【作者】 吴俊强；

【导师】 胡松华；

【作者基本信息】 浙江大学 ， 预防兽医学， 2010， 博士

【摘要】 乳腺炎是奶牛最常见的疾病之一,发病率高,造成奶牛业巨大经济损失。奶牛隐性乳腺炎没有明显的临床症状,表现为牛奶体细胞数升高,产奶量下降,牛奶的成份发生改变,从而影响牛奶的品质。若不及时治疗或治疗不当会发展为临床型乳腺炎,甚至引起瞎奶头,严重时导致奶牛淘汰。由于发病率高和牛奶抗生素残留等问题,隐性乳腺炎一般不治疗。当因隐性乳腺炎使桶奶中的体细胞含量大幅度升高而影响牛奶出售时,仍然要采用抗生素进行治疗。但抗生素治疗易引起奶牛乳腺炎致病菌株产生抗药性、造成牛奶中药物残留及奶牛体内药物蓄积,从而危害人类健康。因此,研制高效、低毒以及无有害残留的新型抗菌药物成为奶牛隐性乳腺炎研究的热点。本论文首先对某奶牛场隐性乳腺炎的病菌进行调查,然后研究了Nisin对奶牛乳腺的刺激性、对奶牛隐性乳腺炎尤其是由金黄色葡萄球菌引起病例的治疗效果。1.杭州近郊某奶牛场隐性乳腺炎主要病原菌的调查为了解杭州近郊某奶牛场奶牛隐性乳腺炎的主要病原菌,首先用杭州乳腺炎快速诊断试剂(HMT)对该牧场的两个牛棚进行隐性乳腺炎检测,采集检测结果为“+++”强阳性乳区奶样89个进行细菌学检验,并对分离的主要病原菌进行药敏试验以及牛奶中SCC和NAGase酶活性的测定。结果显示：在该牧场分离的奶牛隐性乳腺炎的主要病原菌为金黄色葡萄球菌(25.0%)、无乳链球菌(34.7%)和凝固酶阴性葡萄球菌(31.9%)。金黄色葡萄球菌对青霉素G、氨苄西林和苯唑青霉素均有较高的耐药率(88.8%,83.3%和72.2%)；无乳链球菌对以上常规的抗生素保持着较高的敏感性；而凝固酶阴性葡萄球菌对青霉素G、氨苄西林、苯唑青霉素和链霉素均有较高的耐药率(86.4%,63.6%,77.3%和68.2%)。因此,该场引起奶牛隐性乳腺炎的主要致病菌为金黄色葡萄球菌、无乳链球菌以及凝固酶阴性葡萄球菌,葡萄球菌对青霉素类药物已经产生耐药性。2. Nisin Z对奶牛乳腺的刺激性Nisin作为治疗奶牛隐性乳腺炎的药物在应用之前,必须要对其进行药物刺激性试验,以便筛选出治疗奶牛隐性乳腺炎的最佳剂量。本试验选择1头处于泌乳中期且四个乳区均健康的荷斯坦奶牛,分别将三个不同的剂量组(5.0×106IU、2.5×106IU、1.25×106IU)的Nisin制剂于挤奶结束后分别灌注于右前、右后以及左后乳区,左前乳区灌注250mL生理盐水作为对照组,然后在灌注结束后24h、48h、72h、96h以及1周时进行SCC及NAGase酶活性的检测。试验结果表明,除了5.0×106IU剂量组有明显的临床症状外,其他剂量组与对照组均无明显的临床症状。灌注三种不同剂量的药物后,三个灌注乳区牛奶体细胞数在24h时均明显升高,但是24h后又逐渐下降,至1周时恢复正常水平,而对照组乳区则无显著变化。牛奶中NAGase酶活性检测结果表明,灌注药物后24h后,两个高剂量组(5.0×106IU、2.5×106IU) NAGase酶活性均升高,然后在24～72h保持稳定,随后NAGase酶活性恢复至正常水平；而1.25×106IU剂量组与对照组在灌注后24h、48h与72h时,NAGase酶活性则略微升高,随后便恢复至正常水平。上述结果表明,2.5×106IU与1.25×106IU对奶牛乳腺的刺激性最小,结合前期Nisin乳房灌注剂治疗奶牛隐性乳腺炎的抑菌效果,选择2.5×106IU作为治疗奶牛隐性乳腺炎的治疗剂量。3. Nisin Z乳房灌注剂对奶牛隐性乳腺炎治疗效果90头泌乳中期其患隐性乳腺炎的荷斯坦奶牛(90个患病乳区),被随机分成试验组(46个患病乳区),对照组(44个患病乳区)。试验组奶牛乳腺内灌注Nisin乳房灌注剂(2.5×106IU),每天一次,连续三天,对照组则按照牧场常规不采取治疗。通过检测治疗前后HMT变化、细菌学检查、牛奶体细胞计数、以及牛奶中NAGase酶活性变化,确定Nisin乳房灌注剂对奶牛隐性乳腺炎的治疗效果。细菌学检查结果可以看出,患病乳区分离的主要病原菌为金黄色葡萄球菌(27.8%)、凝固酶阴性葡萄球菌(31.1%)和无乳链球菌(33.3%)；经过Nisin治疗后第2周后,试验组与对照组细菌学转阴率分别为65.2%和15.9%,统计学差异极显著(P<0.01)；其中各试验组与对照组主要致病菌在治疗后第2周其细菌学转阴率分别为：金黄色葡萄球菌为50.0%和18.2%,无乳链球菌为90.9%和1.40%,凝固酶阴性葡萄球菌为58.8%和27.3%,两组细菌学转阴率同期比较,金黄色葡萄球菌与凝固酶阴性葡萄球菌均无统计学差异,而无乳链球菌则差异极显著(P<0.01); NAGase酶活性检测结果表明,试验组在经Nisin乳房灌注剂治疗第1、2周后,牛奶中在NAGase酶活性均显著减低(P<0.01),而对照组则无显著差异；牛奶体细胞计数结果显示,试验组在治疗后第1、2周牛奶中体细胞≤5.0×105个／mL的百分比为41.3%和32.6%,而对照组则分别为18.2%和9.1%,两组同期比较差异均极显著(P<0.01)。因此,Nisin乳房灌注剂治疗奶牛隐性乳腺炎具有良好的效果。4.Nisin Z治疗奶牛隐性乳腺炎降低桶奶体细胞含量本试验在安徽试验基地选择两个牛棚,其中试验组牛棚含85头,对照组牛棚含90头,选择的奶牛均为处于泌乳中期的荷斯坦奶牛。3～7月对两个牛棚进行如下内容检查：桶奶体细胞数、桶奶乳成份、桶奶细菌数。8～12月用Nisin乳房灌注剂(2.5x106IU)对试验组中隐性乳腺炎病例进行治疗,每天一次,连续三天,对照组则按照牧场常规不采取治疗。然后每个月分别检测桶奶体细胞数、桶奶细菌数、桶奶中乳成份以及利用ECLIPSE50检测桶奶中Nisin Z的残留,以确定Nisin乳房灌注剂是否能够降低桶奶体细胞数。试验组隐性乳腺炎病例采取治疗措施后,桶奶体细胞数8～12月份均显著低于对照组(P<0.05)；桶奶细菌数显著低于对照组(P<0.05)；桶奶中蛋白质、脂肪、乳糖与非脂质固体的含量均高于对照组。当试验牛群用Nisin灌注剂同时治疗隐性乳房炎病例少于10个时,桶奶ECLIPSE50法试验为阴性。因此,Nisin乳房灌注剂对牛群中奶牛隐性乳腺炎病例治疗后能够有效的降低桶奶体细胞数。