内蒙古地区健康与患炎症奶牛阴道菌群的比较研究

目的对健康与患炎症奶牛阴道菌群进行比较研究,为研制开发防治奶牛阴道炎症制剂奠定基础。方法本实验从内蒙古呼和浩特屠宰场采集健康奶牛的阴道40个,其中未配种奶牛阴道20个,配种期奶牛20个;并再采集患炎症的奶牛阴道20个。采用经典的微生物学方法,将阴道内黏膜以及粘液中的细菌分离、纯化、鉴定及计数,并对它们进行比较。结果未配种奶牛阴道内菌体生长极少,配种期奶牛阴道内菌群数量比例趋势为乳酸菌大肠菌群金黄色葡萄球菌链球菌表皮葡萄球菌其他菌群,患炎症奶牛阴道菌群内优势菌体为金黄色葡萄球菌,其次为大肠菌群与乳酸菌,而后为链球菌、表皮葡萄球菌和其他菌群。结论实验表明健康奶牛阴道内优势菌群为乳酸菌,患炎症奶牛阴道优势菌群为金黄色葡萄球菌,金黄色葡萄球菌比其他菌株更易在阴道粘液中生存,而大肠菌群虽为条件性致病菌,但是在患炎症的奶牛阴道中并未有很高的菌株分离数。     

一株源自人体阴道抗真菌的优势乳杆菌的分离、筛选及鉴定

目的 筛选并鉴定出可以抗真菌且益生特性优良的乳杆菌菌株,为研制预防和治疗人体真菌性阴道炎症的微生态制剂奠定坚实的理论基础。方法 利用经典的微生物方法,将健康人体阴道内乳杆菌分离出来;利用发酵工程学、细胞生物学以及药理学方法,将分离出的乳杆菌中益生特性优良且可以抑制白假丝酵母菌的优势菌株筛选出来,利用分子生物学方法,将筛选出的优良乳杆菌菌株进行鉴定。结果 筛选出1株产酸性能优良,产过氧化氢,具有较强黏附能力,且能够抑制白假丝酵母菌的优势乳杆菌菌株,此菌株经鉴定为Lactobacillus crispatus。结论 本实验从健康人体阴道内分离、筛选并鉴定出的1株可抗真菌性能优良乳杆菌菌株SQ004,具有制备预防和治疗人体真菌性阴道炎症微生态制剂主要菌株的条件。     

内蒙古呼伦贝尔地区传统发酵乳中乳酸菌的多样性分析

【目的】对内蒙古呼伦贝尔地区传统发酵乳制品中乳酸菌资源的生物多样性进行研究。【方法】采用纯培养和16S rRNA基因序列分析法对内蒙古呼伦贝尔地区传统发酵乳中的乳酸菌进行多样性分析。【结果】从8份传统发酵乳制品(6份酸牛奶和2份酸马奶)样品中分离到24株乳酸菌,通过16S rRNA基因序列分析和系统进化关系分析将24株乳酸菌鉴定为2株Lactobacillus kefiranofaciens、2株Lactobacillus kefiri、5株Lactobacillus paracasei、3株Lactobacillus plantarum、1株Lactobacillus rhamnosus、6株Lactococcus lactis subsp.lactis、2株Leuconostoc mesenteroides subsp.dextranicum、2株Streptococcus thermophilus和1株Enterococcus faecium。【结论】Lactococcus lactis subsp.lactis为内蒙古呼伦贝尔地区传统发酵乳制品的优势菌种,占总分离株的25%,其次为Lactobacillus paracasei,占总分离株的20.83%。     

