Kruth P S, Whale J, Léveillé A N, Brisbin J, Barta J R
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada.
Ceva Animal Health Inc, Guelph, ON, Canada.
Parasitol Res. 2024 Dec 28;123(12):422. doi: 10.1007/s00436-024-08429-1.
Coccidiosis is caused by apicomplexan parasites of the genus Eimeria, which infect epithelial cells of the intestinal tract causing diarrhea and negatively impacting production in the poultry industry. The self-limiting and highly immunogenic nature of infection by Eimeria spp. make live vaccination an effective means of coccidiosis control. Paramount to vaccine efficacy is the ability to administer precise numbers of viable oocysts. Unfortunately, no rapid and accurate method for determination of oocyst viability is available presently. This study presents the development of a qPCR-based assay for assessment of Eimeria tenella Tyzzer, 1929 oocyst viability. Transcriptome sequencing supported identification of three viability assay target transcripts based on significant increase in abundance with heat-stimulation. Measurement of shifts in target abundances in response to heat stimulation in oocysts, that ranged from high viability to non-infectious, was achieved via qPCR. Omission of DNase treatment supported use of background DNA in RNA samples for normalization for parasite numbers and oocyst disruption efficiency, while spike in of exogenous RNA supported normalization for variations in RNA recovery and reverse transcription efficiency. The assay demonstrated strong correlation with oocyst viability as confirmed through live infection trials, showing the highest predictive value for a transcript encoding a putative partial translationally controlled tumor protein, XM_013379639.1. This assay provides results in hours and could reduce the reliance on time-consuming and expensive live-infection trials in oocyst viability testing and could improve the accessibility and efficacy of coccidiosis vaccines. Future iterations may facilitate multivalent vaccine quality control and environmental monitoring.
球虫病由艾美耳属的顶复门寄生虫引起，这些寄生虫感染肠道上皮细胞，导致腹泻，并对家禽业的生产产生负面影响。艾美耳属物种感染具有自限性和高度免疫原性，这使得活疫苗接种成为控制球虫病的有效手段。疫苗效力的关键在于能够精确接种活卵囊数量。不幸的是，目前尚无快速准确测定卵囊活力的方法。本研究提出了一种基于qPCR的检测方法，用于评估1929年泰泽艾美耳球虫的卵囊活力。转录组测序支持基于热刺激下丰度显著增加鉴定出三种活力检测靶转录本。通过qPCR测量卵囊（活力范围从高到无感染性）对热刺激的靶丰度变化。省略DNase处理支持使用RNA样本中的背景DNA对寄生虫数量和卵囊破坏效率进行标准化，而外源RNA的掺入支持对RNA回收率和逆转录效率的变化进行标准化。经活感染试验证实，该检测方法与卵囊活力具有很强的相关性，对编码假定部分翻译控制肿瘤蛋白XM_013379639.1的转录本显示出最高的预测价值。该检测方法在数小时内即可得出结果，可减少卵囊活力检测中对耗时且昂贵的活感染试验的依赖，并可提高球虫病疫苗的可及性和效力。未来的改进可能有助于多价疫苗的质量控制和环境监测。
