牛支原体P48蛋白卵黄抗体的制备

为了研究牛支原体P48抗原蛋白的免疫学功能及相应卵黄抗体的生物学特性,探究其抗体效价随免疫时间的变化规律,试验采用牛支原体P48蛋白疫苗免疫健康产蛋母鸡,定时采血并收集鸡蛋,用水稀释法结合硫酸铵盐析法制备特异性卵黄抗体,利用间接ELISA法检测血清抗体(IgG)和卵黄抗体(IgY)效价。结果表明,低、中、高剂量组均能诱导蛋鸡产生有效免疫应答,血清抗体效价与卵黄抗体效价的变化趋势基本一致,但卵黄抗体的产生滞后于血清抗体,在首次免疫后8~10周Ig Y效价达到峰值,其中中剂量组的免疫效价最高,最高效价为1:212。经SDS-PAGE检测,制备所得的纯度达86%以上,得率在8.6 mg/m L以上。说明采用牛支原体P48蛋白免疫蛋鸡可制备高效价、高纯度的抗牛支原体P48蛋白卵黄抗体,从而为卵黄抗体在牛支原体的检测与防控方面的研究奠定了基础。     

奶牛乳腺炎病原菌的分离、耐药性分析及其特异性卵黄抗体的体外抑菌效果

试验从新疆石河子地区3个建设兵团采集60份临床型乳腺炎患牛的奶样。细菌分离纯化后,在VITEK 2微生物自动分析系统基础上,利用16S r DNA序列分析对乳腺炎主要致病菌进行菌种鉴定,并对病原菌进行药敏试验。结果表明,60份奶样中共分离14种细菌,其中无乳链球菌(S.agalactiae)最多(20%),其次为金黄色葡萄球菌(S.aureus)(15.56%)。选择14种常用抗生素,采用Kithy-Bauer纸片法,对分离出的病原菌进行药敏试验,无乳链球菌和金黄色葡萄球菌对青霉素的耐药性最强(耐药菌株最多)。通过免疫蛋鸡制备无乳链球菌和金黄色葡萄球菌的特异性卵黄抗体(Ig Y),体外抑菌试验表明,10mg/m L的特异性Ig Y能够有效抑制两种细菌的生长,为卵黄抗体替代抗生素防治奶牛乳腺炎提供一定理论基础。     

多重PCR快速检测奶牛乳房炎3种主要病原体

奶牛乳房炎是引起奶牛业经济损失的一种重要疫病,目前还没有快速、特异检测奶牛乳房炎主要致病原的方法。本试验根据金黄色葡萄球菌、无乳链球菌、大肠杆菌各自保守的16S或23S rRNA基因序列,合成了3对特异性引物,建立了三重PCR检测方法。特异性试验表明,该方法对所有参与测试的金黄色葡萄球菌、无乳链球菌和大肠杆菌都能扩增出各自的阳性条带,而对所有参与测试的对照菌株则不能扩增出任何条带。敏感性试验表明该方法能检测到4个菌的金黄色葡萄球菌、无乳链球菌和2个菌的大肠杆菌。对送检的乳房炎奶样36份直接进行PCR检测,金黄色葡萄球菌阳性7份,无乳链球菌阳性2份,大肠杆菌阳性6份。     

牛乳腺炎无乳链球菌表面蛋白pgk抗原优势区的原核表达及其间接ELISA检测方法的建立

为评价牛乳腺炎无乳链球菌重组表面蛋白(pgk)免疫原性,并通过建立检测抗体的间接ELISA方法对免疫抗体效价进行评价,本研究根据GenBank登录的无乳链球菌pgk基因序列,设计一对引物,以临床分离菌株基因组DNA为模板PCR扩增出pgk蛋白抗原优势区的编码基因序列。将其插入pET-30a(+)中,并在大肠杆菌中表达。Western blot鉴定表明,表达的重组蛋白与阳性血清具有良好的反应原性。采用纯化的表达产物作为包被抗原建立了检测无乳链球菌抗体的间接ELISA方法,确定抗原最佳包被浓度为3.115μg/mL,血清最佳稀释度为1∶160,酶标二抗最适稀释度为1∶4 000。初步应用于检测无乳链球菌、化脓性链球菌、大肠杆菌、金黄色葡萄球菌小鼠阳性血清的检测。结果表明牛乳腺炎无乳链球菌pgk亚单位抗原具有良好的抗原性,抗体检测灵敏度达到1∶4 000,同时表现良好的特异性。     

