Frey Caroline F, Regidor-Cerrillo Javier, Marreros Nelson, García-Lunar Paula, Gutiérrez-Expósito Daniel, Schares Gereon, Dubey Jitender P, Gentile Arcangelo, Jacquiet Philippe, Shkap Varda, Cortes Helder, Ortega-Mora Luis M, Álvarez-García Gema
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040, Madrid, Spain.
Institute of Parasitology, Vetsuisse Faculty, University of Bern, Länggass-Strasse 122, 3012, Bern, Switzerland.
Parasit Vectors. 2016 Feb 29;9:115. doi: 10.1186/s13071-016-1405-9.
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.
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.
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).
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.
由原生动物贝氏贝诺孢子虫（Besnoitia besnoiti）引起的牛贝诺孢子虫病会降低受感染牛群的生产力和繁殖力。贝诺孢子虫病在欧洲仍在蔓延，目前尚无有效的控制手段。迫切需要实验模型。在此，我们首次描述了贝氏贝诺孢子虫裂解周期标准化体外模型的动力学。这将有助于研究该疾病的发病机制，筛选潜在的疫苗靶点以及对治疗贝诺孢子虫病有用的药物。
我们比较了1株来自芬兰的塔氏贝诺孢子虫（B. tarandi）和7株贝氏贝诺孢子虫分离株（Bb-西班牙1、Bb-西班牙2、Bb-以色列、Bb-埃武拉03、Bb-德国1、Bb-法国、Bb-意大利2）在MARC-145细胞培养中的侵袭和增殖情况。在感染后4、6、8和24小时（hpi）研究宿主细胞侵袭，并在24、48、72、96、120和144 hpi比较增殖特性。
在贝诺孢子虫属中，决定顺序性黏附-侵袭、增殖和逸出步骤的关键参数与相关顶复门寄生虫刚地弓形虫（Toxoplasma gondii）和犬新孢子虫（Neospora caninum）明显不同。贝诺孢子虫属的宿主细胞侵袭是一个相当缓慢的过程，因为在与宿主细胞接触3-6小时后，仅发现50%的寄生虫在细胞内，并且在24小时后仍有侵袭发生。Bb-法国、Bb-埃武拉03和Bb-以色列的侵袭效率显著更高。此外，内二分裂发生的时间跨度长达18-35小时。根据72 hpi时速殖子产量确定，Bb-以色列和塔氏贝诺孢子虫分离株繁殖能力最强。速殖子总产量既不能通过与侵袭相关的参数（速度和侵袭半衰期）预测，也不能通过增殖参数（延迟期和倍增时间（dT））预测。贝诺孢子虫的裂解周期是异步的，这通过存在三种不同的形成噬斑的速殖子类别（裂解噬斑、大小不同的寄生泡）得以证明。
本研究首次深入了解了贝氏贝诺孢子虫分离株的裂解周期，并提供了一个标准化体外模型，可用于筛选治疗贝诺孢子虫病的候选药物。
