Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, Wusterhausen, Germany.
Vet Parasitol. 2011 Jun 10;178(3-4):208-16. doi: 10.1016/j.vetpar.2011.01.038. Epub 2011 Feb 15.
Bovine besnoitiosis, an economically important disease in cattle in some countries of Africa and Asia, is emerging in Europe. The definitive host of Besnoitia besnoiti, the causative agent of bovine besnoitiosis, is unknown and the transmission of the parasite is not completely understood. Sensitive and quantitative DNA detection methods are needed to determine whether serologically positive animals are infectious and to examine the role of vectors (e.g. haematophagous insects) in the transmission of the parasite. To this end, we established two different 5'-nuclease quantitative assays to detect B. besnoiti infection in cattle and to estimate the parasite load in samples (BbRT1 and BbRT2). These PCRs are based on the sequence of the internal transcribed spacer region 1 (ITS-1) of the ribosomal RNA gene. Tests with serial dilutions of B. besnoiti genomic DNA in a buffer containing 100 ng/μl bovine DNA revealed a detection limit of 0.01 pg genomic B. besnoiti DNA. Reliable quantification was possible in samples containing ≥1 pg B. besnoiti genomic DNA with a coefficient of variation of ≤ 2%. To estimate the diagnostic sensitivity of the tests, skin biopsies and scrapings from the mucous membrane of the vestibulum vaginae (vaginal scrapings) were taken from cattle with clinical signs of chronic besnoitiosis. Regardless of the real time PCR assay used, 90.7% (39/43) of these animals were positive in at least one of two samples (skin or vaginal scrapings). Antibody titers, as determined by an immunofluorescent antibody test, and the threshold cycle values of the real time PCR obtained for skin samples and vaginal scrapings, were significantly correlated. The specificity of the PCRs was confirmed using genomic DNA from related parasites, including genomic DNA of Besnoitia spp., Neospora caninum, Toxoplasma gondii, Hammondia hammondi, Hammondia heydorni, Isospora spp., Sarcocystis spp., Eimeria bovis, Cryptosporidium parvum, and Trypanosoma brucei brucei. Since the sequence of the ITS-1 region of B. besnoiti is identical with that of Besnoitia species isolated from donkeys (Besnoitia bennetti), and reindeer (Besnoitia tarandi), both real time PCRs detected also DNA of these parasites. One of the B. besnoiti real time PCRs, BbRT1, but not BbRT2, cross-reacted with Besnoitia darlingi, Besnoitia oryctofelisi, and Besnoitia neotomofelis when large amounts of genomic DNA (10 ng) were used. The other B. besnoiti real time PCR assay (BbRT2) was specific for B. besnoiti, B. bennetti and B. tarandi, but did not react when 10 ng DNA of other related parasite species from the genus Besnoitia or other genera were subjected to analysis.
牛贝氏贝虫病是一种在非洲和亚洲一些国家对牛具有重要经济意义的疾病，正在欧洲出现。贝氏贝虫病的病原体贝氏贝虫的终末宿主尚不清楚，其传播方式也不完全清楚。需要敏感和定量的 DNA 检测方法来确定血清学阳性动物是否具有传染性，并研究载体（如吸血昆虫）在寄生虫传播中的作用。为此，我们建立了两种不同的 5'-核酸酶定量检测方法，用于检测牛的贝氏贝虫感染，并估计样品中的寄生虫载量（BbRT1 和 BbRT2）。这些 PCR 基于核糖体 RNA 基因的内部转录间隔区 1（ITS-1）的序列。在含有 100ng/μl 牛 DNA 的缓冲液中对 B. besnoiti 基因组 DNA 的系列稀释进行测试，显示出检测 0.01pg 基因组 B. besnoiti DNA 的检测限。在含有≥1pg B. besnoiti 基因组 DNA 的样品中，可进行可靠的定量，变异系数≤2%。为了估计检测的诊断灵敏度，从患有慢性贝氏贝虫病临床症状的牛中采集皮肤活检和阴道前庭粘膜刮片（阴道刮片）。无论使用何种实时 PCR 检测方法，这些动物中有 90.7%（39/43）在至少一种两种样本（皮肤或阴道刮片）中呈阳性。通过免疫荧光抗体试验确定的抗体滴度与从皮肤样本和阴道刮片中获得的实时 PCR 的阈值循环值显著相关。使用相关寄生虫的基因组 DNA，包括 Besnoitia spp.、Neospora caninum、Toxoplasma gondii、Hammondia hammondi、Hammondia heydorni、Isospora spp.、Sarcocystis spp.、Eimeria bovis、Cryptosporidium parvum 和 Trypanosoma brucei brucei 的基因组 DNA，证实了 PCR 的特异性。由于 B. besnoiti 的 ITS-1 区序列与从驴（贝氏贝虫）和驯鹿（贝氏贝虫）中分离的贝氏贝虫的序列相同，因此这两种实时 PCR 也检测到了这些寄生虫的 DNA。其中一种 B. besnoiti 实时 PCR 检测方法（BbRT1），但不是 BbRT2，当使用大量基因组 DNA（10ng）时，与 Besnoitia darlingi、Besnoitia oryctofelisi 和 Besnoitia neotomofelis 发生交叉反应。另一种 B. besnoiti 实时 PCR 检测方法（BbRT2）对 B. besnoiti、B. bennetti 和 B. tarandi 具有特异性，但当分析来自 Besnoitia 属或其他属的其他相关寄生虫物种的 10ng DNA 时，不会发生反应。