罗非鱼无乳链球菌特异性IgM抗体ELISA检测方法的建立及对疫苗免疫效果的评估

为建立检测罗非鱼无乳链球菌特异性抗体的方法及评价疫苗的免疫效果,本研究首先制备纯化罗非鱼无乳链球菌特异性IgM和兔抗罗非鱼无乳链球菌IgM的IgG,利用原核表达纯化的无乳链球菌ScpB蛋白作为包被抗原,通过优化反应条件,建立检测无乳链球菌特异性IgM抗体的ELISA方法,并通过比较测定无乳链球菌疫苗免疫后血清抗体水平变化与攻毒后相对免疫保护率的关系来评估该方法的有效性。结果表明:该方法仅能够特异性的检测罗非鱼的血清抗体,而与其它鱼类的血清抗体无交叉反应,特异性良好,其敏感性为1∶800,批内、批间变异系数均小于10%,具有较好的重复性,并且抗体水平的变化与相对免疫保护率(RPS)变化具有平行相关性,变化趋势基本一致。本研究首次建立了检测罗非鱼无乳链球菌特异性IgM抗体的ELISA方法,该方法可以作为评估无乳链球菌疫苗免疫效果的有效手段,也为罗非鱼无乳链球菌病的检测提供技术支撑。     

奶牛乳房炎常见致病菌多重PCR检测方法的建立

为建立同时快速检测奶牛奶样中无乳链球菌、停乳链球菌、乳房链球菌和金黄色葡萄球菌的方法,根据无乳链球菌sip基因、停乳链球菌isp基因、乳房链球菌pauA基因和金黄色葡萄球菌nuc基因各设计1对特异性引物,建立多重PCR检测体系。结果显示,该检测方法具有高特异性,无乳链球菌、停乳链球菌、乳房链球菌和金黄色葡萄球菌敏感性分别为105、104、105、105 CFU/mL。对临床采集的460份奶样检测结果表明,建立的多重PCR体系可以用于临床上无乳链球菌、停乳链球菌、乳房链球菌和金黄色葡萄球菌感染引起的奶牛乳房炎的检测。     

奶牛乳房炎致病菌多重Taq-Man荧光PCR检测方法的建立

为同时检测大肠杆菌、无乳链球菌和金黄色葡萄球菌,提高对奶牛乳房炎的诊断率,本试验针对3种病原菌的保守序列设计合成了3套特异性引物,通过优化PCR反应体系和反应条件,建立了多重Taq-Man荧光定量PCR检测方法。结果显示,该检测方法具有高特异性,与肠炎沙门菌、肺炎克雷伯菌及鲍曼不动杆菌等无交叉反应;大肠杆菌、金黄色葡萄球菌和无乳链球菌的最低检测浓度分别为10~(3 )、10~2和10~(4 )拷贝数/μL,敏感度较高。DNA各个拷贝数浓度的变异系数均低于2.02%,表明重复性良好。对24份临床采集奶样的检测结果表明,建立的多重Taq-Man荧光定量PCR检测方法可用于临床大肠杆菌、无乳链球菌和金黄色葡萄球菌感染引起的奶牛乳房炎的检测,具有良好的推广应用价值。     

奶牛乳房炎多联苗抗原配比及抗人工感染试验的研究

为了进一步提高乳房炎多联苗免疫效果及评价疫苗免疫后的抗感染效果,试验从临床型乳房炎患牛奶样中分离、筛选出毒力及交叉免疫原性强的无乳链球菌(W271)、停乳链球菌(T2531)和金黄色葡萄球菌(J84184)3株优势血清型菌株,制备每剂量(5 mL)中含3种菌的抗原浓度分别为2.5×109CFU、5×109Cfu、1×1010CFU和2×1010CFU的4种氢氧化铝灭活多联苗,将每种疫苗于肩胛部肌肉免疫4头牛,每头牛免疫2次(0天和14天),每次5 mL,分别于免疫前后第28天采血,用间接ELISA方法检测免疫前后血清抗体水平.结果表明:免疫剂量与抗体效价具有显著相关性,随着菌苗中3种菌抗原浓度的增加,免疫后的3种菌血清抗体水平亦增加;用检验用的无乳链球菌(W117)、停乳链球菌(T1030)和金黄色葡萄球菌(J88014)进行免疫攻毒保护试验,结果血清抗体效价与免疫攻毒保护效果具有显著相关性,当免疫后抗体水平达到免疫前的2倍以上时,免疫后的奶牛即具有较好的保护效力.     

