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Gema Alvarezgarcia – 1st expert on this subject based on the ideXlab platform

  • 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, 2020
    Co-Authors: Alejandro Jimenezmelendez, Chandra Ramakrishnan, Adrian B Hehl, Giancarlo Russo, Gema Alvarezgarcia


    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.

  • lytic cycle of Besnoitia besnoiti tachyzoites displays similar features in primary bovine endothelial cells and fibroblasts
    Parasites & Vectors, 2019
    Co-Authors: Alejandro Jimenezmelendez, Luis Miguel Ortegamora, Maria Fernandezalvarez, Alexandra Calle, Miguel Angel Ramirez, Carlos Diezmadiaz, Patricia Vazquezarbaizar, Gema Alvarezgarcia


    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.

  • repurposing of commercially available anti coccidials identifies diclazuril and decoquinate as potential therapeutic candidates against Besnoitia besnoiti infection
    Veterinary Parasitology, 2018
    Co-Authors: Alejandro Jimenezmelendez, Luis Miguel Ortegamora, Andrew Hemphill, Vreni Balmer, Laura Ricosan Roman, Gema Alvarezgarcia


    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.

Gereon Schares – 2nd expert on this subject based on the ideXlab platform

  • Serological survey of Neospora spp. and Besnoitia spp. in horses in Portugal.
    Veterinary Parasitology: Regional Studies and Reports, 2020
    Co-Authors: Helga Waap, Uillians Volkart De Oliveira, Telmo Nunes, Jacinto Gomes, Tiago Gomes, Andrea Bärwald, Alexandre Dias Munhoz, Gereon Schares


    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.

  • 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, 2020
    Co-Authors: Gereon Schares, Andrea Bärwald, J P Dubey, Benjamin M Rosenthal, Mareen Tuschy, Franz Josef Conraths


    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.

  • development and characterization of monoclonal antibodies against Besnoitia besnoiti tachyzoites
    Parasitology, 2019
    Co-Authors: Paula Garcialunar, Gereon Schares, Javier Regidorcerrillo, Alejandro Jimenezmelendez, Andrew Hemphill, A Sanzfernandez, I Garciasoto, Ivan Pastorfernandez, M Fernandezalvarez, L M Ortegamora


    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.

Helder Cortes – 3rd expert on this subject based on the ideXlab platform

  • Besnoitia besnoiti impact on fertility of cattle exploited in Mediterranean pastures (Alentejo)
    , 2020
    Co-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. Marques


    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.

  • Besnoitia besnoiti and toxoplasma gondii two apicomplexan strategies to manipulate the host cell centrosome and golgi apparatus
    Parasitology, 2014
    Co-Authors: Rita M Cardoso, Helder Cortes, Alexandre Leitao, Sofia Nolasco, Joao Goncalves, Helena Soares


    : 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.

  • a review on bovine besnoitiosis a disease with economic impact in herd health management caused by Besnoitia besnoiti franco and borges
    Parasitology, 2014
    Co-Authors: Helder Cortes, Alexandre Leitao, Bruno Gottstein, Andrew Hemphill


    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.