Fernández-García A, Risco-Castillo V, Pedraza-Díaz S, Aguado-Martínez A, Alvarez-García G, Gómez-Bautista M, Collantes-Fernández E, Ortega-Mora L M
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain.
J Parasitol. 2009 Apr;95(2):474-6. doi: 10.1645/GE-1772.1.
Besnoitia besnoiti was isolated from a skin biopsy of a chronically infected cow from central Spain. Zoites released from macroscopic cysts were adapted to its culture in vitro on a MARC-145 cell monolayer. Tachyzoites produced in vitro were either cryopreserved or used for genomic DNA isolation. A 2206 nt sequence containing 18S ribosomal RNA gene, internal transcribed spacer 1 (ITS 1), and a partial sequence of 5.8S ribosomal RNA gene was amplified by PCR and sequenced. This sequence showed a 99-100% identity to 18S, ITS1, and 5.8S sequences of B. besnoiti published in databases. After analysis by transmission and scanning electron microscopy of isolated bradyzoites and tachyzoites, it was observed that their ultrastructural morphology coincided with B. besnoiti. The isolate characterized in this study was identified as B. besnoiti on the basis of the disease produced, molecular characteristics, and morphology. The B. besnoiti isolate was denoted as BbSpain-1; it is the first isolate obtained and characterized in Spain and one of the first European isolates adapted to grow in vitro. The isolation and in vitro production of this B. besnoiti isolate offers a good opportunity to study general aspects of bovine besnoitiosis, including epidemiology, pathogenesis, and diagnosis of this re-emergent disease.
贝氏贝诺孢子虫是从西班牙中部一头慢性感染奶牛的皮肤活检样本中分离得到的。从肉眼可见的囊肿中释放出的速殖子在MARC - 145细胞单层上进行体外培养适应性培养。体外产生的速殖子要么进行冷冻保存，要么用于基因组DNA提取。通过PCR扩增并测序得到一段包含18S核糖体RNA基因、内部转录间隔区1（ITS 1）以及5.8S核糖体RNA基因部分序列的2206 nt序列。该序列与数据库中已公布的贝氏贝诺孢子虫的18S、ITS1和5.8S序列具有99 - 100%的同一性。对分离出的缓殖子和速殖子进行透射电子显微镜和扫描电子显微镜分析后，观察到它们的超微结构形态与贝氏贝诺孢子虫一致。基于所引发的疾病、分子特征和形态学，本研究中鉴定的分离株被确定为贝氏贝诺孢子虫。该贝氏贝诺孢子虫分离株被命名为BbSpain - 1；它是在西班牙获得并鉴定的首个分离株，也是最早适应体外生长的欧洲分离株之一。该贝氏贝诺孢子虫分离株的分离和体外培养为研究牛贝诺孢子虫病的一般方面提供了良好契机，包括这种再度出现的疾病的流行病学、发病机制和诊断。
