Bovine abortions 16S rRNA

Abortion in cattle causes significant economic losses for cattle farmers worldwide. The diversity of abortifacients makes abortiondiagnostics a complex and challenging discipline that additionally is restrained by time and economy. Microbial culture hastraditionally been an important method for the identification of bacterial and mycotic abortifacients. However, it comes with theinherent bias of favoring the easy to culture bacteria, e.g. those that do not require cell culture, pre-enrichment, a variety ofselective growth media, or different oxygen levels for in vitro growth. Molecular methods like polymerase chain reaction (PCR)and next generation sequencing have been established as alternatives to traditional microbial culturing methods in severaldiagnostic fields including abortion diagnostics. Fluorescence in situ hybridization (FISH), a bridging microscopy technology thatcombines molecular accuracy with culture-independence, and spatial resolution of the pathogen-lesion relation, is also gaininginfluence in several diagnostic fields. In this study, qPCR, 16S rDNA amplicon sequencing, and FISH were applied separately and incombination in order to: i) identify potentially abortifacient bacteria without the bias of culturability, ii) increase the diagnosticrate using combined molecular methods, iii) assess the role of the difficult-to-culture zoonotic agents Coxiella burnetii, Chlamydiaspp., and Leptospira spp. as bovine abortifacients in Denmark.Tissues from 162 aborted or stillborn bovine fetuses and placentas submitted for routine diagnostics were screened for pathogenicbacteria using 16S rDNA amplicon sequencing. Subsequently, species-specific FISH assays were used to assess the lesion associationof a selection of abortifacients in the tissues. The presence of Chlamydia spp. and Chlamydia-like organisms was assessed usingqPCR and the role of fungal infections was determined by FISH screening for lesion-associated fungal elements.The 16S rDNA amplicon sequencing-guided FISH approach was suitable for enhancing abortion diagnostics, i.e. the diagnostic ratefor cases with tissue lesions (n=115) was increased from 46% to 53% when compared to routine diagnostic methods. Identificationof Bacillus licheniformis, Escherichia coli, and Trueperella pyogenes accounted for the majority of additional cases with anestablished etiology. No evidence for emerging or epizootic bacterial pathogens was found. The difficult-to-culture abortifacientswere either not detected or not identified as abortifacients.

牛流产对全球肉牛养殖户均造成了显著的经济损失。流产诱导因子的多样性使得流产诊断成为一门复杂且极具挑战性的学科，同时还受到时间与经济成本的双重限制。长期以来，微生物培养法一直是鉴定细菌性与真菌性流产诱导因子的重要手段，但该方法存在固有偏倚，更易分离培养易生长的细菌——例如无需细胞培养、预富集、多种选择性生长培养基，或不同体外培养氧浓度条件的菌株。诸如聚合酶链式反应（polymerase chain reaction, PCR）与下一代测序（next generation sequencing, NGS）在内的分子技术，已在包括流产诊断在内的多个诊断领域成为传统微生物培养法的替代方案。荧光原位杂交（Fluorescence in situ hybridization, FISH）作为一种衔接型显微技术，兼具分子检测的精准性与非培养依赖性，同时可解析病原体与病灶的空间关联，目前也在多个诊断领域逐渐得到应用。本研究分别单独与联合应用了实时定量PCR（qPCR）、16S rDNA扩增子测序与FISH三种技术，以达成以下目标：① 无需依赖培养偏倚，鉴定潜在的流产相关细菌；② 通过联合分子技术提升诊断检出率；③ 评估难以培养的人畜共患病原体——贝氏柯克斯体（Coxiella burnetii）、衣原体属（Chlamydia spp.）与钩端螺旋体属（Leptospira spp.）在丹麦作为牛流产诱导因子的作用。本研究针对162份送检至常规诊断流程的牛流产或死产胎儿及胎盘组织，通过16S rDNA扩增子测序筛查致病性细菌；随后，利用物种特异性FISH检测试剂盒，评估部分流产诱导因子在组织中的病灶关联性；同时通过qPCR检测衣原体属及类衣原体生物的存在情况，并通过FISH筛查病灶关联真菌成分，以明确真菌感染在牛流产中的作用。基于16S rDNA扩增子测序引导的FISH联合检测方案可有效优化流产诊断流程：相较于常规诊断方法，伴组织病灶的病例（n=115）的诊断检出率从46%提升至53%。新增确诊病例中，多数可归因于地衣芽孢杆菌（Bacillus licheniformis）、大肠埃希氏菌（Escherichia coli）与化脓隐秘杆菌（Trueperella pyogenes）的检出。未检出新发或流行性细菌性病原体。难以培养的流产诱导因子要么未被检出，要么未被认定为流产致病因素。