奶牛乳房炎性无乳链球菌的PFGE分型及其致病机制研究

Bovine mastitis is mostly caused by Streptococcus agalactiae, and is becoming an emerging threat to the dairy industry all over the world. S. agalactiae caused moderate or serious clinical cases with apparent milk loss, mammary gland necrosis and poor response to antibiotic therapy, it could cause large economic loss. In the current study, 1011 mastitic milk samples from Yunnan and Guangxi provinces indicated that the incidence rate of clinical mastitis was 3.4-8.3%, subclinical mastitis was 21.5-32.1%, furthermore, Streptococcus agalactiae pathogen was isolated from 153 milk samples, the morbidity was 15.1%. This epidemiological results showed that Streptococcus agalactiae is still one of main bovine mastitis pathogens. Nevertheless, little is known about the bacterial PFGE genotyping and host factors that may contribute to the establishment and persistence of intramammary infection by Streptococcus agalactiae. In addition, most aspects of the pathogenesis of infection caused by Streptococcus agalactiae originating exclusively from bovine mastitis have been poorly characterized to date. The aim of this study was to explore the pathogenesis and resistance to antibiotics of Streptococcus agalactiae strains isolated from bovine mastitis cases in China. Epigenetic characteristics will be analysis by PFGE, virulence genes including sip, cpsE and CAMP will be identified, and finally dominant strain be selected. Therefore, On the one hand, the apoptosis of bovine mammary epithelial cells will be verified by observing the morphological change, growth capability of cells and cytokines including ICAM-1, TNF-ɑ and IL-1 with Streptococcus agalactiae. On the other hand, the bovine mastitis model will be performed on the udder of dairy cow challenging with the colony of Streptococcus agalactiae. Meanwhile, by detecting the SCC, colony count and milking loss and so on, the clinical characteristics of mastitis caused by Streptococcus agalactiae will be demonstrated.

无乳链球菌（GBS）是高度接触性奶牛乳房炎的主要致病菌，可导致奶牛产奶量下降、乳区坏死，给奶牛业造成巨大损失。本课题前期调查滇桂两省1011份奶样，临床型乳房炎发病率达3.4-8.3%，亚临床型乳房炎发病率达21.5-32.1%，153份奶样分离出GBS，占15.1%。但是GBS的基因分型及对奶牛乳腺上皮细胞凋亡和乳房炎病理模型的研究尚未见报道。因此，本课题旨在研究GBS诱导乳腺上皮细胞凋亡和引起奶牛乳房炎的致病机制。采用PFGE分型确定GBS表观遗传学特点，检查Sip、cpsE和CAMP毒力基因分析主要毒力特征，筛选优势菌株。观察GBS优势菌株体外侵染乳腺上皮细胞形态变化、生长活性和细胞周期等，阐述乳腺上皮细胞相关炎症因子包括ICAM-1、TNF-ɑ、IL-1的变化和凋亡情况，进一步建立奶牛乳房炎模型，观察乳腺载菌量、炎症因子和乳腺组织病理变化，揭示GBS导致奶牛乳房炎的致病机理。

无乳链球菌(GBS)感染奶牛引起乳房炎，造成巨大的经济损失。检索文献报道可知，无乳链球菌对乳腺组织呈专嗜性，是传染性奶牛乳房炎致病菌。利用该点，欧洲与北美多国采用“五点法乳房炎防控程序”净化无乳链球菌，然而，近年来我国奶牛乳房炎性无乳链球菌流行率仍居高不下。国内有关无乳链球菌流行亚型、药物敏感性及其致病机理的系统性研究报道相对较少。本项目系统而深入地开展了无乳链球菌流行亚型分析、耐药性检测及其导致奶牛乳房炎的发病机制研究。通过乳房炎的奶牛乳汁分离鉴定牛源无乳链球菌，研究了无乳链球菌的生理生化特性和基因分型特点、检测了无乳链球菌的耐药及毒力基因，建立无乳链球菌体外感染乳腺上皮细胞和体内感染动物的模型，分析无乳链球菌体内外感染时的持续状况、各种病理变化等，揭示了无乳链球菌致奶牛乳房炎的发病机制，为无乳链球菌性奶牛乳房炎的防治提供重要的理论依据。通过本项目发表和已接收发表论文10篇，其中中文核心期刊论文7篇，SCI期刊论文2篇，培养研究生7名。研究结果达到了预期目标。本项目研究结果为奶牛乳房炎研究提供素材和理论依据，体现了重要的理论价值和实践意义。