饲料乳酸菌的分离鉴定及优良菌株筛选

对从饲料玉米、高粱、麦秆及棉花中筛选出的乳酸菌进行分类鉴定和综合性分析。用MRS+CaCO3固体培养基从棉花中分离出乳酸菌18株、高粱中30株、饲料玉米中18株、麦秆中18株。经形态学、生理生化试验进行初步鉴定并按产酸试验,耐盐及耐酸试验挑选出32株产酸率强的乳酸菌对其进行16S rDNA分子鉴定。结果显示,32株菌都具有良好的耐盐、耐酸能力;经生理生化和16S rDNA基因序列鉴定可知32株乳酸菌分属于两个属,即乳杆菌属、肠球菌属,4个种,即干酪乳杆菌(Lactobacilluscasei)、肠道球菌(Entercoccus faecium)、植物乳杆菌(Lactobacillus plantarum)、海氏肠球菌(Entercoccus hirae)。4种饲料原料中肠道球菌普遍存在。除了这种乳酸菌以外,棉花有干酪乳杆菌、植物乳杆菌、海氏肠球菌,玉米和麦秆内有植物乳杆菌。从饲料中筛选出4株具有较强产酸能力的乳酸菌,可进一步研发成青贮饲料添加剂。     

内蒙古原始森林桦树皮内生和表生乳酸菌分离鉴定及其特性

【背景】随着乳酸菌产业的快速发展,不同环境乳酸菌群落结构的分析和菌种资源的开发利用已经成为该领域的研究热点。从不同环境、不同来源分离、保藏乳酸菌对于优良菌种的筛选和开发利用具有重要意义。【目的】阐明内蒙古原始森林桦树皮中乳酸菌的组成,并获得丰富的野生型乳酸菌资源,为优良菌株的筛选和工业化应用提供核心菌株,从原始森林桦树皮中分离、鉴定和筛选优良乳酸菌菌种,并研究其特性。【方法】通过传统纯培养方法对20份桦树皮中的内生和表生乳酸菌进行分离纯化,运用16SrRNA基因序列分析以及系统发育关系研究进行种属鉴定,并对样品中的优势菌株进行筛选,得到2株具有优良特性的菌株。【结果】16S rRNA基因序列分析结果表明,分离到的112株乳酸菌鉴定为3个属7个种,其中树皮表生分离株共53株,内生分离株为59株,包括Enterococcusmundtii(27株)、Enterococcusfaecium(16株)、Enterococcusfaecalis(12株)、Enterococcusdurans(1株)、Lactococcuslactis(37株)、Lactococcusgarvieae(18株)、Lactobacillus fermentum (1株)。通过初筛和复筛比较不同乳酸菌在同一条件下的pH和产酸量,从37株Lactococcus lactis中得到2株产酸速率较高、pH较低的乳酸菌,其编号为IMAU98457和IMAU98428。对菌株IMAU98457和IMAU98428进行生物学特性研究,发现其最适生长温度在37°C,适宜pH范围为5.5-7.5,菌株在37°C发酵18 h后菌液OD600值分别高达2.531 0和2.518 2,pH低至4.36和4.34 (总酸)。菌株IMAU98457和IMAU98428有一定的盐耐受性,当NaCl浓度大于6%时,菌株生长受到明显抑制。【结论】20份桦树皮中分离出的112株乳酸菌,Lactococcus lactis (37株)为内蒙古赤峰市桦树皮样品的优势菌株,占总分离株的33%。桦树皮表生样品与内生样品相比,内生样品较表生样品的菌株种类资源更为丰富,二者的优势菌属具有较大的差异,并从优势菌株Lactococcus lactis中筛得2株繁殖速度快、产酸速率较高、pH较低的菌株IMAU98457和IMAU98428。     

葡萄藤叶青贮中乳酸菌的分离与鉴定

为了筛选出性状优良的乳酸菌菌株,本试验以实验室纯培养方式从葡萄藤叶自然发酵的青贮中分离鉴定乳酸菌。经过菌落形态、细胞形态、生理生化鉴定和16S r DNA基因序列以及系统发育树的分析,从葡萄藤叶青贮中分离出5株乳酸菌,其中菌株P-1. 2和P-2. 2为戊糖乳杆菌(Lactobacillus pentosus),菌株P-1. 7为戊糖片球菌(Pediococcus pentosaceus),菌株P-2. 1为粪肠球菌(Enterococcus faecium),菌株P-2. 3为短乳杆菌(Lactobacillus brevis)。测定产酸速率和生长速率,发现菌株P-2. 1的产酸能力和生长性能最好。     

