title: Besnoitia besnoiti lytic cycle in vitro and differences in invasion and intracellular proliferation among isolates.
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 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.
  
  
  METHODS
  We compared invasion and proliferation of one B. tarandi (from Finland) and seven B. besnoiti 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 h 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 24 h. 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 neither by invasion-related parameters (velocity and half time invasion) nor by 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, large and small parasitophorous vacuoles).
  
  
  CONCLUSIONS
  This study provides first insights into the lytic cycle of B. besnoiti isolates and a standardised in vitro model that allows screening of drug candidates for the treatment of besnoitiosis.
authors.affiliation: SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain. caroline.frey@vetsuisse.unibe.ch.|Institute of Parasitology, Vetsuisse Faculty, University of Bern, Länggass-Strasse 122, 3012, Bern, Switzerland. caroline.frey@vetsuisse.unibe.ch.
authors.forename: Caroline F
authors.initials: CF
authors.lastname: Frey
authors.affiliation: SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain. jregidor@ucm.es.
authors.forename: Javier
authors.initials: J
authors.lastname: Regidor-Cerrillo
authors.affiliation: Centre for Fish and Wildlife Health, Vetsuisse Faculty, University of Bern, Länggass-Strasse 122, 3012, Bern, Switzerland. nelson.marreros@vetsuisse.unibe.ch.
authors.forename: Nelson
authors.initials: N
authors.lastname: Marreros
authors.affiliation: SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain. paulagarcialunar@vet.ucm.es.
authors.forename: Paula
authors.initials: P
authors.lastname: García-Lunar
authors.affiliation: SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain. dgexposito@pdi.ucm.es.
authors.forename: Daniel
authors.initials: D
authors.lastname: Gutiérrez-Expósito
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, Greifswald, Insel Riems, Germany. gereon.schares@fli.bund.de.
authors.forename: Gereon
authors.initials: G
authors.lastname: Schares
authors.affiliation: United States Department of Agriculture, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, Beltsville, MD, 20705-2350, USA. jitender.dubey@ars.usda.gov.
authors.forename: Jitender P
authors.initials: JP
authors.lastname: Dubey
authors.affiliation: Department of Veterinary Medical Sciences, University of Bologna, Ozzano dell'Emilia, Italy. arcangelo.gentile@unibo.it.
authors.forename: Arcangelo
authors.initials: A
authors.lastname: Gentile
authors.affiliation: Institut National Polytechnique (INP), Ecole Nationale Vétérinaire de Toulouse (ENVT), UMR1225, IHAP, Equipe « Besnoitiose et vecteurs », Université de Toulouse, Toulouse, France. p.jacquiet@envt.fr.
authors.forename: Philippe
authors.initials: P
authors.lastname: Jacquiet
authors.affiliation: Division of Parasitology, Kimron Veterinary Institute, P.O. Box 12, Bet Dagan, 50250, Israel. shkapvarda@gmail.com.
authors.forename: Varda
authors.initials: V
authors.lastname: Shkap
authors.affiliation: Laboratório de Parasitologia Victor Caeiro, Núcleo da Mitra, ICAAM, Universidade de Évora, Apartado 94, 7000-554, Évora, Portugal. hcec@uevora.pt.
authors.forename: Helder
authors.initials: H
authors.lastname: Cortes
authors.affiliation: SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain. luis.ortega@vet.ucm.es.
authors.forename: Luis M
authors.initials: LM
authors.lastname: Ortega-Mora
authors.affiliation: SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain. gemaga@vet.ucm.es.
authors.forename: Gema
authors.initials: G
authors.lastname: Álvarez-García
mesh_terms: D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D004847:Epithelial Cells / Q000469:parasitology*; D000882:Haplorhini; D008954:Models, Biological; D021745:Sarcocystidae / Q000254:growth & development*