【作者】 曹立亭；

【导师】 胡松华；

【作者基本信息】 浙江大学 ， 预防兽医学， 2009， 博士

【摘要】 尽管国内外学者对奶牛乳房炎进行了大量研究,但该病仍然是引起奶牛养殖业经济损失最为严重的疾病之一。目前,治疗奶牛乳房炎的常用方法是乳房内灌注抗生素。随着抗生素大量、广泛应用,乳房炎致病菌的耐药性越来越严重,临床治疗效果越来越不理想。用抗菌药物治疗奶牛乳房炎容易造成药物在牛奶中的残留,影响乳品质量,给奶牛养殖业和乳品加工业造成巨大的经济损失；同时,残留在牛奶及乳制品中的抗生素还对人类健康造成威胁。因此,研制无有害残留的非抗生素制剂成为奶牛乳房炎研究方面的一个热点。此外,除了细菌的耐药性之外,很少有学者从细菌和药物在乳房内的分布探讨影响药物疗效的因素。本论文首先对乳房炎主要致病菌和常用抗菌药物在牛奶中的分布进行研究,探讨影响抗菌药物疗效的因素；然后系统研究了Nisin Z对奶牛临床型乳房炎自然发病病例的治疗效果。1.乳房炎主要致病菌在牛奶中的分布研究为观察乳房炎主要致病菌在牛奶中的分布情况,首先对一次挤奶过程不同时段挤出牛奶的主要乳成份进行了分析。选取健康泌乳盛期荷斯坦奶牛4头,于挤奶前期、挤奶中期和挤奶末期无菌操作采集乳区牛奶进行乳成份分析。结果表明,随着挤奶过程的进行,乳脂肪含量显著升高(P<0.05),挤奶末期最高；乳蛋白、非脂乳固体和乳糖含量先升后降,挤奶末期最低,与挤奶前期和中期相比差异显著(P<0.05)。牛奶中添加一定量的乳房炎致病菌金黄色葡萄球菌或大肠杆菌,4℃静置8、12、24 h后分割成上、中、下三等份,采用平板菌落计数法测定各部分牛奶中的细菌数。结果发现,两种致病菌在牛奶中的菌落数从下部到上部呈现上升趋势,且上部牛奶中的菌落数显著高于中、下部(P<0.001),而中、下部之间差异不显著(P>0.05)。从本研究结果推测,乳房炎致病菌经乳头管侵入后可以随牛奶中的脂肪滴上浮,侵入乳腺的深层组织。2.常用抗菌药物在牛奶中的分布研究在牛奶中添加一定浓度的常用抗菌药物,4℃静置8、12、24h后分割成上中、下三等份,采用琼脂扩散法对不同部位牛奶中抗菌药物对指示菌的抑菌作用进行分析比较。结果显示：大部分抗菌药物在牛奶中的抑菌作用从牛奶上部到下部逐渐增强,其中青霉素、庆大霉素、盐酸林可霉素和氨苄西林钠4种极性较强的抗生素三部分之间变化明显；抗菌多肽Nisin Z在牛奶中呈现较好的分布趋势,上、中、下三部分牛奶中Nisin Z的抑菌作用基本一致,且上部牛奶的抑菌作用略高于中、下部,提示Nisin Z可分布于乳腺的深部组织,适于制成乳房内灌注制剂。3. Nisin Z对金黄色葡萄球菌的体外药敏试验从临床型乳房炎病乳中分离到的37株金黄色葡萄球菌,仅有6株(16.2%)对青霉素G敏感,22株(59.5%)完全耐药；6株(16.2%)对庆大霉素耐药；而所有菌株均对Nisin Z敏感。4. Nisin乳房灌注剂对奶牛临床型乳房炎的治疗效果观察92头临床型乳房炎自然发病病例(107个患病乳区)随机分为试验组(48头,51个乳区)和对照组(44头,56个乳区),试验组乳房内灌注含Nisin Z2,500,000IU的Nisin乳房灌注剂,对照组乳房内灌注含量为0.8g的庆大霉素注射液,用量均为2次/d。通过测定治疗前和停药后第1、2周患病乳区细菌学变化、体细胞数以及酸奶发酵情况,确定Nisin乳房灌注剂对奶牛临床型乳房炎的治疗效果。试验结果表明,两组的临床治愈率基本一致(90.2%和91.1%),但Nisin乳房灌注剂治疗组的微生物治愈率高于庆大霉素治疗组,分别为60.8%和44.6%,统计学比较差异不显著(P>0.05). Nisin乳房灌注剂治愈后第1、2周,分别有50.0%和47.8%的患病乳区SCC恢复到500,000个/mL以下,略高于庆大霉素治疗组的33.3%和37.3%。患病乳区的细菌学分析发现,主要致病菌为无乳链球菌和金黄色葡萄球菌,分别占总发病乳区的37.4%和15.9%；其次是凝固酶阴性葡萄球菌(CNS),占总发病乳区的9.3%。Nisin乳房灌注剂治疗后第2周,金黄色葡萄球菌感染乳区转阴率为54.5%(6对11),而庆大霉素治疗组仅为33.3%(2对6)；无乳链球菌感染乳区,两组转阴率分别为83.3%(15对18)和50.0%(11对22)。Nisin乳房灌注剂治疗病例,在停药后12h用药乳区牛奶中NisinZ效价仅为4.5 IU/mL,远低于国家标准规定的乳制品中Nisin的最大添加量(0.5g/kg,约合500 IU/mL);用药乳区牛奶酸奶发酵过程受到轻度抑制,并持续到停药后36 h,非用药乳区混合牛奶在整个采样时间内发酵正常。庆大霉素治疗病例,用药乳区牛奶在停药后60h内发酵过程受到严重抑制,停药后72h发酵恢复正常,非用药乳区混合牛奶在停药后60h内酸奶发酵过程也受到不同程度的抑制。上述结果表明,Nisin乳房灌注剂对奶牛临床型乳房炎具有较好的治疗效果,仅在短时间内影响酸奶的发酵。5. ECLIPSE 50对Nisin乳房灌注剂治疗后牛奶中Nisin Z残留检测ECLIPSE 50是一种广泛用于检测牛奶中抗生素和抑制剂的试剂盒。为探讨ECLIPSE 50对牛奶中Nisin Z残留检测的可行性,在无抗奶中添加Nisin标准品,测定ECLIPSE 50对牛奶中Nisin的检测极限,并对Nisin乳房灌注剂治疗乳房炎病例牛奶中的Nisin Z残留进行了分析。试验结果表明：ECLIPSE 50对牛奶中Nisin的检测极限为5×10-4-5×10-3mg/mL,折合成Nisin效价为0.5-5IU/mL,即牛奶中允许Nisin的最大残留量为5 IU/mL。Nisin乳房灌注剂治疗后8h,灌注乳区牛奶ECLIPSE 50检测阳性,无抗奶稀释5,000倍后检测阴性；非用药乳区混合牛奶ECLIPSE 50阴性。本研究结果提示,ECLIPSE 50可用于牛奶中Nisin残留的检测。6. Nisin乳房灌注剂治疗临床型乳房炎后对奶牛泌乳性能的影响选取临床型乳房炎自然发病病例9头,乳房内灌注Nisin乳房灌注剂进行治疗,通过检测日产奶量以及停药后2、7、14、21d的主要乳成份指标,确定Nisin乳房灌注剂对临床型乳房炎病例泌乳性能的影响情况。结果显示,患病乳区乳腺组织得到一定程度的修复,日产奶量逐渐恢复,停药后第8d的日产奶量与发病前第6d相比,平均降幅2.5kg。治愈后患病乳区牛奶乳脂肪、乳蛋白、乳糖和非脂乳固体含量都呈现上升趋势,但与同期相比,略低于非用药乳区混合牛奶；其中,乳糖含量增加幅度较快,除停药后第2d的治疗乳区外,乳糖含量均在4.7%以上,提示奶牛乳房炎病例经Nisin乳房灌注剂治疗后,受损乳腺组织修复较快,乳腺上皮细胞合成乳成份的能力大幅度提高。综上所述,乳房炎主要致病菌和常用抗菌药物在牛奶中分别向上下两极移动,这种分布上的分离现象导致抗菌药物和致病菌不能充分接触可能是导致临床上乳房炎治疗失败的一个重要原因；而NisinZ在牛奶中具有较好的分布趋势,可研制成乳房内灌注制剂用于治疗奶牛乳房炎。Nisin Z体外可有效抑制奶牛乳房炎主要致病菌金黄色葡萄球菌的生长繁殖,即使对常规抗生素耐药菌株也有较好的抑菌效果。用Nisin乳房灌注剂治疗奶牛临床型乳房炎,微生物治愈率为60.8%,略高于常规抗生素治疗组(44.6%). Nisin乳房灌注剂治愈病例感染乳区体细胞数明显下降,牛奶中乳脂肪、蛋白质、乳糖和非脂乳固体含量呈现上升趋势,停药36h后乳区牛奶酸奶发酵正常。更多还原

