【作者】 吴旧生；

【导师】 周继勇； 胡松华；

【作者基本信息】 浙江大学 ， 微生物学， 2007， 博士

【摘要】 本文从奶牛隐性乳腺炎的流行病学、影响纸片保存奶样中乳糖、乳蛋白等生化指标测定的因素、纸片保存奶样中无乳链球菌、金黄色葡萄球菌的PCR检测方法及其适用性进行了探索、最后对牛奶蛋白的SDS-PAGE电泳在隐性乳腺炎诊断的应用作了探讨，并对各种方法与标准诊断方法进行了比较分析。对杭州等地4388份奶样体细胞计数值与季节、奶产量、泌乳周期、乳脂和脂蛋白等因素的关系进行了调查，利用限制性最大似然估计(REML)模型进行了分析，结果表明采样月份(F=8.83，p＜0.01)、日奶产量(F=212.12，p＜0.01)、胎次(F=16.86，p＜0.01)、乳脂率(F=71.87，p＜0.01)、乳蛋白(F=78.92，p＜0.01)等因素对奶样体细胞数均有显著影响。利用混合线性模型的最大似然估计(REML)方法建立了体细胞计数响应模型，该模型可以有效地预测体细胞数的变化规律。在此基础上利用Logistic回归模型分析了采样月份、胎次、泌乳周期和日产奶量等因素对隐性乳腺炎发病率的影响程度，建立了相应的预测模型，经共线性检验，模型预测值与原始数据吻合良好(p＞0.05)模型分析提示，每年的8-9月份体细胞水平显著升高(p＜0.05)。日产奶量在20-30kg，乳脂率在3.5-5.0％，蛋白含量在2.5-3.1％时奶牛隐性乳腺炎发病机率最低。纸片保存奶样中乳糖含量的变异系数略大于液态奶样，但乳糖水平无显著差异(t=0.389，p＞0.05)。保存期(F=1.004，p＞0.05)、保存温度(F=0.004，p＞0.05)对纸片保存奶样的乳糖水平没有显著影响，其乳糖含量仅受奶样本身体细胞数的影响(F=162.42，p＜0.01)。提示纸片保存奶样适用于进行牛奶乳糖指标的测定。纸片保存奶样的蛋白含量约为液态奶样的83.6％，其变异系数为4.41％。保存时间和预处理介质对纸片保存奶样蛋白含量有显著影响(p＜0.01)，并受牛奶体细胞数的影响(p＜0.05)。保存温度对其影响不显著(p=0.052)。保存时间和预处理介质对纸片保存奶样的蛋白水平有协同影响(p＜0.01)。初始值和保存1、2、4周的样品间均有统计学显著差异(p＜0.05)；随着时间的延长，样品中蛋白含量逐渐下降，DDW、Tris盐酸缓冲液预处理对纸片保存奶样蛋白水平均有显著影响，EDTA对测定结果没有显著影响(p＞0.05)。提示纸片保存奶样蛋白质含量稍有下降，但在1-4周内可以保持相对稳定。对人工污染无乳链球菌的纸片奶样进行复苏培养，结果表明，纸片预处理介质(p＜0.01)和保存期(p＜0.01)对纸片保存无乳链球菌复苏率有显著影响，而保存温度对结果的影响较小(p＞0.05)。纸片保存温度(p＜0.05)、预处理方法(p＜0.01)和保存期(p＜0.01)对金黄色葡萄球菌的培养成功率有显著影响。针对无乳链球菌16-23S rRNA基因间区序列的引物，可扩增出唯一的270bp的PCR产物，而对人工污染的金黄色葡萄球菌、停乳链球菌、乳房链球菌和大肠杆菌的液态奶和纸片保存样品均无扩增产物形成。该引物对无乳链球菌的PCR检测灵敏度为2×10~2cfu／ml，与液态奶的检出结果相似。预处理方法对PCR结果没有显著影响，所有处理均可获得满意的PCR结果。以细菌培养为参照，PCR诊断的敏感性为96.15％，特异性为98.60％，准确率为98.06％，可以有效地从纸片保存样品中检出无乳链球菌。金黄色葡萄球菌引物SAISR1和SAISR2可分别从纸片保存样品中扩增出约500bp和250bp的特异性PCR产物，而对人工污染无乳链球菌等奶牛隐性乳腺炎主要病原无特异性扩增。SAISR1和SAISR2对纸片样品的检测灵敏度分别为2×10~4CFU／mL和2×10~3CFU／mL；若采用聚丙烯酰胺凝胶电泳结合硝酸银染色，SAISR2的灵敏度可提高至2×10~1CFU／mL。纸片预处理介质对PCR结果没有显著影响。以细菌为标准，SAISR2的灵敏度为89.19％，特异性为98.08％，诊断准确率96.37％，可有效地从纸片保存奶样中检出金黄色葡萄球菌。利用SDS-PAGE研究了纸片保存奶样乳蛋白成分变化，分子量较小的成分得到较好的分离(R_f＞0.2)，呈现为独立的条带群。分子量较大的成分滞留在上样孔附近，迁移率很小(R_f＜0.2)。迁移率较大的成分主要形成R_f值在0.51(A)，0.62(B)，0.72(C)，0.78(D)，0.84(E)和0.98(F)等6个条带群，其中C、D和E三个条带群，可因奶样不同而呈现不同的亮度或缺少，经与HMT、SCC和主要病原菌分离培养等试验结果联合分析，C、D、E三个条带群与SCC(r=0.795，p＜0.01)、HMT(r=0.471，p＜0.01)和主要病原菌的存在(r=0.389，p＜0.01)有显著相关。与细菌分离培养对比，SDS-PAGE方法对隐性乳腺炎诊断的灵敏度为100％，特异性为55.56％，若将分离到凝固酶阴性葡萄球菌(CNS)也计入病原菌，则SDS-PAGE的灵敏度为90％，特异性为70.59％，与细菌培养的结果高度一致(kappa=0.622，p＜0.05)，PAGE方法比常用的HMT方法具有更好的灵敏度和特异性，与SCC法相当。综上所述，纸片保存奶样的隐性乳腺炎诊断可以采用乳糖、蛋白成分分析和PCR检测等方法进行，其中以PCR方法的特异性较高，而以蛋白质成分分析的灵敏度最高。更多还原

【Abstract】 Filter paper was widely used to collect and store liquid biological samples for its portable, safety and economic properties. This study was conducted in order to systematically evaluate the suitability, sensitivity and specificity of milk samples collected on filter paper disks. The filter paper disks (5.0 mm in diameter) were made from Xinhua No.3 Chromatographic filter paper. Each filter paper disks can absorb 24.99±0.05μL milk samples. A series diagnosis protocols based on lactose, protein and bacteria information of milk samples collected on filter paper disks were proposed.A regional investigation was conducted in Hangzhou, China to estimate the prevalence of bovine mastitis (BM) and its association with the seasons, parity, period postpartum, milk fat, milk protein and milk production. A total of 4388 milk samples were analyzed for the SCC. A linear mixed