【作者】 邵颖；

【导师】 祁克宗；

【作者基本信息】 安徽农业大学 ， 动物病原生物学， 2025， 博士

【摘要】 生猪养殖业作为现代农业经济的重要支柱,在提升生产效率的同时,也面临着猪肠道微生物组失衡、抗生素耐药性基因传播及新发/再发病毒性疫病频发等多重挑战。这些风险因子不仅制约行业的可持续发展,还可能通过食物链和环境传播对公共卫生安全构成潜在风险。肠道微生物群作为宿主的“第二基因组”,在营养代谢、免疫调控及病原防御中发挥核心作用。然而,集约化养殖模式下,猪肠道微生物组失衡现象普遍,进而引发宿主代谢紊乱和免疫功能异常。同时,猪粪中富集的细菌、病毒和真菌等微生物组分成为病原传播和耐药基因水平转移的重要载体。皖江地区作为我国长江中下游核心生猪产区,其病毒性疫病呈现高发态势,不仅造成严重的经济损失,还可能通过病原跨宿主传播对公共卫生安全构成潜在威胁。尽管近年来关于猪肠道微生物多样性及其与宿主互作机制的研究已取得重要突破,但区域特异性微生物群落多样性及功能特征、毒力和耐药基因的传播规律以及发病猪群的病毒感染特征等关键科学问题仍需深入探究。皖江区域特有的高温高湿气候与集约化高密度养殖模式的协同作用,显著影响了微生物群落的生态位分布和功能特征,为毒力因子和抗生素耐药基因的水平转移以及病原微生物的传播扩散创造了有利条件。值得注意的是,病毒作为外源性病原体,其感染过程与肠道微生物群存在双向互作:一方面,肠道菌群通过调节宿主免疫应答影响病毒感染进程;另一方面,病毒感染可通过改变肠道微环境重塑菌群结构与功能。基于此,本研究选取皖江区域8个具有代表性的规模化养殖场(均饲养杜洛克×长白×大白三元杂交商品猪)作为研究对象,通过解析猪肠道微生物结构及功能特征,为区域特征性微生物研究提供了重要数据支撑;通过深入探究毒力和耐药基因的分布及其与病原菌的关联,为制定区域化防控策略提供了理论参考;同时,运用病毒宏基因组学技术分析了皖江区域发病猪群的病毒组感染情况,揭示了区域内病毒性疾病的感染谱系和流行特征;并针对区域内高流行病原:猪圆环病毒2型(PCV2)、猪圆环病毒3型(PCV3)及猪细小病毒7型(PPV7),建立了一种基于Taq Man探针技术的能同时检测三种病原的多重q PCR检测方法,为猪病的快速诊断和防控提供了科学依据。本文主要研究内容与结果如下:1.皖江区域猪肠道微生物组结构与功能的宏基因组学特征本研究利用细菌宏基因组学技术,以皖江区域8个典型规模化猪场160日龄以上的健康三元杂交商品猪为研究对象,解析其肠道微生物群落的多样性及功能特性,深入探究肠道微生物与宿主健康、养殖环境及公共卫生的潜在关联。结果表明:(1)细菌群落以厚壁菌门((47.64%)、变形菌门((28.27%)和拟杆菌门((13.70%)为优势菌门,其中梭菌纲((27.16%)、γ-变形菌纲((26.41%)、芽孢杆菌纲((15.64%)和拟杆菌纲((12.60%)为优势菌纲,梭菌纲与γ-变形菌纲的丰度分布呈负相关趋势。变形菌门(假单胞菌科、莫拉氏菌科和肠杆菌科)及不动杆菌属的高丰度占比提示潜在的养殖生态压力,其携带的耐药基因和毒力因子可能引发感染并造成耐药基因水平传播,对公共卫生安全构成威胁。(2)病毒群落以有尾噬菌体纲、未分类的有尾噬菌体纲目级病毒及未分类的有尾噬菌体纲科级病毒占据优势。此外,未分类病毒的高占比不仅揭示了猪肠道病毒组中仍存在大量未表征的病毒分类单元,同时也提示了潜在的致病风险,后续研究需进一步探讨病毒群落结构与猪群疾病发生的关联性。2.皖江区域猪肠道微生物中毒力与耐药基因组的分布特征本研究基于肠道微生物组的结构特征,进一步揭示了皖江区域猪粪便微生物中毒力基因(VFGs)和抗生素耐药基因(ARGs)的分布特征及其与特定病原菌的关联,为区域化抗生素使用管理和养殖场生物安全防控提供了科学依据。结果表明:(1)在毒力基因方面,以侵袭性毒力因子(65.17%)占比最高,其中黏附素((32.14%)和分泌系统((20.25%)占主导地位,表明猪粪便微生物群落具有较强侵袭性。肠杆菌科是毒力因子的主要贡献者,其次为链球菌科和假单胞菌科,凸显了这些菌群在猪肠道致病过程中的关键作用。(2)在耐药基因方面,四环素类(23.51%)、氨基糖苷类(21.18%)和MLS类(18.48%)ARGs构成核心耐药类型,其中Erm B、APH(3’)-IIIa、flo R、tet W、sul2、APH(6)-Id、tet Q、ANT(3’’)-IIa为优势耐药基因,这一耐药特征为制定区域抗生素使用策略提供了重要依据。3.基于宏基因组学的皖江区域发病猪群病毒组特征解析本研究基于宏病毒组学技术对皖江区域232份发病猪组织样本(合并为37份混样)进行病毒组测序分析,揭示了该地区猪群病毒感染特征及新发病毒风险。结果表明:(1)共鉴定到23种病毒,其中,猪圆环病毒(PCV)、Porcine associated porprismacovirus、猪细环病毒(TTSu V)和猪细小病毒(PPV)为优势病毒,占总病毒序列的87.66%。(2)在皖江区域检出猪粪相关环状病毒(Po SCV)、Wuhan large pig roundworm virus 1、非典型猪瘟病毒1型(APPV-1)和SPa V-1等新发病毒。(3)获得PCV2、PCV3、PPV4、PPV7和TTSu V2五种病毒的全基因组序列,分子流行病学分析显示PCV2b和PCV2d基因型流行特征与全球趋势一致,TTsu V2-2b为本研究的主要流行株。4.PCV2、PCV3和PPV7 Taq Man三重q PCR检测方法的建立本研究基于宏病毒组测序结果,针对PCV和PPV高丰度病原,开展了37份临床样本的PCV2/3与PPV4～7的混合感染特征研究,并成功建立了可同步检测PCV2、PCV3和PPV7的Taq Man三重q PCR方法,为临床诊断和流行病学监测提供了高效检测工具。结果表明:(1)PCV2在皖江区域呈现100%的高流行特征,PCV3检出率为43.24%,PPV4～PPV7检出率为8.11%～32.43%;同时,多种病原混合感染现象突出,67.57%样本存在双重及以上病原感染,其中PCV2/PCV3/PPV7三重病原混合感染率达16.22%。(2)建立了可同步检测PCV2、PCV3和PPV7的Taq Man三重q PCR方法,灵敏度达到10~2 copies/μL,同时兼具高特异性和良好重复性,并进一步通过临床样本检测验证了该方法的高效和实用性。综上所述,本研究以皖江区域8个典型规模化猪场的肠道微生物为研究对象,利用细菌宏基因组学技术解析了猪肠道微生物中细菌与病毒群落的多样性及潜在功能特性;基于猪肠道微生物组结构特点,进一步阐明了VFGs和ARGs的分布特征及其与病原菌的关联性;基于病毒宏基因组学与分子流行病学分析,揭示了该区域发病猪群的病毒流行特征及混合感染模式;针对皖江区域规模化猪场高发病原,建立了高效Taq Man三重q PCR检测方法,为临床病毒性疫病的快速诊断与精准防控提供了技术支撑。本研究结果为优化区域养殖管理并制定针对性的疫病防控策略提供了科学依据,对该区域生猪健康养殖水平的提升具有重要意义。更多还原