【Abstract】 Despite the fact that much research and effort has been dedicated to mastitis prevention and treatment for so many years, bovine mastitis is one of the most prevalent and costly diseases in dairy industries around the world. At present, intramammary administration of antimicrobial drugs is the main approach used for treatment of bovine mastitis. However, poor responses to antibiotic treatments have become very common in veterinary practice, because antimicrobial resistance of mastitis pathogens increased following so many kinds of antibiotics widely used. And the extensive use of antibiotics on dairy farm may cause antibiotic residues in milk which often brought great economic losses to dairy industry because of bad quality of milk products. Antibiotic residues in milk also have the potential threats to consumers’health. Therefore, it was necessary to develop a non-antibiotic preparation for the treatment of bovine mastitis, which was not harmful to public health. This study was designed to analyze the distribution of major mastitis-causing bacteria and common antimicrobial drugs in milk in vitro. And then therapeutic efficiency of nisin-based formulation in the treatment of clinical mastitis in lactating dairy cows was analyzed.1. Distribution of major mastitis-causing bacteria in milkTo investigate the distribution of major mastitis-causing bacteria in milk, the variations in major constituents of milk during milking in healthy dairy cows were analyzed. Quarter milk samples were collected from 4 Hostein dairy cows during the first, middle and last period of milking and their major constituents were analyzed. Results indicated that milk fat concentration gradually increased during the progress of milking, and was significantly higher in the milk collected during the last period than those collected during other periods of milking (P＜0.05). The concentration of milk protein, solids-not-fat and lactose had a rise trend at first, and then declined. There ware significant differences in the milk collected between the last period and other periods of milking (P＜0.05). After adding a certain amount of Staphylococcus aureus or Escherichia coli in milk and standing for 8,12,24 h at 4℃, Sta. aureus and E. coli were found largely in upper portion of milk, and the colony forming units (CFU)/mL of both isolates were significantly higher than those in middle and lower portions (P<0.001). However, there was no difference between the middle and lower portions (P>0.05). It is suggested that bacteria getting entrance into teat canal may float up through attaching to fat globes and invade the deep mammary tissues.2. Distribution of common antimicrobial drugs in milkIn order to analyzed the distribution of common antimicrobial drugs in mammary gland tissues after intramammary infusion, the distribution of common antibiotics and nisin Z in milk in vitro was investigated by measuring their inhibitory effect on indicator bacteria, after adding a certain concentration of common drugs and standing for 8,12,24 h at 4℃. The majority of all selected antimicrobial drugs were found to have higher inhibitory activities in lower portion of milk than in middle and upper portions. Four antibiotics including penicillin, gentamicin, lincomycin and ampicillin which had higher polarity had