Department of Microbiology, National Veterinary Institute, SE-75189 Uppsala, Sweden.
Department of Animal Health and Antimicrobial Strategies, SE-75189 National Veterinary Institute, Uppsala, Sweden.
J Med Microbiol. 2019 Jul;68(7):1003-1011. doi: 10.1099/jmm.0.001016. Epub 2019 Jun 7.
The present study aimed to establish pretreatment protocols as well as real-time and droplet digital polymerase chain reaction (PCR) methodologies to detect and quantify Erysipelothrix rhusiopathiae (ER) DNA in blood samples from infected chickens, as tools for routine diagnostics and monitoring of experimental infections. Chicken blood is a problematic matrix for PCR analysis because nucleated erythrocytes contribute large amounts of host DNA that inhibit amplification.
Using artificially spiked samples of fresh chicken blood, as well as blood samples from three experimental infection studies, the performance of pretreatment protocols, including choice of blood stabilization agent, centrifugation speeds and Ficoll gradient separation, was evaluated. The results were compared with those from traditional culture-based protocols combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).Results/Key findings. Simple preparations producing cell-free samples performed well on artificial spike-in samples, providing high sensitivity. However, performance was poor in clinical samples or artificial samples where the bacteria were incubated for 4 h or more in fresh blood prior to DNA extraction. In these samples, a Ficoll separation protocol that creates samples rich in lymphocytes, monocytes and thrombocytes prior to DNA extraction was far more effective.
Our results indicate that ER bacteria undergo rapid phagocytosis in chicken blood and that analysis of a blood fraction enriched for phagocytic cells is necessary for reliable detection and quantification. The presented results explain the poor performance of PCR detection reported in previously published experimental ER infection studies, and the proposed solutions are likely to have broader implications for PCR-based veterinary diagnostics in non-mammalian host species such as poultry and fish.
本研究旨在建立预处理方案以及实时和液滴数字聚合酶链反应（PCR）方法，以检测和定量感染鸡血液样本中的红斑丹毒丝菌（ER）DNA，作为常规诊断和实验感染监测的工具。鸡血液是 PCR 分析的一个有问题的基质，因为有核红细胞会贡献大量宿主 DNA，从而抑制扩增。
使用新鲜鸡血液的人工添加样本以及来自三个实验感染研究的血液样本，评估预处理方案的性能，包括血液稳定剂的选择、离心速度和菲可尔梯度分离。将结果与传统的基于培养的方案结合基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）进行比较。
结果/关键发现：简单的制备方法可以产生无细胞样本，在人工添加样本中表现良好，具有高灵敏度。然而，在临床样本或在新鲜血液中孵育细菌 4 小时或更长时间后才进行 DNA 提取的人工样本中，性能较差。在这些样本中，在 DNA 提取之前使用菲可尔分离方案创建富含淋巴细胞、单核细胞和血小板的样本要有效得多。
我们的结果表明，ER 细菌在鸡血液中迅速被吞噬，并且需要分析富含吞噬细胞的血液部分，才能进行可靠的检测和定量。所提出的解决方案可能对兽医 PCR 诊断在非哺乳动物宿主（如家禽和鱼类）中具有更广泛的意义。