model with restricted maximum likelihood (REML) was applied to analyse the association between somatic cell counts (SCC) with the factors mentioned above. The results presented that month (p < 0.01), parity (p < 0.01), milk production (p < 0.01), milk fat (p = 0.049), and milk protein (p < 0.01) show significantly associated with the SCC. A SCC response model was established. It is turned out an efficient model tested with a colineartic diagnostics. The predicted values by it were similar to the origin (p > 0.05). A logistic regression analysis demonstrated that the factors (month, parity, milk fat, milk protein, milk production) including in this survey contribute to the risk of mastitis in Holstein cows (p < 0.05). Based on these analysis, the related risk estimation models were worked out. The models revealed that in Aug and Sept the cows are prone to develop mastitis, while in the suitable milk production (in 20-30 kg/d), milk fat(in 3.5-5.0%) and milk protein(2.6-3.1%) patterns, the mastitic risk are very low.To evaluate the influence of time and temperature for storage on the lactose of milk samples on filter paper disks, a DNS assay was employed to analyze lactose concentration under different stored conditions. The results indicated that intra-sample reproducibility of lactose concentration in the milk samples collected on filter papers from the same individual was found to be 13.6 percent, while for the tube sampling milk was 4.7 percent. There was no significant difference between results obtained from tube sampling milk and filter paper disk collected milk (p > 0.05) . Storage at different temperatures (p > 0.05) and for various periods (p > 0.05) showed little change in lactose concentration of milk sample preserved in filter paper disks. There was a little decrease in concentrations of lactose in milk on the filter paper when compared to tubes collected milk and a high standard deviation probably because of loss in the extraction process. Milk collected on filter paper disks could be used to screen subclinical mastitis cases using milk lactose as an indicator.Bradford protein assay was employed to evaluate the accuracy, suitability and coefficient of variation of the milk protein collected on filter paper disks. The intra-sample coefficient of variation (CV) was 4.41%. The protein level significantly decreased in comparison with the results of raw milks and only 83.6% protein could be recovered. The results presented that the protein concentration was significantly changed by time for storage and pretreatment with media. The protein levels on filter paper disks gradually decreased with the time increasing. The results presented that the filter paper disks pretreated with different media give a great variation in the protein level of milk samples kept on it.To acquire the viable Strep. agalactiae and Staph. aureus and PCR to use artificially contaminated milk samples collected on filter paper disks, milk samples to analyze were dried and kept one to four weeks at room temperature. After that, they were cultured for PCR analysis. The results revealed that the recovery rate of Strep. agalactiae and Staph. aureus on dried filter paper disks was influenced