利用条形码标记的同基因菌株对鸡中肠炎沙门氏菌传播途径进行定量追踪：概念验证研究。
Quantitative Tracking of Enteritidis Transmission Routes Using Barcode-Tagged Isogenic Strains in Chickens: Proof-of-Concept Study.
作者信息
Yang Yichao, Ricke Steven C, Tellez Guillermo, Kwon Young Min
机构信息
Department of Poultry Science, University of Arkansas , Fayetteville, AR , USA.
Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, USA; Department of Food Science, University of Arkansas, Fayetteville, AR, USA; Center of Food Safety, University of Arkansas, Fayetteville, AR, USA.
出版信息
Front Vet Sci. 2017 Feb 14;4:15. doi: 10.3389/fvets.2017.00015. eCollection 2017.
is an important foodborne bacterial pathogen, however, a fundamental understanding on transmission routes within a poultry flock remains unclear. In this study, a series of barcode-tagged strains were constructed by inserting six random nucleotides into a functionally neutral region on the chromosome of . Enteritidis as a tool for quantitative tracking of transmission in chickens. Six distinct barcode-tagged strains were used for infection or contamination at either low dose (10 CFUs; three strains) or high dose (10 CFUs; three strains) in three independent experiments (Experiment 1 oral gavage; Experiment 2 contaminated feed; Experiment 3 contaminated water). For all chick experiments, cecal and foot-wash samples were collected from a subset of the chickens at days 7 or/and 14, from which genomic DNA was extracted and used to amplify the barcode regions. After the resulting PCR amplicons were pooled and analyzed by MiSeq sequencing, a total of approximately 1.5 million reads containing the barcode sequences were analyzed to determine the relative frequency of every barcode-tagged strain in each sample. In Experiment 1, the high dose of oral infection was correlated with greater dominance of the strains in the ceca of the respective seeder chickens and also in the contact chickens yet at lesser degrees. When chicks were exposed to contaminated feed (Experiment 2) or water (Experiment 3), there were no clear patterns of the barcode-tagged strains in relation to the dosage, except that the strains introduced at low dose required a longer time to colonize the ceca with contaminated feed. Most foot-wash samples contained only one to three strains for the majority of the samples, suggesting potential existence of an unknown mechanism(s) for strain exclusion. These results demonstrated the proof of concept of using barcode tagged to investigate transmission dynamics of in chickens in a quantitative manner.
是一种重要的食源性病原体，然而，对于家禽群体内传播途径的基本认识仍不清楚。在本研究中，通过将六个随机核苷酸插入肠炎沙门氏菌染色体上的功能中性区域，构建了一系列条形码标记菌株，作为定量追踪鸡体内传播的工具。在三个独立实验（实验1口服灌胃；实验2污染饲料；实验3污染水）中，使用六种不同的条形码标记菌株以低剂量（10 CFU；三种菌株）或高剂量（10 CFU；三种菌株）进行感染或污染。对于所有雏鸡实验，在第7天或/和第14天从一部分雏鸡中采集盲肠和足部冲洗样本，从中提取基因组DNA并用于扩增条形码区域。将所得的PCR扩增产物合并后通过MiSeq测序进行分析，共分析了约150万个包含条形码序列的读数，以确定每个样本中每个条形码标记菌株的相对频率。在实验1中，高剂量口服感染与相应接种鸡以及接触鸡盲肠中菌株的更高优势相关，但程度较小。当雏鸡接触污染饲料（实验2）或水（实验3）时，条形码标记菌株与剂量之间没有明显的模式，除了低剂量引入的菌株需要更长时间才能在污染饲料的情况下定殖于盲肠。大多数足部冲洗样本在大多数样本中仅包含一至三种菌株，这表明可能存在未知的菌株排除机制。这些结果证明了使用条形码标记以定量方式研究鸡体内传播动态的概念验证。
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