腹泻新生仔猪肠道微生物群落宏基因组学分析

Diarrhea is one of the most important problems in neonatal piglets in China. The morbidity and mortality of diarrhea in neonatal piglets have dramatically increased recent years, which can be as high as 100% and 80%, respectively. Huge economic losses to the pig industry have been caused by this disease. Diarrhea can be caused by infectious factors and non-infectious factors or a combination of these. Pathogenic bacteria, molds, parasites, and viruses are major causative agents of diarrhea. Of which, the viral pathogens are the most critical etiologic agents causing pig diarrhea. Presently porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and rotavirus（PRV） are most frequently detected viral pathogens from diarrheal piglets. Recently, several new potential pathogens, including the porcine bocavirus (PBoV), were isolated from diarrheal pigs. However, due to the limitation of the spectrum of current detecting assays as well as the possible occurrence of novel variants or/and emerging pathogens, we might fail to detect some known/unknown pathogenic agents. Therefore, a more comprehensive analysis for the etiological causes of diarrhea, especially the severe form, is necessary. Novel high-throughput-sequencing-based microbial metagenomics along with random primer amplification and up to dated bioinformatics provides a powerful tool and thus enables us to pursue answers for piglet diarrhea at the genetic and functional levels of microbiota and virome. Hence, we propose to analyze gut microbita profiles of diarrheal neonatal piglets, healthy pigs, and gnotabiotic piglets in context of microbial metagenomics so that potential new pathogens can be explored. The expected findings to be achieved will provide insight into diarrheal causes, healthy and diarrheal piglet gut microbiomes, and their functional genomics which will benefit the development of new diagnostic assays, vaccine candidates, and the discovery of new targets for anti-diarrheal drugs/antibiotics.

仔猪腹泻一直是困扰我国养猪业的重要疾病之一。近年来，该病呈严重上升的趋势，仔猪的发病率和死亡率可分别高达100%和80%,给养猪业造成巨大损失。导致猪只腹泻原因有传染性因素和非传染性因素。细菌、霉菌及其毒素、寄生虫、病毒等传染性因素是导致猪只腹泻的主要原因。目前已知猪流行性腹泻病毒（PEDV)、传染性胃肠炎病毒（TGEV）和轮状病毒（PRV）是腹泻病中检出率最高的病原；2011年从腹泻病猪中检测出了猪博卡病毒(PBoV)等新的可能病原。然而，我们认为，导致仔猪腹泻的更深层次因素还有待于进一步揭示。本研究拟采用随机引物扩增结合高通量鸟枪法测序为基础的微生物宏基因组学、生物信息学等技术，分析和比较7日龄以下腹泻仔猪、健康仔猪以及悉生（Gnotabiotic）仔猪的微生物宏基因组学（包括Microbiota and virome)；以期确定微生物群落构成、多样性、丰度、表型以及与腹泻的关系。

本项目对猪腹泻相关病毒进行了分子流行病学和基因组学分析，采用宏基因组学及生物信息学方法系统分析比较了健康与腹泻仔猪肠道微生物群落结构和功能。结果表明，猪流行性腹泻病毒（PEDV）在腹泻猪群中的检出率最高（58.38%）；新现猪丁型冠状病毒（PDCoV）检出率次之（30.26%）；而胃肠炎病毒（TGEV）和猪轮状病毒（PoRV）的检出率均低于3%。回溯性研究表明最早可从2012年的腹泻样品中检出新现PDCoV，基因组分析表明，江西省流行的PDCoV毒株与其它国家或地区的PDECoV及鸟类δ冠状病毒均在同一进化分支内，与α、β和γ冠状病毒分属不同的进化分支；PDCoV不同地区毒株间各基因的同源性在97%以上，说明目前流行的各PDCoV毒株遗传学高度相似。病毒宏基因组学分析结果表明共检测出多余28种的病毒属和种；正常/无症状猪只、腹泻猪肠道病毒群落结构和丰度具有明显差异，腹泻猪肠道致病性病毒群落结构和丰度高于正常/无症状猪只肠道内病毒群落；腹泻猪样品中PEDV读长比例显著高于健康猪PEDV读长的比例；腹泻后康复母猪虽然不腹泻，但其体内依然存在高丰度的PEDV和PDCoV等腹泻相关病毒，与腹泻猪同窝的无腹泻症状的仔猪肠道也具有高丰度的腹泻相关病毒。基于16S rRNA的细菌菌落结构分析表明，腹泻可引起肠道菌群从门到属，甚至种水平的改变。产气荚膜梭菌等炎症或腹泻相关细菌的丰度均在腹泻母猪肠道内显著升高，而丁酸盐产生菌丁酸梭菌等的丰度显著降低。在仔猪样品中，腹泻及同窝无症状仔猪肠道中普氏菌属等产生短链脂肪酸的细菌及丁酸梭菌的丰度显著下降，但肠球菌科及产气荚膜梭菌等致病菌的丰度显著上升。说明PEDV的感染可能会导致肠道有益菌的丰度降低，而致病菌丰度的升高，从而进一步加剧了腹泻及其导致的肠炎和损伤。基于细菌宏基因组学分析也获得了与16S rRNA-based分析所获结果相一致的结果，但是具有更高的分辨率。PEDV导致的低日龄仔猪腹泻不仅改变了仔猪肠道菌群结构和丰度，而且影响了仔猪肠道细菌的功能。本项目在探寻潜在的新的肠道致病原，发挖掘肠道微生物结构、多样性、丰度、功能与腹泻的关系等方面将为防控此次腹泻提供了科学的理论基础和技术手段。