停乳链球菌全基因组测序分析及新型毒力基因致病力研究

Bovine mastitis is one of the most frequent and costly diseases of the dairy industry that results in major economic losses, including reduction in milk production and deteriorating the milk quality, therapeutic interventions, premature culling, and issues with animal welfare. Different pathogens, mainly the bacterial, are involved in the etiology of bovine mastitis; nevertheless, Streptococcus dysgalactiae (S. dysgalactiae) is among the important mastitis causing pathogens which are imparting considerable financial losses to the dairy herds. S. dysgalactiae is a gram positive, beta-haemolytic, coccal bacterium which is belonging to the family Streptococcaceae. It is capable of infecting both humans and animals, but is most frequently encountered as a commensal of the alimentary tract, genital tract, or less commonly, as a part of the skin flora. Several different animal species are susceptible to infection by S. dysgalactiae; however, bovine mastitis have been most frequently reported. S. dysgalactiae have the unique characteristic of being considered both con¬tagious and environmental pathogens. The organ¬ism can spread from cow to cow at milking time and are also commonly found in the cow’s environment. A recent increase in virulence of pathogenic S. dysgalactiae has been widely proposed. Such an increase may be partly explained by the acquisition of new virulence traits by horizontal gene transfer from related streptococci such as Streptococcus pyogenes and Streptococcus agalactiae. Bovine mastitis is still a biggest challenge to the dairy industry. In our previous study we collected 346 (10.5%) Streptococcus dysgalactiae strains from 3,288 quarter milk samples in 161 herds located in 21 different provinces of China that showed variety of antibiotic resistance pattern. These changes vary from region to region in their applicability and impacts. However, in case of bovine mastitis causing S. dysgalactiae, the pathogenesis, virulence determinants, evolutionary relationships and genetic basis of host tropism of S. dysgalactiae are still unclearly established. Therefore, the objective of the present project is to investigate the genetic relationships, the virulence gene profiles and cell invasiveness as well as pathogenesis of S. dysgalactiae isolated from bovine mastitis milk samples and from human being samples. Using PFGE we will select the genetically related strains which will be further studied for whole genome sequence analysis. From whole genome sequencing we will explore the novel virulent determinants. To study the pathogenesis of clinical strains of S. dysgalactiae, the bovine mammary epithelial cells (bMECs) will be used for in vitro study, while in vivo study we will use animal model for the pathogenesis of these novel isolates in our designed study. This project will be summarized some of the most noteworthy technique for study the pathogenesis of bovine mastitis pathogen that might be helpful for control of mastitis in China and other part of the world and pave the way for the modern udder health management programs. Moreover, this will also improve our understanding of the cytokine response to intramammary infection, will highlight recent findings identifying differences in the cytokine response to various bacterial pathogens, and discuss future research directions that will increase our knowledge of the role of inflammatory mediators in predicting and governing the outcome of mastitis.

停乳链球菌是奶牛乳房炎的主要致病菌之一，在牛群中广泛分布，引起奶牛养殖业巨大的经济损失。本项目组前期研究表明：我国21省161个大型牧场的3288份奶牛乳房炎乳样中分离获得停乳链球菌346株，占比高达10.5%；同时，该菌呈现多种抗生素耐药性。停乳链球菌造成人类感染的病例呈逐年上升的趋势。目前，未见报道奶牛乳房炎源性和人源性停乳链球菌的遗传进化分析，也没有证实停乳链球菌是否可作为食源性致病菌（牛奶摄入）引起人类感染。因此，本课题拟对奶牛源性和人源性停乳链球菌进行全基因组测序分析，确定其遗传进化方向，并筛选出乳房炎相关的新型毒力基因。利用基因敲除技术，构建小鼠攻毒模型和乳腺上皮细胞体外感染模型，研究所选毒力基因的致病力。本研究将明确停乳链球菌在奶牛和人类之间的传播特性，具有重要的公共卫生学意义；同时，证实其新型毒力基因的乳腺致病作用，为相关毒力基因缺失疫苗的研制提供理论基础。

停乳链球菌性奶牛乳房炎是由停乳链球菌引起奶牛乳腺组织炎症和乳质下降为特征的一种疾病。我们通过14省76个规模化的奶牛场奶牛乳房炎奶样1309份，分离获得的停乳链球菌高达7.1%。对常见抗菌药物表现出很强的耐药率，分别为红霉素（50.5%）、克林霉素（41.9%）、头孢氨苄（32.2%）和四环素（31.2%）。停乳链球菌在奶牛场之间的发病率和耐药性还存在显著的差异，需对奶牛场进行抗生素耐药性精准监测，确保最佳的治疗效果。但是停乳链球菌致奶牛乳房炎的致病机制仍不清楚。本项目旨在对奶牛乳房炎性停乳链球菌停乳亚型（SDSD）分离株进行基因型和表型分析，选择优势株进行全基因组测序分析，发掘SDSD关键毒力基因，通过奶牛乳腺上皮细胞（bMECs）开展停乳链球菌粘附侵袭，炎性损伤，氧化应激和其对bMECs TLR2/NF-κB通路调控炎症的致病特性研究。结果显示在β-内酰胺类药物的环境压力下，停乳链球菌的生物学特性和生物膜形成能力均发生适应性变化，出现多基因位点突变、插入和缺失，使其耐药性增强，并增加对奶牛乳腺上皮细胞的致病能力。16S rRNA系统发育分析，51株SDSD菌被分为4个簇，多位点序列分型的系统发育分析显示，51株SDSD菌被鉴定为11个亚型，包括新发现的5个STs（ST521、ST523、ST526、ST527、ST529），它们属于2个不同的克隆复合体和4个克隆单体。全基因组测序分析，12株SDSD菌中检测出108种毒力因子基因和23种耐药基因。泛基因组、核心基因组和辅助基因组分别由2663、1633和699个基因组成。SDSD菌强毒株和弱毒株均能黏侵袭bMECs。但强毒株SDSD感染bMECs引起炎性因子释放量和MDA含量显著增加，GSH-px和SOD的活性显著降低。TLR2/NF-κB信号通路介导炎性因子的表达。本研究为阐明停乳链球菌奶牛乳腺炎的分子机制提供依据。研究结果于2020年获宁夏省政府科技进步二等奖，获批1个计算机软件著作权登记证书，发表5篇研究论文，其中SCI 收录的论文4篇，累计影响因子15.026分，累计被引58频次，单篇最高引用38次。培养研究生5名，其中博士研究生3名，硕士研究生2名。