Periyasamy Dhinesh, Huang Yanyun, Hill Janet E
Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
Prairie Diagnostic Services, Inc., Saskatoon, Saskatchewan, Canada.
J Clin Microbiol. 2025 Jan 31;63(1):e0143324. doi: 10.1128/jcm.01433-24. Epub 2024 Dec 10.
Bovine reproductive failure, which includes infertility, abortion, and stillbirth in cattle, leads to significant economic losses for beef and milk producers. Diagnosing the infectious causes of bovine reproductive failure is challenging as there are multiple pathogens associated with it. The traditional stepwise approach to diagnostic testing is time-consuming and can cause significant delays. In this study, we have developed a syndromic next-generation sequencing panel (BovReproSeq) for the simultaneous detection of 17 pathogens (bacteria, virus, and protozoa) associated with bovine reproductive failure. This targeted approach involves amplifying multiple pathogen-specific targets using ultra-multiplex PCR, followed by sequencing with the Oxford Nanopore platform and subsequent analysis of the data using a custom bioinformatic pipeline to determine the presence or absence of pathogens. We tested 116 clinical samples and found that BovReproSeq results matched with current diagnostic methods for 93% of the samples, and most of the disagreements occurring in samples with very low pathogen loads (Ct >35). At the optimal read-count threshold of 10 reads (minimum number of reads to classify the sample as positive), the clinical sensitivity of the assay was approximately 82%, while clinical specificity was 100%. The overall accuracy of the assay was 98.8%. Matthews correlation coefficient (correlation coefficient of binary classification) was approximately 0.90 and F1 score (harmonic mean of precision and recall) was 0.90, indicating excellent overall performance. Our study presents a significant advancement in detecting the infectious agents associated with bovine reproductive failure and the BovReproSeq panel's ability to detect 17 pathogens makes it a promising tool for veterinary diagnostics.IMPORTANCEBovine reproductive failure causes substantial economic losses to beef and milk producers, and infectious disease contributes significantly to this syndrome. Etiologic diagnosis is complicated since multiple pathogens can be involved and infections with some pathogens are asymptomatic or cause similar clinical signs. A stepwise approach to diagnostic testing is time-consuming and increases the risk of missing the correct diagnosis. BovReproSeq is a next-generation sequencing-based diagnostic panel that allows detection of 17 reproductive failure pathogens simultaneously.
牛繁殖障碍包括牛的不育、流产和死产，给牛肉和牛奶生产者带来了巨大的经济损失。诊断牛繁殖障碍的感染原因具有挑战性，因为与之相关的病原体有多种。传统的逐步诊断测试方法耗时且可能导致显著延迟。在本研究中，我们开发了一种综合征下一代测序检测板（BovReproSeq），用于同时检测与牛繁殖障碍相关的17种病原体（细菌、病毒和原生动物）。这种靶向方法包括使用超多重PCR扩增多个病原体特异性靶点，然后使用牛津纳米孔平台进行测序，并使用定制的生物信息学流程对数据进行后续分析，以确定病原体的存在与否。我们测试了116份临床样本，发现BovReproSeq的结果与当前诊断方法对93%的样本匹配，大多数不一致发生在病原体载量非常低（Ct>35）的样本中。在最佳读数阈值为10次读数（将样本分类为阳性的最小读数数量）时，该检测方法的临床敏感性约为82%，而临床特异性为100%。该检测方法的总体准确率为98.8%。马修斯相关系数（二元分类的相关系数）约为0.90，F1分数（精确率和召回率的调和平均数）为0.90，表明总体性能优异。我们的研究在检测与牛繁殖障碍相关的感染因子方面取得了重大进展，BovReproSeq检测板检测17种病原体的能力使其成为兽医诊断的一个有前途的工具。
重要性
牛繁殖障碍给牛肉和牛奶生产者造成了巨大的经济损失，传染病对该综合征有重大影响。病因诊断很复杂，因为可能涉及多种病原体，并且一些病原体感染无症状或引起相似的临床症状。逐步诊断测试方法耗时且增加了错过正确诊断的风险。BovReproSeq是一种基于下一代测序的诊断检测板，可同时检测17种繁殖障碍病原体。
