Veterinary Parasitology, 2012Co-Authors: D Gutierrezexposito, Paula Garcialunar, J P Dubey, Luis Miguel Ortegamora, Alvin A Gajadhar, Gema AlvarezgarciaAbstract:Abstract Bovine besnoitiosis, caused by Besnoitia besnoiti, is considered to be emergent in Europe and responsible for severe economic losses due to the chronic and debilitating course of the disease but has not been reported in North America. Besnoitia tarandi is a related species and it has been reported in reindeer and caribou from different locations of the Arctic Pole, including North America. Diagnosis of clinical besnoitiosis is largely based on the recognition of dermal grossly visible tissue cysts of Besnoitia. Nothing is known of cross reactivity between B. besnoiti and B. tarandi species. Here, we evaluated the use of serological tests employed in the diagnosis of bovine besnoitiosis for the detection of Besnoitia spp. infections in different wild ruminant species (caribou, elk, mule-deer, white-tailed deer, moose, muskox and bison) from Canada and investigated cross-reactivity between B. besnoiti and B. tarandi species by indirect immunofluorescence antibody test and Western blot. For this, species-specific antibodies were obtained in rabbits experimentally infected with B. besnoiti and B. tarandi. Marked cross reactivity was found between B. besnoiti and B. tarandi. For the first time, antibodies to Besnoitia spp. infection were found in 16 of 20 caribou (Ranginfer tarandus), seven of 18 muskox (Ovibos moschatus), one of three bison (Bison bison), but not in 20 elk (Cervus canadensis), 20 white tailed deer (Odocoileus virginianus), and 20 moose (Alces alces) in Canada; results were similar using B. besnoiti and B. tarandi as antigen. There was no cross reactivity between the two Besnoitia species, Neospora caninum and Toxoplasma gondii with the cut-offs applied that prevented to observe it. The present study provides evidence that the serological assays can be useful to accomplish large scale prevalence studies in caribou and other wildlife species. Further studies are needed to study sylvatic and domestic cycle of B tarandi and B. besnoiti.
