【作者】 彭宇；

【导师】 杭苏琴；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2011， 硕士

【摘要】 子宫内膜炎是奶牛产后常见的繁殖疾病,常造成子宫和卵巢机能恢复减慢,产犊间隔延长,受胎率下降,淘汰率上升等,严重影响了奶牛生产的经济效益。为了给临床诊断和治疗提供更多的理论依据,本试验通过体外培养和分子生物学方法集中研究了奶牛产后子宫内菌群的动态变化及其与子宫颈粘液性状的关系。一奶牛产后子宫内菌群动态变化及子宫粘液性状与细菌种类关系的研究选取分娩日期临近、产后子宫粘液性状不同的荷斯坦奶牛42头,于分娩后第10d、20d、30d和40d采集子宫粘液,微生物体外培养的方法研究奶牛产后子宫内优势菌群随时间的动态变化及细菌种类与子宫粘液性状之间的关系,以便于临床的快速诊断及治疗。结果显示,奶牛产后子宫内分离率最高的细菌为大肠杆菌(85.7%)、变形杆菌(64.3%)和葡萄球菌(61.9%)；白色或米白色粘液、黄色或土黄色粘液中每头牛细菌分离株数(5.56±0.38,5.50±1.40)高于清亮透明粘液(3.48±1.58),其中沙门氏菌、变形杆菌、链球菌和化脓隐秘杆菌产后30d和40d可能与白色或黄色脓性粘液的出现相关；产后40d黄色或土黄色化脓性粘液的出现可能与产气荚膜梭菌的出现相关；而粪肠球菌和嗜酸乳杆菌产后10d清亮粘液中分离数较不正常粘液高,产后30d和40d在脓性粘液中分离数高于清亮粘液,说明其作为有益菌可能在分泌不正常粘液的子宫内具有抵抗病原菌的作用。研究结果表明奶牛产后子宫内优势菌群呈动态变化,子宫粘液性状与特异病原菌的出现相关,为临床治疗药物种类和剂量选择提供了理论依据。二奶牛产后子宫内细菌多样性的研究采集健康和子宫内膜炎奶牛产后10d和40d子宫粘液,利用细菌通用引物8F/1510R,建立其16S rRNA基因克隆库,研究产后不同时间、不同生理状态奶牛子宫内菌群多样性。试验共挑取297个克隆,结果发现奶牛产后子宫内细菌主要属于Bacteroidetes、Fusobacteria、Firmicutes、Tenericutes和Proteabacteria.产后10d健康奶牛(H10)和子宫内膜炎奶牛(M10)克隆序列均主要来自于Bacteroidetes、Fusobacteria和Firmicutes,所占比例及最相似菌具有差异。H10的71个克隆中64个(90.14%)属于Bacteroidetes,序列比对分析显示有36个(50.70%)最相似于Bacteroides pyogenes;M10的79个克隆中52个(65.82%)属于Bacteroidetes,其中31个(39.24%)最相似于Bacteroides heparinolyticus;17个(21.52%)属于Firmicutes细菌中11个(13.92%)最相似于Peptostreptococcus sp.,结果提示Bacteroides heparinolyticus和Peptostreptococcus. sp.可能是引起子宫内膜炎的主要病原菌。产后40d健康奶牛(H40)Bacteroidetes克隆数量较产后10d明显下降(5个,7.14%),克隆序列主要属于Tenericutes(39个,55.71%),全部最相似于Ureaplasma diversum(98%);产后40d子宫内膜炎奶牛(M40)77个克隆中32个(42.86%)属于Firmicutes,16个最相似于Parvimonas sp.(95～96%)；26个(33.77%)属于Proteabacteria的克隆均最相似于Histophilus somni(99-100%)。结果表明,H40菌群多样性较H10下降,Tenericutes细菌成为子宫内优势菌群；M40中Bacteroidetes克隆仍维持较高数量,新增Proteabacteria门克隆。结果提示Bacteroidetes门和Proteabacteria门的部分细菌可能是导致子宫复原时间延长的主要细菌。三奶牛产后子宫内菌群结构和数量的研究选取产后健康(H)和子宫内膜炎(M)奶牛各3头,采集产后10d、20d、30d和40d子宫粘液,运用基于16SrRNA基因的PCR/DGGE技术和Real-time PCR定量技术探讨奶牛产后子宫内菌群结构和数量变化及不同性状粘液子宫内菌群结构。DGGE结果表明,坏死梭杆菌(Fusobacterium necrophorum)、解肝素拟杆菌(Bacteroides heparinolyticus)和普雷沃氏菌属(Prevotella sp.)产后30d,化脓隐秘杆菌(Arcanobacterium pyogenes)产后40d只出现子宫内膜炎奶牛子宫内,可能为子宫内膜炎致病菌；健康奶牛子宫内较子宫内膜炎奶牛更早出现棒状杆菌属(Corynebacterium sp.),可能为子宫内有益菌群；相似性分析发现H组10d与M组10-40d之间具有较高的相似性,说明子宫内膜炎奶牛产后20、30和40天子宫内菌群结构没有因自净而改变,可能是子宫疾病的原因之一。坏死梭杆菌与棕黑色、黄色或白色等不正常粘液相关,解肝素拟杆菌和普雷沃氏菌与白色或黄色粘液出现相关,化脓隐秘杆菌产后40d与白色粘液相关,相同性状粘液间具有较高相似性。定量结果表明H组总细菌、消化链球菌属和梭杆菌属细菌数量产后30和40天较产后10天均显著降低,M组产后10d和30d三种细菌数量均显著高于H组,总细菌、消化链球菌属和梭杆菌属细菌数量可能是导致子宫内膜炎的一个原因；H组和M组拟杆菌随时间均显著降低,M组产后10d显著高于H组,但H组拟杆菌占总菌比例产后30d恢复至30%左右,说明拟杆菌属细菌中部分有益菌的大量增殖可能有利于子宫内膜的恢复；产后10天和30天健康奶牛子宫内大肠杆菌数量显著高于子宫内膜炎奶牛,健康和子宫内膜炎奶牛体内牛链球菌数量差异不显著,结果提示这两种细菌可能不是子宫炎症的主要致病菌。更多还原