【Abstract】 The swine industry,as a key pillar of modern agricultural economics,faces multiple challenges while striving to improve production efficiency,including imbalances in the swine gut microbiome,the spread of antibiotic-resistant genes,and the frequent occurrence of emerging/re-emerging viral diseases.These risk factors not only hinder the industry’s sustainable development but may also pose potential risks to public health safety through food chains and environmental transmission.The gut microbiome,often referred to as the host’s“second genome,”plays a central role in nutrient metabolism,immune regulation,and pathogen defense.However,under intensive farming systems,gut microbiota imbalance is widespread,leading to host metabolic disorders and immune dysfunction.Additionally,microbial components such as bacteria,viruses,and fungi enriched in pig manure serve as important carriers for pathogen transmission and horizontal gene transfer of antibiotic resistance.As a core pig production area in the middle and lower reaches of the Yangtze River,the Wanjiang region faces a high incidence of viral diseases,which not only cause significant economic losses but may also pose potential threats to public health safety through pathogen transmission across hosts.Although significant breakthroughs have been made in recent years in the study of pig intestinal microbial diversity and its interaction mechanisms with the host,key scientific questions such as regionally specific microbial community diversity and functional characteristics,the transmission patterns of virulence and antibiotic resistance genes,and the viral infection characteristics of diseased pig populations still require further investigation.The unique high-temperature and high-humidity climate of the Wanjiang region,combined with intensive high-density farming practices,significantly influences the ecological niche distribution and functional characteristics of microbial communities,creating favorable conditions for the horizontal transfer of virulence factors and antibiotic resistance genes,as well as the spread and dissemination of pathogenic microorganisms.Notably,viruses,as exogenous pathogens,exhibit bidirectional interactions with the intestinal microbial community during infection:on one hand,the intestinal microbiota influences the course of viral infection by regulating the host immune response;on the other hand,viral infection can reshape the structure and function of the microbiota by altering the intestinal microenvironment.Based on this,this study selected eight representative large-scale pig farms in the Wanjiang region(all raising Duroc×Landrace×Large White three-way crossbred commercial pigs)as research subjects.By analyzing the structural and functional characteristics of pig intestinal microbiota,the study provided important data support for regional characteristic microbiota research;by deeply exploring the distribution of virulence and antibiotic resistance genes and their association with pathogenic bacteria,the study provided theoretical references for formulating regional prevention and control strategies;Additionally,viral metagenomics technology was employed to analyze viral infection patterns in diseased pig populations in the Wanjiang region,revealing the infection spectrum and epidemiological characteristics of viral diseases within the region.Specifically targeting highly prevalent pathogens in the region:Porcine Circovirus Type 2(PCV2),Porcine Circovirus Type 3(PCV3),and Porcine Parvovirus Type 7(PPV7),a multiplex q PCR detection method based on Taq Man probe technology was established to simultaneously detect the three pathogens,providing scientific basis for the rapid diagnosis and control of