5. Nisin Z治疗奶牛隐性型乳腺炎后在牛奶中的残留本试验通过选择3头泌乳中期患隐性乳腺炎的奶牛,用Nisin乳房灌注剂(2.50×106IU)治疗患病乳区,每天一次,连续三天；未治疗的相邻乳区作为对照。治疗结束后24h、48h、72h与96h后利用国标法检测治疗乳区与对照乳区的Nisin Z残留。结果表明,Nisin灌注剂治疗后24h牛奶中Nisin Z的残留量为75.81-36.50 IU／mL,远低于国家标准规定的乳制品中Nisin的最大允许添加量(0.5g／Kg,约合500IU／mL),而相邻乳区的混合奶样,则无Nisin检出。发酵培养试验结果表明,在治疗后24h采集治疗乳区牛奶经发酵培养会被抑制,但24h后检测均没有抑制发酵；而对照乳区采集的混合奶样经发酵培养均不会被抑制。本试验说明Nisin治疗奶牛隐性乳腺炎后24h,治疗乳区牛奶中的Nisin Z的残留量远低于国家标准,但灌注乳区的牛奶在治疗后24h会抑制发酵培养。6. Nisin Z在不同炎症程度牛奶中的降解选择牛奶SCC在0～2.0×105个／mL的正常奶牛、SCC在1.0～2.0×10~6个／mL的隐性乳腺炎奶牛以及临床型乳腺炎奶牛各3头,然后分别采集正常乳区与患病乳区的牛奶各10mL,首先测定了三种不同炎症程度牛奶中NAGase酶的活性,然后在牛奶中加入Nisin (104IU),在37℃培养。分别检测0h、2h、4h、6h与8h后Nisin在牛奶中的残留量。结果表明：Nisin在不同牛奶中的降解速度不一,从高到低依次为：正常牛奶、隐性乳腺炎牛奶和临床型乳腺炎牛奶。随着时间的增加,Nisin活性减少。7. Nisin与金黄色葡萄球菌疫苗联合应用治疗奶牛隐性乳腺炎选择处于泌乳中期的荷斯坦奶牛75头,每头牛有一个隐性乳腺炎乳区。奶牛被随机分成三组,Nisin与金黄色葡萄球菌疫苗联合治疗组,Nisin治疗组,对照组,且每组各25头。在Nisin与疫苗联合治疗组中,首先在挤奶结束后于奶牛乳腺上淋巴结注射金黄色葡萄球菌疫苗2mL,1周后对患病乳区开始灌注Nisin Z制剂(2.5×106IU／mL),每天一次,连续三天,灌注结束后1周第二次在乳腺淋巴结注射疫苗2mL; Nisin治疗组则是每天灌注Nisin Z制剂一次(2.5×106IU／mL),连续三天；对照组则不采取治疗。然后在处理前与治疗后第2、4和6周采集样品,分别检测HMT强阳性率、牛奶体细胞数、NAGase酶活性以及细菌学检查。两个治疗组经治疗后第2、4和6周后其体细胞数≤5.0×105个／mL的百分比分别为(56.0%、48.0%、100%)、(64.0%、52.0%、100%)和(60.0%、48.0%、100%),两个治疗组同期比较均均无差异(P>0.05),但与治疗前及对照组比较差异极显著(P<0.01)。两个治疗组与对照组在处理后第2、4和6周后其NAGase酶的活性分别为(34.2±9.8U／L、35.5±12.5U／L、56.5±17.8U／L)、(30.3±12.0U／L、31.7±13.5 U／L、56.0±16.3U／L)和(29.9±14.2U／L、32.2±15.4U／L、57.0±17.3U／L),两个治疗组同期比较均无统计学差异(P>0.05),但是与治疗前及对照比较差异均极显著。细菌学检查表明,Nisin与金黄色葡萄球菌疫苗治疗组、Nisin治疗组与对照组在第2、4和6周的细菌学治愈率分别为(68.0%、44.0%、0),(72.0%、40.0%、0)和(72.0%、40.0%、0),而两个治疗组同期比较,其中第2周比较差异不显著(P>0.05),第4周和第6周差异均显著(P<0.05),而与对照组比较差异均极显著(P<0.01)。对治疗前后牛奶中乳成份的变化进行分析可以看出,治疗后第2、4和6周牛奶中蛋白质、脂肪、乳糖与总固体的含量均呈升高趋势,去治疗前以及对照组相比较差异均为显著(P<0.01),但是两个治疗组同期比较差异均不显著(P>0.05)。上述试验结果表明,利用Nisin与金黄色葡萄球菌疫苗联合治疗奶牛隐性乳腺炎病例,尤其是由金黄色葡萄球菌引起的隐性乳腺炎病例,效果要优于单独用Nisin治疗。综上所述,杭州近郊某奶牛场奶牛隐性乳腺炎的主要致病菌为金黄色葡萄球菌、无乳链球菌与凝固酶阴性葡萄球菌,且葡萄球类细菌已经对青霉素类药物产生耐药性。Nisin Z制剂对奶牛乳腺的刺激性试验表明,2.50×106IU适合作为治疗奶牛隐性乳腺炎的剂量。利用Nisin乳房灌注剂治疗奶牛隐性乳腺炎均有良好的治疗效果,而且在治疗后24h牛奶中Nisin Z残留量低于国家允许在食品中添加的量,不影响酸奶发酵。在牛群中用Nisin乳房灌注剂治疗隐性乳腺炎病例,可以降低桶奶体细胞数、桶奶细菌数,同时使牛奶中脂肪、蛋白、乳糖及非脂质固体的含量升高；Nisin Z在炎症牛奶中的降解速度比正常牛奶快,有利于治疗后及时排出。用Nisin与金黄色葡萄球菌疫苗联合应用治疗奶牛隐性乳腺炎时,治愈率要高于单独使用Nisin Z制剂。更多还原

【Abstract】 Mastitis, one of widespread disease for dairy cows, made a great loss to dairy production with high incidence. Subclinical mastitis can result in an elevated milk SCC and decrease milk yield, change the composition of milk to influnce milk quality. If not treated in time, it may lead to clinical mastitis, even teat blind or culling. In general, subclinical mastitis was not treated because of high incidence and antibiotic residues in milk. When bulk milk somatic cell count was obviously increased to