distinct differences among 3 parts. Nisin Z was distributed more evenly but a little higher concentration in the upper portion, suggesting that nisin Z have a good distribution in mammary tissues and be suitable to make an intramammary preparation.3. Inhibitory effects of nisin Z on Sta. aureus in vitroOf 37 Sta. aureus strains isolated from bovine clinical mastitis,83.8% were resistant to penicillin, and 16.2% to gentamicin (GM), but none to nisin Z.4. Efficacy of nisin-based formulation in treatment of clinical mastitis in lactating dairy cowsA total of 92 cows with 107 naturally occurring clinical mastitic quarters were randomly assigned to nisin-based formulation (48 cows with 51 quarters) and GM (44 cows with 56 quarters) treated groups. In nisin-based formulation treated group, cows received an intramammary infusion of nisin Z at a dose of 2,500,000 IU, in the GM-treated group, intramammary infusion of GM was administrated at a dose of 0.8 g. The treatment was performed twice daily (administrating after morning and afternoon milkings) until the inflammatory signs disappeared and mammary secretion became visibly normal. The treatments were repeated 4.1±0.2 and 6.1±0.3 times in groups of nisin-based formulation and GM, respectively. Nisin-based formulation offered a clinical cure rate similar to GM (90.2% vs.91.1%) and no difference in a higher bacteriological cure rate than GM-treated group (60.8% vs.44.6%, respectively). Proportions of the quarters with milk somatic cell counts lower than 500,000 cell/mL were no differences in nisin-based formulation treated group (50.0% and 47.8%) compared with GM treated group (33.3% and 37.3%) 1 and 2 wk after treatment. Of the bacteria isolated from all the clinical mastitis cases, Streptococcus agalactiae (Str. agalactiae) and Sta. aureus were the predominant causative pathogens, being isolated from 37.4% and 15.9% of 107 cases, respectively. Coagulase-negative staphylococci seemed less important and accounted for 9.3% of all cases. Nisin-based formulation therapy eliminated 54.5% (6 of 11) of Sta. aureus intramammary infection (IMI), whereas GM eliminated 33.3% (2 of 6). For Str. agalactiae mastitis, 83.3% (15 of 18) and 50.0%(11 of 22) became bacteriologically negative in the cases treated by intramammary infusion of nisin-base formulation and GM, respectively. Nisin Z in milk (4.5±0.8 IU/mL) was detected only in the treated quarters at 12 h following intramammary infusion, which was much lower than the upper limit (0.5 g/kg, equivalent to 500 IU/mL) allowed as preservative in milk products by the China authority. Reduced fermentation with low acidity from nisin-based formulation treated cows with clinical mastitis was found in milk collected from the treated quarters until 36 h following the last intramammary infusion, whereas no inhibition was detected in fermentation of the milk from untreated quarters throughout the experiment. But for GM treated cows, the inhibited fermentation was found in milk collected from treated and untreated quarters until 72 h following the last intramammary infusion, with more inhibitory activity detected in the milk from the treated quarters. All data indicated that nisin-based formulation has a better