swine diseases.The main research content and results of this study are as follows:1.Metagenomic characteristics of the structure and function of the porcine gut microbiome in the Wanjiang regionThis study employed bacterial metagenomics technology to investigate the diversity and functional characteristics of intestinal microbial communities in healthy ternary hybrid commercial pigs aged over 160 days from eight typical large-scale pig farms in the Wanjiang region.The research further explored the potential correlations between intestinal microbiota and host health,farming environments,and public health.The results demonstrated that:(1)The bacterial community was dominated by Firmicutes(47.64%),Proteobacteria(28.27%),and Bacteroidetes(13.70%),with Clostridia(27.16%),Gammaproteobacteria(26.41%),Bacilli(15.64%),and Bacteroidia(12.60%)being the predominant classes.The abundance distribution of Clostridia and Gammaproteobacteria exhibited a negative correlation trend.The high relative abundance of Proteobacteria(including Pseudomonadaceae,Moraxellaceae,and Enterobacteriaceae)and Acinetobacter suggested potential ecological pressures in farming environments.These bacteria may carry antibiotic resistance genes and virulence factors,potentially leading to infections and horizontal gene transfer of resistance genes,thereby posing a threat to public health security.(2)The viral community was predominantly composed of Caudoviricetes,unclassified Caudoviricetes at the order level,and unclassified Caudoviricetes at the family level.Additionally,the high proportion of unclassified viruses not only revealed the existence of numerous uncharacterized viral taxa in the porcine intestinal virome but also indicated potential pathogenic risks.Further research is needed to investigate the association between viral community structure and disease occurrence in pig populations.2.Distribution characteristics of virulence and antibiotic resistance genes in porcine gut microbiome in the Wanjiang regionBased on the structural characteristics of the gut microbiome,this study further elucidated the distribution patterns of virulence genes(VFGs)and antibiotic resistance genes(ARGs)in swine fecal microbiota from the Wanjiang region,as well as their associations with specific pathogenic bacteria.The findings provide a scientific basis for regional antibiotic use management and biosecurity control in swine farms.The results demonstrated that:(1)Virulence genes:Offensive virulence factors accounted for the highest proportion(65.17%),with adhesins(32.14%)and secretion systems(20.25%)being the most dominant,indicating a highly invasive potential in the swine fecal microbial community.Enterobacteriaceae were the primary contributors to virulence factors,followed by Streptococcaceae and Pseudomonadaceae,highlighting the critical role of these bacterial groups in porcine intestinal pathogenesis.