奶牛源乳酸菌的分离、鉴定及其微生态制剂的研究

为筛选出能用于防治奶牛子宫内膜炎的益生性乳酸菌菌株并以其为原材料制作微生态制剂,采集产后30～50 d健康奶牛的子宫颈口处分泌物,利用MRS选择性培养基厌氧培养方式进行乳酸菌初步筛选。纯化得到的单株菌株进行染色和镜检,将典型的乳酸菌进行生化鉴定。提取其DNA,扩增16S rDNA全序列并测序,将所得序列与NCBI核酸数据库进行同源性比对,明确各分离菌株种属归属。将鉴定得到的单株乳酸菌过滤以及离心获取上清,得到的上清进行抑菌实验。采用琼脂扩散法检测对大肠埃希菌的抑菌活性。以得到的良好抑菌能力的菌株为原材料进行混合抑菌,进行微生态制剂最优比例的探究,并研究这些菌株的生长曲线和产酸能力。结果显示,经染色镜检、生化鉴定和16S rDNA测序分析和单株抑菌实验得到6株较好的抑菌能力的乳酸菌,其中鼠李糖乳杆菌、魏斯式细菌和粪肠球菌的混合上清抑菌效果最好,具有较强的生长和产酸能力。研究得到了可用于防治奶牛子宫内膜炎益生菌株,可进一步用于临床试验探究。     

应用rep-PCR分型技术筛选潜在治疗性乳杆菌

分离鉴定阴道弯曲乳酸杆菌并对其进行基因分型分析和产H2O2能力测定,初步筛选具有潜在防治女性生殖道感染的弯曲乳酸杆菌菌株。将健康妇女阴道分泌物接种到de Man,Rogosa and Sharpe (MRS) 培养基,分离培养乳酸杆菌。通过16S rRNA序列进行乳酸杆菌分类鉴定,重复序列片段PCR扩增方法进行弯曲乳酸杆菌的基因分型,并进一步采用pH直接测酸法和辣根过氧化物酶催化四甲基联苯胺与过氧化氢反应显色法检测了10株弯曲乳酸杆菌产酸和产H2O2能力。经过序列比对鉴定,共得到65株乳酸杆菌。其中,弯曲乳杆菌Lactobacillus crispatus 19株,詹氏乳杆菌Lactobacillus jensenii 17株,发酵乳杆菌Lactobacillus fermentum 12株;rep-PCR分型发现不同种类的乳酸杆菌和同一种弯曲乳酸杆菌均表现为不同的带型指纹图;10株弯曲乳酸杆菌均产酸,其中T22-3和T29-5两株弯曲乳酸杆菌产H2O2量最高。结果表明个体阴道内乳酸杆菌分布具有差异,弯曲乳酸杆菌具有种内多样性,产H2O2丰富的T22-3和T29-5两株弯曲乳酸杆菌有可能作为防治女性生殖道感染的有益菌株。     

大熊猫粪便乳酸菌的分离鉴定

大熊猫作为国家保护动物,其健康问题备受瞩目。为了维护大熊猫的肠道健康,本研究从大熊猫肠道内分离出适宜于大熊猫肠道环境的乳酸菌菌株,有望将其制成熊猫肠道微生物制剂,从而改善大熊猫肠道菌群环境。从雅安市宝兴县蜂桶寨自然保护区选取圈养与野生大熊猫的粪便,通过体外培养分离出9个菌株。分离菌株经过革兰氏染色镜检、过氧化氢产气、菌落形态观察等方法与技术初步鉴定为乳酸菌。对这9株乳酸菌进行耐酸试验、耐胆盐试验、抑菌能力试验和产酸能力等测试,筛选出了3个适应性较强,有望制成调节大熊猫肠道内环境平衡作用的微生态菌剂的菌株。16S rRNA基因序列分析表明:分离菌株J1、J2和J4分别为融合魏斯氏菌（Weissella confusa）,海氏肠球菌（Enterococcus heynei）和非解乳糖链球菌（Streptococcus alactolyticus）,有望被应用于大熊猫肠道微生态制剂的研究。     

广西传统发酵米粉中乳酸菌的分离鉴定

对广西传统米粉发酵液中的乳酸菌进行分离筛选,获得6株纯培养优势菌株。通过形态学鉴定及16S rDNA序列分析,结果表明其中4株乳酸菌属于戊糖片球菌(Pediococcus pentosaceus),另外2株鉴定为植物乳杆菌(Lactobacillus plantarum)。这2种乳酸菌均为对人类及动物安全的益生菌,该结果将为传统发酵米粉中有益微生物资源的挖掘和利用奠定基础。     