influence milk sale, it was still treated with antibiotics. However, this could increase the resistance of mastitis pathogens, residues in milk and accumulation in vivo, which did harm to human health. As a result, it was necessary to develop a non-antibiotic preparation for the treatment of bovine subclinical mastitis, which was more highly-efficient, low toxicity and residues free. This study was designed to identify the pathogens with the sensitive test for routine antibiotics, and use nisin to treat bovine subclinical mastits, especially Staphylococcus aureus subclinical mastitis.1. The patogens of bovine subclinical mastitis was investigated in Jin Jiang Dairy FarmTo investigate the main pathogen of bovine subclnical mastitis, a survey had been conducted for 232 cows with HMT in a dairy farm of Hangzhou suburb.89 milk samples with HMT "+++" were collected to detect main pathogens, SCC and milk samples. Results showed the main pathogens were Staphylococcus aureus (25.0%), CNS (31.7%) and Streptococcus agalactiae (34.9%) respectively. S. aureus and CNS had been resistant to Penicillin G, Ampicillin and Oxacillin, and the resistant rates were (88.8%,86.4%), (83.3%,77.3%) and (72.2%,68.2%), while Streptococcus agalactiae was sensitive to these routine antibiotics. This showed the main pathogens in the dairy farm were Staphylococcus aureus, CNS and Streptococcus agalactiae, which had been resitant to Penicillin Q Ampicillin and Oxacillin except Streptococcus agalactiae.2. The irritancy test of Nisin for bovine quartersIn order to select the optimum dose to treat bovine subclinical mastitis, Nisin based formulation was evaluated for the dairy quarters before treatment. One heathy Holstein cow was selected to make an irritant test with three doses of nisin-based formulation (5.0×106IU、2.5×106IU、1.25×106IU), saline (250mL) as a control. Results indicated that the visible symptoms were observed in the quarter after treatment at the dose of 5.0×106IU alone. The acticity of NAGase in milk quarters was increased after 24h with infusion of Nisin for three groups, it was returned to the normal level after one week. All the results indicated that two doses (2.5×106IU, 1.25×106IU) had little irritative for dairy mammary glands. According to the efficacy of nisin-based formulation for clinica mastitis, the dose of 2.5×106IU was suitable to treat bovine subclinical mastitis.3. Efficacy of nisin-based formulation in treatment of subclinical mastitis in lactating dairy cowsA total of 90 cows with 90 subclinical mastitic quarters were randomly assigned to Nisin treated group and control group. In nisin-based