therapeutic effect for bovine mastitis and reduced fermentation was found in a short time after treatment.5. ECLIPSE 50 test for the detection of nisin Z in milk after nisin-based formulation treatmentECLIPSE 50 is a test for the detection of inhibitors and antibiotics in milk. To analyze the feasibility of this kit for detecting nisin Z residue in milk, detection limit of this kit for standard nisin in milk was established. Then ECLIPSE 50 test was used to detect nisin Z in milk samples collected from nisin-based formulation treated cows. Results indicated that the detectable limit of ECLIPSE 50 test for nisin in cow’s milk was 5×10-4-5×10-3 mg/mL (equal to 0.5-5 IU/mL). The maximum residue limit (MLR) of nisin in milk was 5 IU/mL. Milk samples collected from all the treated quarters were positive at 8 h after receiving an intramammary infusion of nisin-based formulation at a dose of 2,500,000 IU. After diluted 1:5000 with non-antibiotic milk, ECLIPSE 50 test became negative. For the milk collected from untreated quarters, ECLIPSE 50 test was negative throughout the experiment. Therefore, ECLIPSE 50 can be used for the detection of nisin Z in milk.6. Effect of nisin-based formulation treatment on lactating performance of clinical mastitic cowsNine dairy cows with clinical mastitis were selected and intramammary administrated 6 times with nisin-based formulation. Daily milk production and major milk constituents were analyzed to evaluate the effect of nisin-based formulation treatment on lactating performance. Daily milk productions of all the mastitic cases gradually recovered, but the milk production at 8 d after treatment decreased by an average of 2.5 kg compared with 6 d before infection. Milk fat, protein, lactose and solids-non-fat contents of milk collected from treated quarters increased after intramammary infusion of nisin-based formulation, but they were lower than those from untreated quarters (composite milk samples). The concentration of milk lactose was above 4.7% in all treated quarters except for 2 d after intramammary infusion, suggesting that mammary tissues damage may recover quickly and the synthesis of epithelia cells increase in large extent.In summary, major mastitis-causing bacteria and common antibacterial drugs may not contact each other sufficiently due to their contra-direct movements in mammary gland, resulting in poor responses of mastitic cases to antibacterial therapy. Nisin Z which was more efficient against Sta. aureus isolated from clinical mastitis in vitro was distributed more evenly in milk, suggesting that it be suitable to make an intramammary preparation. Nisin-based formulation had better therapeutic effects on bovine clinical mastitis in vivo with the bacteriological cure rate of 60.8% (Gentamicin 44.6%). Quarter milk SCC gradually declined and lactating performance including daily milk production and major constituents recovered more quickly after nisin-based formulation administrated. Small amounts of nisin Z residue and reduced fermentation were found in milk collected from treated quarters in short time after treatment. Nisin-based formulation will be widely used as a new medicine to treat bovine mastitis, as well as its safety in humans.更多还原