Vaginal bacterial microflora modifications during the growth of healthy cows

The aim of this work was first, to determine the predominant groups capable of colonizing the vagina and maintaining high numbers with time. The normal microbial flora of the cow's vagina and its evolution from weaning to service was then studied using standard microbiological methods. The results show that the most dominant bacteria belong to the streptococci, followed by the staphylococci, with similar levels during the whole study period. Enterobacteriaceae and lactobacilli were present at very low levels, the latter increasing during the cow's growth, suggesting some kind of hormonal influence. The results will allow the selection of micro-organisms with probiotic characteristics, classified as GRAS (Generally Regarded as Safe), to be used in the prevention of infections in the vaginal tract of cows, such as metritis, which produces delayed periods between partum and conception, and consequent economic losses.     

阴道乳酸杆菌的分子生物学鉴定方法研究

目的鉴定女性生殖道乳杆菌种类及各种乳杆菌在阴道中的分布差异。方法通过扩增和测序乳酸杆菌16S rRNA序列中特异性区段（8f926r）,并利用Vector NTI 8.0序列分析软件对各菌株的基因序列与标准菌株比对进行。结果 26株乳酸菌,L1株、L25株和L46株为乳酸杆菌属中的格氏乳酸杆菌（Lactobacillus gasseri）;L2株、L5株、L6株、L7株、L8株、L11株、L13株、L15株、L19株、L20株、L24株、L26株、L28株、L29株、L31株、L32株、L33株、L34株、L35株、L36株、L40株与L45株均为乳酸杆菌属中的卷曲乳酸杆菌（Lactobacillus crispatus）,而L21株未鉴定为乳酸球菌。结论女性阴道乳酸杆菌的优势菌种是卷曲乳杆菌、格氏乳杆菌。     

Mycoflora of cervicovaginal fluids in dairy cows with or without reproductive disorders

Fungal infection of reproduction system of dairy cattle have not been received much more attention. The aim of this study was to determine the fungal infection of Holstein dairy cows with reproductive disorders or healthy. Fungal isolates of cervicovaginal fluids of 176 Holstein dairy cows were collected by using the double rod swabs for cervix and the sterilized cotton swabs for discharges of vagina. They were evaluated for fungal infections. The treatment group included 70 dairy cows with reproductive diseases, such as abortion, repeat breeder, endometritis, metritis, retention of fetal membrane, dystocia, cervicitis, and vaginitis. The control groups were included 42 healthy non-pregnant cows and 64 pregnant cows. Isolates of fungi were obtained from cervix and vagina of 27.1% and 28.6% of treatment group, 26.7% and 31.2% of pregnant cows, and 33.3% and 21.4% of healthy non-pregnant cows, respectively, indicating no significant differences. It is showed that the cervix and vagina of the treatment group have been infected by six different mycotic isolates. However, the cervix and vagina of pregnant and non-pregnant healthy cows in control group were infected with 5, 6 and 5, 4 different fungal agents, respectively. Penicillium and yeast were the most common isolated agents. Regarding to the result of this study, it is concluded that fungal infections can occur in cervicovaginal cavity of Holstein dairy cows with or without reproductive diseases.     