formulation treated group, the cows were received an intramammary infusion of Nisin Z at the dose of 2.5×106IU, the control group without treatment. The treatment was performed once daily for 3 days in the nisin treatded group. The main pathogens in all cases were Staphylococcus aureus (27.78%), CNS (31.11%) and Streptococcus agalactiae (33.33%) respectively. After treatment, the bacteriological cure rate of nisin-based group and control group were 65.2%and 15.9%in the 2nd week with significant difference (P<0.01) in the 2nd wk. And the bacteriological cure rates of the main pathogens in the nisin-treated group and control group were (50.0%,18.2%), (90.9%,1.40%) and (58.8%,27.3%) respectively including Staphylococcus aureus, CNS and Streptococcus agalactiae. No significant differences were observed for S.aureus and CNS between the two groups, while significant difference for S.agalactia. The NAGase acticvity in the nisin-treated group were decreased significantly (P<0.01), while no difference in the control group (P>0.05). And the percentage of SCC≤5.0×105/mL in the two groups were (41.3%, 18.2%) and (32.9%,9.1%) in the 1st and 2nd week after treatment, the differences at the same time point were both P<0.01. All data indicated that nisin-based formulation had a better therapeutic effect for bovine subclinical mastitis.4. Efficacy of nisin-based formulation to decrease bulk tank somatic cell count after treatment of bovine subclinical mastitis in lactating dairy cows.Two cowsheds were selected to evaluat nisin-based formulation to decrease BTMCC, total 85 and 90 cows in nisin and control group. Before the treatment, a survey was conductecd to detect BTSCC, bulk bacteria and bulk milk composition from March to July. The high BTSCC herd was selected as the nisin-based group, and the low BTSCC herd was the control group. From August to December, nisin-based formulation was used to treat the subclinial cases once daily for 3 days in the treated group, while the control group without treatment. The efficacy of nisin-based formulation to treat subclinical mastitis was evaluated with BTSCC, bulk bacteria, bulk milk composition and Nisin Z residues detected by ECLIPSE50.Results indicated that BTSCC, bulk bacteria in the treated group were significantly lower than the control group (P<0.05), while bulk milk composition including fat, protein, and lactose was increased in the nisin-based formulation group. Nisin Z could not be detected by ECLIPSE50 in bulk tank milk when the number of quarters infused with nisin-based formulation was less 10. All results showed that nisin-based formulation could decrease bulk tank