河北省奶牛乳房炎主要致病菌多重PCR检测方法的建立

为同时检测奶牛源金黄色葡萄球菌、无乳链球菌和绿脓杆菌,本试验根据GenBank中3种病原菌的nuc、ef-tu和eta基因保守序列分别合成了3对特异性引物,通过对反应条件、反应体系优化,建立了多重PCR检测方法。特异性试验结果表明,该方法与大肠杆菌、克雷伯杆菌、表皮葡萄球菌、巴氏杆菌无交叉反应,敏感性试验结果表明,多重PCR检测方法对金黄色葡萄球菌、无乳链球菌和绿脓杆菌的检出限为10~(-3) mg/L。应用该方法对150份临床样品进行检测,结果金黄色葡萄球菌与无乳链球菌混合感染检出率为28.7%(43/150),金黄色葡萄球菌与绿脓杆菌混合感染检出率为14%(21/150),无乳链球菌与绿脓杆菌混合感染检出率为11.3%(17/150),3种菌混合感染检出率为6%(9/150)。应用多重PCR检测结果与分离培养诊断方法结果进行比较,均能检测出样品中的病原菌,2种检测方法的总符合率分别为金黄色葡萄球菌92.6%,无乳链球菌91.3%,绿脓杆菌94.6%。该检测方法对奶牛乳房炎的防控、临床监测及疾病诊治具有重要意义。     

检测牛轮状病毒抗体间接ELISA方法的建立

利用CsCI密度梯度离心化的牛轮状病毒作为包被抗原,建立了检测牛轮状病毒抗体的间接酶联免疫吸附试验(EuSA).用牛病毒性腹泻病毒、大肠杆菌、金黄色葡萄球菌、无乳链球菌制备的对照包被抗原与牛轮状病毒阴性和阳性血清反应,其结果均无交叉反应,说明该方法具有良好的特异性,可用于牛轮状病毒抗体的检测.     

Characterization of specific egg yolk immunoglobulin (IgY) against mastitis-causing Escherichia coli

The objective of this study was to estimate the in vitro activity of egg yolk immunoglobulin (IgY) against mastitis-causing Escherichia coli. Specific IgY was produced by hens immunized with formaldehyde killed E. coli O111 in long-standing immunization response (titer > or =6400 for 100 days) and was isolated from yolks with a purity of 86% by water dilution, salt precipitations and ultrafiltration. Enzyme-linked immunosorbent assay (ELISA) indicated the produced IgY specifically targeted E. coli O111 and five other E. coli strains which were isolated from mastitic cows. The growth inhibition activity of the specific IgY to bacteria was dose-dependent with an effective concentration of 20mg purified IgY per milliliter. The phagocytic activity of E. coli either by milk macrophages (MPhi) or by polymorphonuclear neutrophil leukocytes (PMN) in the presence of specific IgY was significantly higher than that with nonspecific IgY or without IgY (p<0.05), suggesting that it enhanced phagocytic activity. The current work suggests that this specific IgY has potential as a therapeutic treatment for mastitis in dairy cows.     

新型磺胺类截短侧耳素衍生物抑菌效果研究

为研究新型磺胺类截短侧耳素衍生物对金黄色葡萄球菌和大肠杆菌的抑菌活性,采用纸片扩散法,用截短侧耳素和磺胺嘧啶作比较,通过测定抑菌圈大小进行判断。结果表明,新型磺胺类截短侧耳素衍生物对金黄色葡萄球菌和大肠杆菌均有抑制作用。     

Characterization of chicken egg yolk immunoglobulins (IgYs) specific for the most prevalent capsular serotypes of mastitis-causing Staphylococcus aureus

