Besnoitia darlingi, B. neotomofelis and B. oryctofelisi are closely related coccidian parasites with cats as definitive hosts. While B. darlingi uses opossums as intermediate hosts, B. neotomofelis and B. oryctofelisi have been described in Southern Plains woodrats (Neotoma micropus) from the USA and in domestic rabbits from Argentina, respectively. A comparison of the Internal Transcribed Spacer-1 (ITS-1) region of the ribosomal DNA (rDNA) of these Besnoitia spp. showed only a few differences. The present study aimed at developing a real-time PCR to detect B. darlingi, B. neotomofelis and B. oryctofelisi in tissues of intermediate and in faeces of definitive hosts in order to support studies of these organisms' epidemiology and pathogenesis. The established PCR was based on primer regions distinct from the ITS-1 sequences of ungulate Besnoitia spp. and made use of a Besnoitia universal probe. To monitor inhibition, a heterologous internal control was established based on the enhanced green fluorescent protein gene. The real-time PCR reacted with B. darlingi, B. neotomofelis and B. oryctofelisi, while the novel PCR did not recognize ungulate Besnoitia spp. (B. besnoiti, B. bennetti, B. tarandi). DNA of Apicomplexa ascribed to other Besnoitia-related genera, including other gut parasites of cats (Cryptosporidium parvum, Giardia duodenalis, Tritrichomonas foetus), was not recognized. The real-time PCR had an analytic sensitivity of less than 1 tachyzoite per reaction. In feline faeces spiked with B. darlingi oocysts, the limit of detection was a DNA amount equivalent to 1 oocyst per PCR reaction. In B. darlingi infected ɣ-interferon knock-out mice, the lung was identified as the predilection organ. In conclusion, this real-time PCR should advance further studies on these parasites and may inspire research on related species, not only in the Americas, but also in other parts of the world.

中文翻译：

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灵敏，定量检测中寄主中的Besnoitia darlingi和相关寄生虫，并评估作为已知和尚未描述的相关寄生虫物种的确定宿主的猫科动物。

达斯尼虫，新芽孢杆菌和稻芽孢杆菌是密切相关的球虫寄生虫，猫为最终宿主。尽管达令吉芽孢杆菌使用负鼠作为中间宿主，但美国的南方平原伍德拉特鼠（Neotoma micropus）和阿根廷的家养兔子中均已描述了新芽孢杆菌和稻芽孢杆菌。这些Besnoitia spp核糖体DNA（rDNA）的内部转录间隔区1（ITS-1）区域的比较。仅显示出一些差异。本研究旨在开发一种实时PCR，以检测定殖宿主中间和粪便组织中的B. darlingi，B。neotomofelis和B. oryctofelisi，以支持对这些生物的流行病学和发病机理的研究。建立的PCR基于不同于有蹄类贝氏菌属物种的ITS-1序列的引物区域。并使用了Besnoitia通用探头。为了监测抑制，基于增强的绿色荧光蛋白基因建立了异源内部对照。实时PCR与达氏芽孢杆菌，新芽孢杆菌和稻芽孢杆菌反应，而新型PCR不能识别有蹄类贝氏菌。（B. besnoiti，B。bennetti，B。tarandi）。不能归因于归因于其他与疟疾相关的属，包括猫的其他肠道寄生虫（小隐孢子虫，贾第鞭毛虫，Tritrichomonas foetus）的蚜虫DNA。实时PCR的每个反应的分析灵敏度小于1速殖子。在掺有达氏芽孢杆菌卵囊的猫粪中，检测极限是每个PCR反应相当于1个卵囊的DNA量。在达氏芽孢杆菌感染的β-干扰素敲除小鼠中，肺被确定为好发器官。总而言之，这种实时PCR应当推动对这些寄生虫的进一步研究，并可能激发相关物种的研究，不仅在美洲，而且在世界其他地区。