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Title:   Author  item
                                            FREY, CAROLINE - Complutense University Of Madrid (UCM)item
                                            REGIDOR-CERRILLO, JAVIER - Complutense University Of Madrid (UCM)item
                                            MARREROS, NELSON - University Of Bernitem
                                            GARCIA-LUNAR, PAULA - Complutense University Of Madrid (UCM)item
                                            GUTIERREZ-EXPOSITO, DANIEL - Complutense University Of Madrid (UCM)item
                                            SCHARES, GEREON - Bundesforschungsinstitut Für Tiergesundheititem
                                            Dubey, Jitenderitem
                                            GENTILE, ARACANGELO - University Of Bolognaitem
                                            JACQUIET, PHILIPPE - National Veterinary School Of Toulouse, Franceitem
                                            SHKAP, VARDA - Kimron Veterinary Instituteitem
                                            CORTES, HELDER - University Of Evoraitem
                                            ORTEGA-MORA, LUIS - Complutense University Of Madrid (UCM)item
                                            ALVAREZ-GARCIA, GEMA - Complutense University Of Madrid (UCM)
  Submitted to: Parasites & Vectors
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 1/20/2016
                                    Publication  Date: 2/1/2016
                                    Citation: Frey, C.F., Regidor-Cerrillo, J., Marreros, N., Garcia-Lunar, P., Gutierrez-Exposito, D., Schares, G., Dubey, J.P., Gentile, A., Jacquiet, P., Shkap, V., Cortes, H., Ortega-Mora, L.M., Alvarez-Garcia, G. 2016. Besnoitia besnoti lytic cycle in vitro and differences in invasion and intracellular proliferation among isolates. Parasites & Vectors. 29:115.Interpretive Summary: Toxoplasmosis, caused by the single-celled parasite, Toxoplasma gondii,  continues to be a public health problem. Its diagnosis from its relatives in the genera , Sarcocystis, Neospora, and Besnoitia is often difficult because of morphological similarities and cross reacting antibodies among these parasites. Besnoitia besnoiti is such a parasite that causes debilitating disease, besnoitiosis in cattle. In the present paper the authors compared growth patterns of several isolates of B. besnoiti and found that Besnoitia parasites grow slower than Toxoplasma and Neospora. The methods described will aid to study the pathogenesis of the disease, in the screening for vaccine targets and drugs potentially useful for the treatment of besnoitiosis. These results will be of interest to parasitologists and biologists.
                                Technical Abstract: 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 standardized 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.

Methods: We compared invasion and proliferation of one B. tarandi (from Finland) and seven B. besnoitia isolates (Bb-Spain1, Bb-Spain2, Bb-Israel, Bb-Evora03, Bb-Ger1, Bb-France, Bb-Italy2) in MARC-145 cell culture. Host cell invasion was studied at 4, 6, 8 and 24 hours post infection (hpi), and proliferation characteristics were compared at 24, 48, 72, 96, 120, and 144 hpi .
Results: In Besnoitia spp., the key parameters that determine the sequential adhesion-invasion, proliferation and egress steps are clearly distinct from those in the related apicomplexans Toxoplasma gondii and Neospora caninum. Besnoitia spp. host cell invasion is a rather slow process, since only 50% of parasites were found intracellular after 3-6 h of exposure to host cells, and invasion still took place after 24h. Invasion efficacy was significantly higher for Bb-France, Bb-Evora03 and Bb-Israel. In addition, the time span for endodyogeny to take place was as long as 18-35 h. Bb-Israel and B. tarandi isolates were most prolific, as determined by the tachyzoite yield at 72 hpi. The total tachyzoite yield could not be predicted either by invasion-related parameters (velocity and half time invasion) or proliferation parameters (lag phase and doubling time (dT)). The lytic cycle of Besnoitia was asynchronous as evidenced by the presence of three different plaque forming tachyzoite categories (lysis plaques, big and small parasitophorous vacuoles).

Conclusions: This study provides first insights into the lytic cycle of B. besnoitia isolates and a standardized in vitro model that allows screening of drug candidates for the treatment of besnoitiosis.


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