Biomarkers for Early Stages of Johne’s Disease Infection and Immunization in Goats. Biomarkers for Early Stages of Johne’s Disease Infection and Immunization in Goats

Background: Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of Johne’s disease, a chronic enteric infection of ruminants. Infection occurs within the first few months of life but remains subclinical for an average of 2-5 years. Current diagnostics to detect early subclinical infections lack diagnostic sensitivity, which hinders disease control resulting in significant economic losses to the dairy industry worldwide. The pathophysiology of early infection with M. paratuberculosis is still not well understood and represents a key hurdle towards the development of better diagnostics. Methods: The present study employed a large-scale RNA-Sequencing technology to better understand early stages of M. paratuberculosis infection and immunization. Specifically, gene expression profiles of peripheral blood mononuclear cells (PBMCs) from infected and vaccinated goats were compared to controls. Results: The transcriptome of the control goats revealed a large number of genes (N = 226, 1018, 1714, 1133) that were differentially expressed compared to the M. paratuberculosis-infected goats, goats vaccinated with live attenuated or inactivated vaccines, and goats both vaccinated and infected. Bioinformatics evaluation of the differentially expressed genes indicated the regulation of a large number of genes with immunity and inflammatory functions including IL-18BP, IFN-γ, IL-17A, NOS2, LIPG and IL-22. Interestingly, a large number of goat genes (N=667) were regulated whether live or inactivated vaccine were used. Some of the regulated genes (e.g. IL-17A, IFN-g) continued its unique transcriptional profile up to 12 months post-challenge. Conclusions: Overall, transcriptome analysis of infected and/or immunized goats identified potential targets for developing early diagnostics for Johne’s disease and a potential approach to differentiate infected from vaccinated animals. A similar approach could be used to analyze later stages of Johne’s disease or other chronic infections. Overall design: One-month post-infection trancriptomic analysis (Illumina HiSeq 2000) of goat PBMC from goats that were vaccinated 60 days before experimental infection with Mycobacterium avium subsp. Paratuberculosis. Three to four animals were included in each group.

背景：副结核分枝杆菌（Mycobacterium avium subsp. paratuberculosis, M. paratuberculosis）是约翰病（Johne’s disease）的病原菌，该病是一类感染反刍动物的慢性肠道传染病。该病原菌通常于宿主出生后数月内发生感染，但平均会经历2~5年的亚临床期。当前用于检测早期亚临床感染的诊断方法诊断灵敏度不足，这一缺陷阻碍了疾病防控工作，给全球乳品养殖业带来了显著的经济损失。目前人们对副结核分枝杆菌早期感染的病理生理机制仍缺乏充分认知，这也是开发更优质诊断方法的核心障碍。

方法：本研究采用大规模RNA测序（RNA-Sequencing）技术，以期更深入地解析副结核分枝杆菌感染与免疫的早期进程。具体而言，研究对比了感染组、免疫山羊以及对照组山羊外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）的基因表达谱。

结果：与副结核分枝杆菌感染山羊、接种弱毒活疫苗或灭活疫苗的山羊，以及同时免疫并感染的山羊相比，对照组山羊的转录组中存在大量差异表达基因（数量分别为226、1018、1714、1133）。对差异表达基因的生物信息学分析显示，大量参与免疫与炎症功能的基因受到调控，其中包括IL-18BP、IFN-γ、IL-17A、NOS2、LIPG及IL-22。值得注意的是，无论接种弱毒活疫苗还是灭活疫苗，均有大量山羊基因（共667个）受到调控。部分受调控的基因（如IL-17A、IFN-γ）在攻毒后长达12个月内仍维持其独特的转录表达模式。

结论：综上，对感染和/或免疫山羊的转录组分析，为约翰病早期诊断方法的开发提供了潜在靶点，同时也为区分感染动物与免疫动物提供了可行思路。该研究方法亦可用于解析约翰病晚期阶段或其他慢性感染性疾病的相关机制。

整体实验设计：于副结核分枝杆菌实验感染前60天进行免疫的山羊，在感染后1个月采集其外周血单个核细胞进行转录组分析（采用Illumina HiSeq 2000测序平台）。每组包含3至4只实验动物。