The objective of this in vitro study was to evaluate the potential of egg yolk immunoglobulins (IgYs) for treating mastitis caused by Staphylococcus aureus. Specific IgY against type 5 (IgY-T5), type 8 (IgY-T8) and type 336 (IgY-T336) S. aureus strains were obtained by immunizing hens with whole cell vaccines and the IgY produced were then purified to around 80% purity using a water dilution method coupled with salting out and ultra-filtration. Enzyme-linked immunosorbent assay indicated that the IgY specifically targeted the three homologous strains. A growth inhibition assay was performed in Columbia broth (non-encapsulated form) and phosphate-buffered saline (encapsulated form) for an 8h incubation. The results showed that IgY-T336 significantly inhibited (but only 1.5 log units; P<0.01) the growth of all three strains at 15 mg/ml in the Columbia broth. In contrast, the same concentrations of IgY-T5 and IgY-T8 did not show obvious bacteriostatic activity against the two homologous strains. In phosphate buffered saline, no inhibition of the two encapsulated strains was observed with IgY-T5, IgY-T8 and IgY-T336. However, IgY-T336 reduced live bacteria by 1.0 log unit against strain 336 compared with the control. An internalization test indicated that all of the specific IgY (at 5mg/ml) significantly (about 3.0 log units of the control; P<0.01) blocked the internalization of their homologous strains by bovine mammary epithelial cells (MAC-T cells) within 6h. These results suggested that research on the application of IgY as a treatment for mastitis caused by S. aureus should be focused on the internalization inhibition activity rather than on the growth inhibition activity of the IgY.     

安普霉素及其联合用药对3种细菌体内抗生素后效应的研究

采用菌落计数法和中性粒细胞减少小鼠股部感染模型,分别测定了安普霉素及其与阿莫西林、氨苄西林联用对金黄色葡萄球菌、沙门氏菌和大肠杆菌的体内抗生素后效应(post antibiotic effect,PAE)。试验结果表明,安普霉素在体内对3种细菌均有较长的体内PAE,且随药物浓度的升高其PAE也相应的延长,呈明显的剂量依赖性。安普霉素与阿莫西林或氨苄西林联用对3种细菌的体内PAE呈现协同作用。     

Efficacy of specific egg yolk immunoglobulin (IgY) to bovine mastitis caused by Staphylococcus aureus

The objective of this study was to estimate the efficacy of specific egg yolk immunoglobulin (IgY) to bovine mastitis caused by Staphylococcus aureus. Eighteen lactating cows with clinical mastitis and 18 lactating cows with experimental mastitis (1 quarter per cow) were randomly assigned to three treatments: IgY (20mg/ml) infusion, penicillin (100mg/ml) infusion and no infusion. Treatments for clinical mastitis and experimental mastitis were performed by a 6-day course of intramammary infusion with a dosage of 10ml at an interval of 12h. Milk samples were collected at morning milking time for testing color, clot, somatic cell counts (SCC) and bacterial count. For most of the cows treated with IgY and penicillin, the milk color and clot recovered to normal form during the therapy course. The milk SCCs and bacterial counts of treated cows decreased compared to those of untreated cows (p<0.05). The cure rates by IgY for experimental and clinical mastitis were 83.3% and 50%, respectively, and those by penicillin were 66.7% and 33.3%, respectively. These results showed the potential of specific IgY to be an alternative therapy for mastitis caused by S. aureus.     

Resistance of broiler chickens to Escherichia coli respiratory tract infection induced by passively transferred egg-yolk antibodies

Egg-yolk antibodies induced by immunizing hens with selected Escherichia coli antigens were evaluated for their ability to protect broiler chickens against respiratory/septicemic disease caused by avian pathogenic E. coli (APEC). Seven groups of broiler breeder hens were vaccinated three times, 1 week apart with live E. coli, killed E. coli, E. coli antigens [lipopolysaccharide (LPS), type 1 pilus adhesin (FimH), P pilus adhesin (PapG), aerobactin outer membrane receptor (IutA)] or phosphate buffered saline (PBS). An O78 APEC strain was used for preparation of all the antigens. Egg yolk immunoglobulins (IgY) were purified from eggs of each group and antibody activity in serum and purified IgY was determined by enzyme-linked immunosorbent assay (ELISA). IgY (100mg) was injected intramuscularly into 11-day-old broiler chickens, which were challenged 3 days later with homologous (O78) or heterologous (O1 or O2) E. coli by the intra-air sac route. Mortality was recorded and surviving chickens were euthanized 1 week after the challenge and examined for macroscopic lesions. Passive antibodies against all antigens except FimH were protective (90-100%) against the homologous challenge, but only anti-PapG and anti-IutA were effective against heterologous challenge. Anti-PapG IgY provided the greatest protection against the three serogroups of E. coli used for challenge. Hence vaccination of broiler breeders to induce anti-PapG and anti-IutA antibodies may provide passive protection of progeny chicks against respiratory/septicemic disease caused by APEC.     