【Abstract】 The uterus infection by bacteria after parturition often causes endometritis. Endometritis delayed the recovery of the uterus, increased intervals from calving to first service or conception, associated with lower conception rates, seriously affected the economic efficiency of dairy production. The objectives of the present study were to determine the bacterial community in uterus from day10to day40after parturition and the relationship between bacteria community and the character of uterus mucus in postpartum dairy cows.1Bacterial community in uterus and the relationship with uterus mucus character of postpartum cowsUterus mucus were collected from42dairy cows from d10to d40after parturition. Bacteria were identified following aerobic and anaerobic culture in vitro. The dominate genus and species bacteria dynamic changes with the postpartum time, and the relationship between mucus character and bacterial community were investigated. The results showed that the most prevalent bacteria in uterine were E. coli (85.7%), Proteus (64.3%) and Staphylococcus sp.(61.9%). The highest isolation rate of bacteria has changed at different postpartum time. The number of bacteria isolated from white or off white mucus and yellow or earthy yellow purulent mucus (5.56±0.38,5.50±1.40) were higher than that isolated from clear mucus (3.48±1.58). Salmonella, Proteus mirabilis, Streptococcus sp. and Arcanobacterium pyogenes were isolated from white or yellow purulent mucus more than clear mucus on d30and d40postpartum. Clostridiales bacterium was associated with yellow or earthy yellow purulent mucus on day40postpartum.2The investigation of the diversity of bacterial community in the uterus of dairy cows after parturitionUterus mucus were collected on day10(H10, M10) and day40(H40, M40) from healthy and endometritis cows after parturition, respectively. Four clone libraries were constructed based on bacterial16S rRNA gene amplified by using universal primers8F/1510R. Total of297positive clones were selected. Sequences belonged to5major groups, Bacteroidetes, Fusobacteria, Firmicutes, Tenericutes and Proteabacteria. The most clone sequences in H10and M10libraries were affiliated with the phylum Bacteroidetes(116), Fusobacteria(14) and Firmicutes(30). The number of36(50.70%) sequences from H10showed high similarity with Bacteroides pyogenes. The ratio of39.24%(31) and13.92%(11) in total sequences from M10library had high identity with Bacteroides heparinolyticus and Peptostreptococcus sp.. Phylogenetic analysis revealed that55.71%and34.29%clones in H40library affiliated Tenericutes and Firmicutes, all the sequences belonged to Tenericutes were related to Ureaplasma diversum. The diversity and abundance of Bacteroidetes from library H40(64,90.14%) significantly decreased than that from library H10(5,7.14%). A total of77positive clones from M40. The ratio of34.29%and33.77%colons belonged to phylum Firmicutes and Proteabacteria,20.78%and33.77%had high identity with Parvimonas sp. and Histophilus somni, respectively.3Shift of bacterial community in uterus of postpartum cows from10days to40days after parturitionMolecular methods of PCR/DGGE and Real-time PCR based on16S rRNA gene were used to study bacterial communities in uterus and changes of bacterial community composition in different character mucus of postpartum cows from d10to d40after parturition. The DGGE profiles of total bacteria community showed that the predominant bands similar to Fusobacterium necrophorum and Bacteroides heparinolyticus only appeared on d30and d40from endometritis cows, the bands similar to Arcanobacterium pyogenes was found only on d40endometritis cows. The similarity index of DGGE profile from20to40days of endometritis cows had high similarity with that d10healthy and endometritis cows. The DGGE profiled from different character uterus mucus reviewed that Fusobacterium necrophorum were associated with brown, yellow or white purulent mucus, Bacteroides heparinolyticus and Prevotella sp. were associated with white or yellow mucus. There is high similarity between the same character mucus. The DGGE profile of Actinobacteria revealed that group Corynebacterium sp. appeared earlier in healthy cows than endometritis, suggested they maybe the beneficial bacteria for uterus. Real-time PCR assay revealed that the total bacteria, Peptostreptococcus spp. and Fusobacterium spp. were all significantly lower in health cows than endometritis cows on d10and d30(P<0.05). Bacteroidetes on20days both health and endometritis cows were significant lower than d10postpartum, while endometritis cows higher than health in10days(P<0.05). The percentages of Peptostreptococcus spp. and Fusobacterium spp. relative to total bacteria were significantly lower in health cows than endometritis cows on d30and d40. A decrease of the number of E.coli were observed both in health and endometrtis cows after30days partutition, while health cows significant higher than endometritis cows on d10and d40(P<0.01).更多还原