somatic cell counts effectively.5. The residues of nisin-based formulation in mik samples after treatment for bovine subclinical mastitis3 Holstein cows with subclinial mastitis were selected to evaluate nisin residues in treated quarters after treatment. The treatment was performed once daily for 3 days in the nisin treatded quarters at the dose of 2.50×106IU, while the control quarters without treatment. The results showed that Nisin Z in milk(75.81±36.50 IU/mL) was detected only in the treated group at 24h following intramammary infusion, which was much lower than the upper limit (0.5g/kg, equivalent to 500IU/mL) allowed as preservative in milk products by the China authority, while no residues of Nisin Z in milk samples from the control quarters. Reduced fermentation with low acidity from treated quarters was found in milk samples at 24h, where no inhibition was detected in fermentation from control quarters throughout the experiment. All the results showed that nisin-based formulation was safe and litte fermentation was found in a short time after treatment.6. Degradation of Nisin Z in normal, subclinically and clinically mastitic milk samples in vitro9 Holstein cows were selected and divided into 3 normal cases (SCC 0-2.0×105cells/mL),3 subclinical cases (SCC 1.0-2.0×106cells/mL) and 3 clinical cases. At first, the NAGase activities in different milk samples were detected after freezing and thawing, then Nisin Z (104IU) was added into lOmL milk samples to detect the residues at different time points (Oh,2h,4h,6h and 8h) after culture at 37℃in vitro. The results indicated that the NAGase activity in milk samples were increased from normal, subclinical and clinical mastitis cases in turn. Nisin residues were detected by National Standards to be decreased gradually after cultivation at 37℃in vitro from normal, subclinical and clinical mastitis cases.7. Efficacy of vaccination in combination with Nisin Z treatment against Staphylococcus aureus mastitis in lactating dairy cowsAlthough nisin-based formulation could have been proved effective on bovine sublinical mastitis, how to improve the efficacy of Nisin Z had been a new concern in the following research. At first, Staphylococcus aureus vaccine was attempted to improve the effect of nisin-based formulation to treat bovine subclinical mastitis.75 Holstein cows with subclinical mastitis were divided into 3 groups, nisin combination with Staphylococcus aureus vaccine group (25), Nisin treated group (25) and control group (25). Staphylococcus aureus vaccine (2mL) was injected into lymph mammary gland, after one week, Nisin-based formulation (2.50×106IU) was infused to the mastitic quarters once daily for 3 days, then one week later,2mL Staphylococcus aureus vaccine for lymph mammary gland again; In the Nisin-treated group, Nisin-based formulation was performed once daily for 3 days with the dose of 2.50×106IU. The indicators of milk samples were HMT, SCC, NAGase, bacteriological exzamination and milk composition after treatment in the 2nd,4th and 6th week.The percentage of SCC≤5.0×105cells/mL was (56.0%,48.0%,100%), (64.0%, 52.0%,100%) and (60.0%,48.0%,100%) among three groups at the same time point. And the NAGase activity in two treated groups were (34.2±9.8U/L,35.5±12.5U/L) (30.3±12.0U/L,31.7±13.5U/L)and(29.9±14.2U/L,32.2±15.4U/L)in the 2nd,4th and 6th wk after infusion. No significant differences were observed in the two treated groups after HMT, SCC and NAGase test at the same time point (P>0.05), while significant differences (P<0.01) compared with pretreatment and control group among three groups.The bacteriological cure rates of three groups were (68.0%,44.0%,0) (72.0%,40.0%,0) and (72.0%,40.0%,0) at the same time point after treatment. No significant difference was observed between two treated groups in the 2nd week after infusion, while significant differences (P<0.05) in the 4th and 6th week after treatment. The percentage of milk fat, protein, lactose and NSF were all increased in the same time point after treatment. Compared with pre-treatment and the control group, significant differences (P<0.01) were observed, while no significant differences (P>0.05) between two treated groups. All the results indicated that Nisin combination with vaccine had a better therapeutic effect than nisin-based formulation alone for subclinical mastitis with Staphylococcus aureus treated.In summary, the main pathogens of the dairy farm were Staphylococcus aureus, Streptococcus agalactiae and CNS, and the Staphylococcus was resistant to Penicillin G, Ampicillin and Oxacillin. After the irritant test of nisin-based formulation for mammary gland, the dose of 2.50×106IU was suitable to treat bovine subclinical mastitis. Nisin-based formulation had a better therapeutic effect for bovine subclinical mastitis, and the residues of Nisin Z in milk after treatment was much lower than the upper limitallowed as preservative in milk products by the China authority. Nisin-based formulation could decrease BTSCC, bulk bacteria, and improve bulk milk compositions. Combination with Staphylococcus aureus vaccine, nisin-based formulation had a better therapeutic effect for subclinical mastitis, especially for those subclinical cases caused by Staphylococcus aureus.更多还原