by the pretreatment with media and storage period. The recovery test suggested that not all the bacteria artificially contaminated milk samples on the filter paper disks could be recovery. The recovery rate was 86.6% and 83.5%, respectively. After that, a PCR based on 16-23S intergenic spacer region of Strep agalactiae was performed. The methods can distinguish the Strep. agalactiae from major pathogens of bovine mastitis at a 2×10~3 colony forming units ( CFU)/mL level, which show similar sensitivity to the liquid milk samples. It also presented that the milk samples collected on filter paper disks could be kept at room temperature for one to four weeks and had little negative effect on its sensitivity and specificity. The field test showed that the sensitivity and specificity of diagnosis was 96.15% and 98.60% respectively. The primers SAISR1 and SAISR2 based on 16-23S intergenic region can distinguish the Staph. aureus from major pathogens of bovine mastitis at 2×10~4 and 2×10~3 CFU/mL level, which show similar sensitivity to the method by liquid milk samples, respectively. It also presented that the milk samples collected on filter paper disks can be kept at room temperature for one to four weeks and have little negative effect on its sensitivity and specificity. The field test based on 193 milk samples showed that the sensitivity and specificity of SAISR2 was 89.19% and 98.08%, respectively. A Kappa analysis presented that PCR method based on milk samples collected on filter paper disks was in good consistent with the bacterial culture in vitro based on correspondent liquid milk. Thus, there can be no doubt that the PCR protocol based on milk samples collected paper disks will be a rapid and economic procedure for the detection of bovine mastitis.SDS-PAGE based on milk samples collected on filter paper disks were conducted to evaluate the modification of milk protein components under subclinical mastitis. The results revealed that the protein with small MW can be good separated (R_f > 0.2), while the molecules with large MW presented low R_f and near the loading portion. The bands with a value of R_f>0.2 formed six main band groups in the PAGE patterns: 0.51(A), 0.62(B) , 0.72(C) , 0.78(D) , 0.84(E) and 0.98(F) . Band groups A, B and F presented no significant correlation with the SCC and major pathogens, while the band groups C, D and E show marked correlation with SCC(r = 0.795, p < 0.01), HMT(r = 0.471, p < 0.01) and the major pathogens(r = 0.389, p < 0.01). The sensitivity and specificity of PAGE was 100% and 55.66% respectively. The PAGE methods is in a moderate consistent with pathogens culture (kappa=0.515, p < 0.05). If the CNS was included in the bacterial culture results, the sensitivity and specificity was 90% and 70.59% respectively, with a Kappa value of 0.622 (p < 0.05). PAGE method showed similar sensitivity and specificity with SCC and more sensitive than HMT in the diagnoses of subclinical bovine mastitis.As described above, lactose level, PCR on major pathogens and PAGE of protein could be used to detect BM using milk samples collected on filter paper disks. In addition, the specificity of PCR and the sensitivity of PAGE were discussed.更多还原