(2)Antibiotic resistance genes:Tetracyclines(23.51%),aminoglycosides(21.18%),and MLS(macrolide-lincosamide-streptogramin,18.48%)resistance genes constituted the core ARG types.The predominant resistance genes included Erm B,APH(3’)-IIIa,flo R,tet W,sul2,APH(6)-Id,tet Q,and ANT(3’’)-IIa.This resistance profile provides crucial evidence for formulating region-specific antibiotic usage strategies.3.Metagenomic-based analysis of the virome characteristics in diseased swine populations in the Wanjiang regionThis study employed metaviromic sequencing technology to analyze 232 diseased porcine tissue samples(pooled into 37 composite samples)from the Wanjiang region,elucidating the viral infection characteristics and emerging viral risks in local swine populations.The results demonstrated that:(1)A total of 23 viruses were identified,with porcine circovirus(PCV),Porcine associated porprismacovirus,porcine torque teno sus virus(TTSu V),and porcine parvovirus(PPV)being the dominant viruses,collectively accounting for 87.66%of total viral sequences.(2)Novel emerging viruses including porcine stool-associated circular virus(Po SCV),Wuhan large pig ascarid virus 1,APPV-1,and SPa V-1 were detected in the Wanjiang region.(3)Complete genome sequences of five viruses(PCV2,PCV3,PPV4,PPV7,and TTSu V2)were successfully obtained.Molecular epidemiological analysis revealed that the prevalent characteristics of PCV2b and PCV2d genotypes were consistent with global trends,while TTsu V2-2b emerged as the predominant strain in this study.4.Establishment of a Taq Man triple q PCR assay for detection of PCV2,PCV3,and PPV7Based on metaviromic sequencing results,this study investigated the co-infection patterns of high-abundance pathogens PCV and PPV by analyzing 37 clinical samples for PCV2/3 and PPV4-7,and successfully developed a Taq Man-based triplex q PCR assay capable of simultaneously detecting PCV2,PCV3,and PPV7,providing an efficient diagnostic tool for clinical surveillance.The results demonstrated that:(1)PCV2 exhibited a 100%prevalence rate in the Wanjiang region,while PCV3 was detected in 43.24%of samples and PPV4-PPV7 showed detection rates ranging from8.11%to 32.43%.Notably,multiple pathogen co-infections were prominent,with 67.57%of samples showing dual or multiple infections.Specifically,the PCV2/PCV3/PPV7triple co-infection rate reached 16.22%.(2)Established a Taq Man triplex q PCR method for synchronous detection of PCV2,PCV3,and PPV7,achieving a sensitivity of 10~2copies/μL with high specificity and excellent reproducibility.Clinical sample validation further confirmed the efficiency and practical utility of this method.In summary,this study investigated the intestinal microbiota of pigs from eight typical large-scale farms in the Wanjiang region.Using bacterial metagenomics,we comprehensively characterized the diversity and potential functional profiles of both bacterial and viral communities in the porcine gut microbiome.Building upon the structural features of the intestinal microbiota,we further elucidated the distribution patterns of virulence genes(VFGs)and antibiotic resistance genes(ARGs)and their associations with pathogenic bacteria.Through metaviromic analysis and molecular epidemiological investigation,we revealed the viral prevalence characteristics and co-infection patterns in diseased pigs within this region.To address the high-prevalence pathogens in Wanjiang’s intensive pig farms,we developed an efficient Taq Man triplex q PCR detection method,providing technical support for rapid diagnosis and precise prevention of clinical viral diseases.These findings offer scientific evidence for optimizing regional farming management and formulating targeted disease control strategies,which holds significant implications for advancing swine health and production standards in this area.更多还原