Abortion in cattle causes significant economic losses for cattle farmers worldwide. The diversity of abortifacients makes abortion diagnostics a complex and challenging discipline that additionally is restrained by time and economy. Microbial culture has traditionally been an important method for the identification of bacterial and mycotic abortifacients. However, it comes with the inherent bias of favoring the easy-to-culture species, e.g., those that do not require cell culture, pre-enrichment, a variety of selective growth media, or different oxygen levels for in vitro growth. Molecular methods such as polymerase chain reaction (PCR) and next-generation sequencing have been established as alternatives to traditional microbial culturing methods in several diagnostic fields including abortion diagnostics. Fluorescence in situ hybridization (FISH), a bridging microscopy technique that combines molecular accuracy with culture independence, and spatial resolution of the pathogen-lesion relation, is also gaining influence in several diagnostic fields. In this study, real-time quantitative PCR (qPCR), 16S rDNA amplicon sequencing, and FISH were applied separately and in combination in order to (i) identify potentially abortifacient bacteria without the bias of culturability, (ii) increase the diagnostic rate using combined molecular methods, (iii) investigate the presence of the difficult-to-culture zoonotic agents Coxiella burnetii, Chlamydia spp., and Leptospira spp. in bovine abortions in Denmark. Tissues from 162 aborted or stillborn bovine fetuses and placentas submitted for routine diagnostics were screened for pathogenic bacteria using 16S rDNA amplicon sequencing. Lesion association of fungal elements, as well as of selection of bacterial abortifacients, was assessed using specific FISH assays. The presence of Chlamydia spp. and chlamydia-like organisms was assessed using qPCR. The study focused on bacterial and fungal abortifacients, because Danish cattle is free from most viral abortifacients. The 16S rDNA amplicon sequencing–guided FISH approach was suitable for enhancing abortion diagnostics, i.e., the diagnostic rate for cases with tissue lesions (n = 115) was increased from 46 to 53% when compared to routine diagnostic methods. Identification of Bacillus licheniformis, Escherichia coli, and Trueperella pyogenes accounted for the majority of additional cases with an established etiology. No evidence for emerging or epizootic bacterial pathogens was found. The difficult-to-culture abortifacients were either not detected or not identified as abortifacients.

牛的流产给全世界的牛农造成了巨大的经济损失。堕胎科学家的多样性使堕胎诊断成为一门复杂而具有挑战性的学科，此外还受到时间和经济的限制。传统上，微生物培养一直是鉴定细菌和霉菌流产学家的重要方法。但是，它带有偏向于易于培养的物种的固有偏见，例如，不需要细胞培养，预富集，各种选择性生长培养基或不同氧含量的易于培养的物种。体外生长。在包括流产诊断在内的多个诊断领域，已经建立了诸如聚合酶链反应（PCR）和下一代测序等分子方法作为传统微生物培养方法的替代方法。荧光性原位杂交（FISH）是一种桥接显微镜技术，结合了分子准确性和培养独立性以及病原体与病变之间关系的空间分辨率，在一些诊断领域也正在受到影响。在这项研究中，实时定量PCR（qPCR），16S rDNA扩增子测序和FISH分别或组合应用，以（i）在不影响可培养性的情况下识别潜在的堕胎细菌，（ii）使用结合分子方法，（iii）研究难于培养的人畜共患病药物的存在衣原体柯氏杆菌 spp。，和 钩端螺旋体spp。在丹麦的牛堕胎中 使用16S rDNA扩增子测序对提交常规诊断的162例流产或死胎的牛胎儿和胎盘组织进行了病原菌筛选。使用特定的FISH分析评估了真菌元素的病变关联以及细菌流产的选择。存在的衣原体spp。使用qPCR评估衣原体样生物。该研究的重点是细菌和真菌的流产剂，因为丹麦的牛没有大多数病毒的流产剂。16S rDNA扩增子测序指导的FISH方法适用于增强流产诊断，即组织损伤病例的诊断率（ñ与常规的诊断方法相比，“ 115”从46％增加到53％。鉴定地衣芽孢杆菌，大肠杆菌， 和 化脓小球藻病因已明确的占其他病例的大多数。没有发现新出现或流行的细菌病原体的证据。难以培养的流产者没有被发现或未被鉴定为流产者。