阴道中益生特性乳杆菌的分离与功能评价

目的 分离健康女性阴道中的乳杆菌并鉴定其益生特性,为开发治疗妇科疾病的复方益生菌制剂提供新型菌株。方法 采集健康女性阴道分泌物并分离筛选乳杆菌,通过16S rDNA序列分析鉴定乳杆菌分离株,并对其产酸性能、产H2O2能力、抑菌能力、产生物膜能力进行检测。结果 从50名健康女性阴道内共分离出179株乳杆菌,其中卷曲乳杆菌101株、詹氏乳杆菌42株、格氏乳杆菌26株、植物乳杆菌5株、唾液乳杆菌3株以及干酪乳杆菌2株。179株乳杆菌中有146株具有产酸能力,发酵液pH值的最低的5株菌分别为卷曲乳杆菌J3、卷曲乳杆菌J8、詹氏乳杆菌J87,植物乳杆菌J75以及格氏乳杆菌J35,其pH分别为4.20、4.23、4.24、4.26及4.36;产H2O2弱阳性菌株有87株、阳性有37株、强阳性有9株,这9株菌分别为卷曲乳杆菌J3、卷曲乳杆菌J8、卷曲乳杆菌J20、詹氏乳杆菌J87,詹氏乳杆菌J90、詹氏乳杆菌J15、格氏乳杆菌J11、植物乳杆菌J75、植物乳杆菌J69以及植物乳杆菌J40;能拮抗大肠埃希菌的菌株有115株、拮抗金黄色葡萄球菌的有84株、拮抗白假丝酵母的有52株;经统计,对三者同时有拮抗作用且作用最强的只有6株,分别为卷曲乳杆菌J3、卷曲乳杆菌J50、卷曲乳杆菌J62、詹氏乳杆菌J87、詹氏乳杆菌J16和格氏乳杆菌J66;不同乳杆菌产生物膜能力数值范围在1.0~5.4,卷曲乳杆菌、詹氏乳杆菌、干酪乳杆菌的生物被膜形成能力显著高于其他三种菌（P<0.05）。在全部179株菌中,卷曲乳杆菌J3和詹氏乳杆菌J87既具有强的产酸能力和产过氧化氢能力,又有较强抑菌活性,同时产生物膜能力也最强。结论 卷曲乳杆菌J3和詹氏乳杆菌J87具有优良的生物学特性,有望成为用于治疗妇科疾病微生态制剂的备选菌株。     

新疆发酵乳品中乳酸菌的分离鉴定及其耐药性

目的对新疆传统发酵乳品中乳酸菌进行分离鉴定并检测其耐药性。方法利用传统形态学鉴定法和生化鉴定等方法对新疆发酵乳中乳酸菌进行鉴定,采用纸片扩散法对分离鉴定的菌进行耐药性分析。结果从新疆发酵乳品中共分离出8株乳酸菌,经鉴定分别为瑞士乳杆菌(Lactobacillus helveticus)、植物乳杆菌(Lactobacillus plantarum)、马乳酒样乳杆菌(Lactobacillus kefianofaciens)、乳酸乳球菌(Lactococcus lactis)、副干酪乳杆菌(Lactobacillus paracasei)、副干酪乳杆菌类坚韧亚种(Lactobacillus paracasei subsp.tolerans)、哈尔滨乳杆菌(Lactobacillus harbinensis)、希氏乳杆菌(Lactobacillus hilgardii),并且发现8株乳酸菌对万古霉素、庆大霉素、阿莫西林、多西环素、环丙沙星、阿奇霉素、头孢他啶、头孢孟多具有一定敏感性。结论新疆发酵乳品中以乳杆菌居多,对常见抗生素具有一定的敏感性。     

Identification and plasmid profiles of Lactobacillus species from the vagina of 100 healthy women

Abstract It was hypothesized that Lactobacillus acidophilus might not be the dominant aerobic or microaerophilic Lactobacillus species in the human vagina, and that the dominant isolates are likely to express a variety of properties associated with colonization. To examine the question, vaginal swabs were collected from 100 healthy premenopausal women and cultured to detect the dominant aerobic or microaerophilic isolates of Lactobacillus . Only eight species were detected, with Lactobacillus jensenii being the dominant colonizers in the highest number (35) of women. Twelve different plasmid profiles were found amongst the isolates, but there was no association between plasmids and vaginal colonization or hydrogen peroxide production. Of the strains tested, few were resistant to the spermicidal agent nonoxynol-9. The data identify species within which strains may possess properties associated with maintenance of a healthy vaginal ecology     

Lactobacilli isolated from vaginal vault of dairy and meat cows during progesteronic stage of estrous cycle