肉桂醛体外抑制奶牛乳房炎主要致病菌的研究

研究天然化合物肉桂醛对奶牛乳房炎主要致病菌——大肠杆菌、金黄色葡萄球菌和链球菌的抑菌效果。采用试管二倍稀释法测得肉桂醛对奶牛乳房炎主要致病菌金黄色葡萄球菌和标准金黄色葡萄球菌的体外最小抑菌浓度(MIC)分别为3.2 mmol/L和1.6 mmol/L;链球菌1.6 mmol/L;大肠杆菌和标准大肠杆菌6.4 mmol/L和3.2 mmol/L,且能完全抑制这三种菌的生长。肉桂醛的最小杀菌浓度(MBC)金黄色葡萄球菌和标准金黄色葡萄球菌分别为25.6 mmol/L和12.8 mmol/L;链球菌12.8 mmol/L;大肠杆菌和标准大肠杆菌25.6 mmol/L和12.8 mmol/L;在7 h内能有效杀灭这三种菌,肉桂醛对奶牛乳房炎主要致病菌有抑菌和杀菌能力,是一种潜在的奶牛乳房炎的临床治疗药物。     

苦荞麦中芦丁提取及体外抑菌活性研究

采用乙醇回流法提取苦荞麦中的芦丁,并进行TLC检测、紫外光谱和红外光谱检测。将芦丁对两种供试菌进行体外最小抑制浓度(MIC)、最低杀菌浓度(MBC)、半数抑菌浓度(IC50)测定。结果表明,其对大肠埃希氏杆菌、金黄色葡萄球菌的MIC分别为0.40mg/mL和0.20mg/mL;MBC分别为0.50mg/mL和0.20mg/mL;IC50分别为0.2605mg/mL和0.1607mg/mL。     

四种“爱丽丝”牌消毒剂对五种细菌的杀菌效果比较

采用定量悬浮试验法,选择大肠杆菌、鼠伤寒沙门氏菌、多杀性巴氏杆菌、金黄色葡萄球菌和无毒炭疽芽胞杆菌等菌株,对四种未知成份（５％浓度）的消毒剂进行了杀菌效果比较。结果表明,Ⅰ号消毒剂对各菌的杀灭效果均很差,Ⅱ号对沙门氏菌和金黄色葡萄球菌的杀灭效果最好,Ⅲ号对多杀性巴氏杆菌的杀灭效果最好,Ⅳ号对大肠杆菌和炭疽杆菌的杀菌效果最好。比较而言,Ⅱ号消毒剂的杀菌效果最好,Ⅲ号、Ⅳ号次之。     

甲氧苄啶对蒲公英体外抗菌增效作用的研究

采用微量棋盘稀释法测定了蒲公英、甲氧苄啶及两药联用对金黄色葡萄球菌、大肠杆菌和沙门氏菌的最小抑菌浓度(MIC),计算蒲公英与甲氧苄啶联用的组分抑菌浓度(FIC)指数。结果表明,蒲公英与甲氧苄啶联用对金黄色葡萄球菌、大肠杆菌和沙门氏菌的FIC指数分别为0.75、0.75和1,呈相加作用。采用平板计数的方法,测定了蒲公英与甲氧苄啶联用作用于3种细菌1、2、4和8 h后的细菌数,并计算杀菌率。利用最小二乘法对杀菌率进行拟合,计算出甲氧苄啶增强蒲公英体外抗金黄色葡萄球菌、大肠杆菌和沙门氏菌作用的最优添加剂量分别为4.0、1.8和2.0 mg/g。