Lactobacilli have been barely studied in cows. We proposed isolate and characterize lactic acid bacteria from dairy cows as compared to those raised for meat production and elucidate the presence of strains with evident probiotic employment's potential. For this, isolation and quantification of LAB mainly lactobacilli were realized from vaginal cattle samples in MRS medium. Each selected microorganism was then briefly characterized. The MATH method was employed using hexadecane, xilene an toluene as solvent. According to the hydrophobic characteristics, strains were classified into three categories: high (71-100%), medium (36-70%) and low (0-35%). Hydrogen peroxide qualitative production was studies too, lactobacilli were streaked onto an MRS agar plate containing 5 mg of 3,3',5,5'-tetramethylbenzidine and 0.20 mg of horseradish peroxidase. Twenty-one sampled cows (78%) were positive for lactic acid microflora, 12 belonging to the dairy group and 17 of the meat group. Total LAB counting including dairy and meat cows were log 2,41 CFU/ml. Of overall identified strains, an 83% corresponded to lactobacilli. Most strains belonged to the heterofermentative facultative group (75%), with L. plantarum as the most frequent specie. The highest proportion of isolated vaginal strains (69%) had low hydrophobicity, the LAB with highest hydrophobic characteristics (3 strains) were found only in meat cows. In the qualitative evaluation of H(2)O(2) production, a positive reaction was observed in 13 of 29 strains (45%). The role of lactobacilli in vaginal microbiota is limited, and therefore the present work is interesting in incorporate knowledge of normal microflora of progesteronic healthy cows, in this case in production animals. The isolation and characterization data obtained are consistent in consider the study of particular strains with great potential in the development of a probiotic for production cows.     

女性阴道中产细菌素的乳杆菌筛选与鉴定

目的对从60例健康女性阴道中筛选出产生细菌素的优势乳杆菌进行鉴定,并为研制开发微生态制剂提供优良可靠菌种。方法利用牛津杯法筛选出19株乳杆菌,其菌株发酵乳酸量高并且产生细菌素。对19株乳杆菌进行了多项理化鉴定。结果19株乳杆菌分别为：格氏乳杆菌9株,唾液乳杆菌1株,卷曲乳杆菌9株。结论筛选的19株乳杆菌是健康女性阴道中的优势有益菌,具有较强的产酸能力,都产生细菌素,其中16株产生过氧化氢,某些菌株具有较高的生产应用价值。     

Bacteriologic studies of bovine genital tracts: The use of dye as an indicator of postmortem contamination of the uterus by vaginal fluids

Non-specific bacteria were more frequently isolated from uteri of culled dairy cattle than beef cows (P <.01). In some studies, food dye was placed in the vagina just prior to slaughter as an indicator of postmortem contamination of the uterus by vaginal fluids and bacteria. The dye was more often found in the uteri of dairy cows than beef cows (P <0.05).Bacteria could always be isolated from uteri totally contaminated by the dye. Bacteria were more frequently isolated from uncontaminated areas of uteri that were partially contaminated by the dye, as compared to uteri in which no dye was found (P <.01).It is hypothesized that an enlarged cervical canal may allow dye and also bacteria to transverse the cervical canal into the uterus during the slaughtering process. It is further hypothesized that an enlarged cervical canal may more readily permit nonspecific bacteria of the vagina to ascend into the uterus in the live animal and cause reproductive failure.     

Evaluation of the Change of Serum Copper and Zinc Concentrations of Dairy Cows with Subclinical Ketosis

Ketosis in dairy cows can lead to poor reproductive success and decreased milk production. Since the serum concentrations of copper (Cu) and zinc (Zn) are closely associated with the health status of cows, we investigated whether serum concentrations of Cu and Zn differed in dairy cows with subclinical ketosis and healthy dairy cows. Blood samples of 19 healthy dairy cows and 15 subclinically ketotic dairy cows were collected from three farms, and the concentrations of β-hydroxybutyrate (BHBA), glucose, non-esterified fatty acids (NEFA), Cu, and Zn were determined. Subclinically ketotic dairy cows had significantly higher BHBA and NEFA levels (p?<?0.01) and lower glucose (p?<?0.01) than healthy dairy cows. Likewise, serum concentrations of Zn were significantly decreased (p?<?0.05) in dairy cows with subclinical ketosis. There was no significant difference observed for serum Cu concentration between healthy and subclinically ketotic dairy cows. This study suggests that a decreased serum Zn concentration could be a cause of decreased reproductive performance in subclinically ketotic dairy cows.