治疗奶牛乳房炎基因药物的环境释放试验

用人溶菌酶cDNA重组质粒治疗奶牛乳房炎的初步试验结果显示,该基因药物对奶牛乳房炎具有良好的预防和治疗效果.为验证该基因药物在大规模推广使用前对环境的安全性,按照农业部颁布的《农业转基因生物安全评价管理办法》要求,选择地处相对偏僻的某奶牛场为试验地,用人溶菌酶重组质粒pcDNAKLYZ分别治疗干奶期和泌乳期患牛,随后对实验牛的血液、乳汁进行质粒残留检测,并对唾液、尿液、粪便和饲养环境中的微生物进行转化菌检测.结果显示,仅71.43%（5/7）的泌乳牛的乳区在治疗后的第1天从奶中检测到质粒,血液检测结果均为阴性;也未发现该重组质粒被其分泌物、排泄物中菌群以及环境中微生物摄取、转化.因此,该重组质粒注射奶牛后,通过乳汁、尿液以及其他分泌物、排泄物向饲养环境泄露的可能性极小,也不可能通过转化菌在环境中水平传播.由此可见,该重组质粒在治疗奶牛乳房炎时对环境是安全的.     

联合注射牛γ干扰素和人溶菌酶表达质粒对奶牛乳房炎的治疗效果

通过乳腺注射牛γ干扰素(BoINF-γ)和人溶菌酶(LYZ)表达质粒,比较单独和联合注射重组质粒与抗生素对奶牛乳房炎的治疗效果。将PCR扩增的BoINF-γc DNA插入真核表达载体pcDNAK,用重组质粒pcDNAK-BoINF-γ转染NIH 3T3细胞,经免疫荧光试验证明,重组质粒能正确表达。将91个奶牛乳房炎乳区分为4组,通过乳腺穿刺法注射,第1组注射400μg pcDNAKLYZ,第2组注射400μg pcDNAK-BoINF-γ,第3组注射400μg pcDNAKLYZ和400μg pcDNAK-BoINF-γ,第4组注射100 IU青霉素和25μg链霉素,次日重复注射1次。在注射后10 d进行临床症状观察、乳汁体细胞检测和细菌培养计数。结果显示:注射pcDNAKLYZ对临床型乳房炎的治疗效果为80%,对隐性乳房炎的治疗效果为79.2%;注射pcDNAK-BoINF-γ对临床型乳房炎的治疗效果为50%,对隐性乳房炎的治疗效果为66.7%;联合注射pcDNAKLYZ和pcDNAKBoINF-γ对临床型乳房炎的治疗效果为100%,对隐性乳房炎的治疗效果为83.5%;青链霉素合剂对临床型乳房炎的治疗效果为50%,对隐性乳房炎的治疗效果为60%。试验结果表明,联合注射人溶菌酶和牛γ干扰素表达质粒对奶牛乳房炎具有更好的治疗效果。     

人溶菌酶基因在奶牛乳腺中的表达试验

为了提高牛奶品质，降低牛奶细菌含量，使其性质更接近于人奶。将表达人溶菌酶基因的动物乳腺特异表达载体注射到奶牛的乳区，然后根据基因注射前、后奶样的C．M．T．试验结果和溶菌酶的表达量判定效果。试验结果表明，重组质粒在奶牛乳腺中能表达有活性的人溶菌酶，表达量达1．96μg／ml。能使奶样的C．M．T．试验转阴。     

重组溶菌酶质粒pcDNAKLYZ治疗泌乳期奶牛乳房炎

通过比较注射人溶菌酶重组质粒pcDNAKLYZ的隐性乳房炎奶牛注射前后的奶样中细菌计数结果,对重组溶菌酶基因工程质粒治疗奶牛乳房炎的效果进行分析。对乳房炎患牛的156个乳区治疗前后312份奶样进行细菌培养,其中在注射前的乳样的培养结果中,阴性率为0（0/156）,菌落数在50以内的比率为12.82%（20/156）,在51～100的比率为16.67%（26/156）,大于等于100的比率为70.51%（110/150）;在注射人溶菌酶的重组质粒pcDNAKLYZ后的奶牛乳样的培养结果中,阴性率为51.92%（81/156）,菌落数在50以内的比率为45.51%（71/156）,菌落数在51～100的比率为0%（0/156）,大于等于100的比率为2.56%（4/156）。因此,从细菌计数结果来看,重组质粒组的阴性及小于50的比率（97.44%）远高于治疗前（12.82%）,重组质粒的抑菌效果显著（P〈0.05）,该重组质粒对奶牛乳房炎具有较好的治疗效果。     

牛源金黄色葡萄球菌单链抗体对奶牛乳腺炎的治疗效果评价

本文首先对患隐性乳腺炎的50头奶牛采取奶样,并对奶样进行了细菌分离鉴定及PCR检测,共检出金黄色葡萄球菌21株;随后对分离到的金黄色葡萄球菌进行了药敏试验,结果显示70%的金黄色葡萄球菌对青霉素产生了耐药性。为了研究针对金黄色葡萄球菌全菌抗原的单链抗体对奶牛乳腺炎的治疗效果,将表达单链抗体的重组质粒通过无针注射器注射到患病奶牛的乳腺(600μg/乳区),48 h后再以相同的剂量注射一次。14 d后,奶样的加利福尼亚乳腺炎试验(California Mastitis Test,CMT)结果显示,治疗奶牛乳腺炎的有效率为83.33%,其中2例完全治愈。定期采集试验奶牛的乳样,并用PCR方法进行检测,发现第二次注射后的14 d内,奶样中未检测到重组质粒,说明重组质粒不能在奶牛体内长期残留。本研究为奶牛乳腺炎治疗抗体的研发提供了理论依据。     

人溶菌酶基因治疗奶牛乳腺炎的初步研究

将两种自行构建的表达人溶菌酶基因的重组质粒注射到患病奶牛的乳腺,经CMT试验证明对临床型和隐性乳房炎具有治疗作用。将其中的p205C3LYZ注射入奶牛乳腺,经微球菌溶解试验证明溶菌酶的表达量达到1．18μg／ml以上,表达至少维持8d。不同注射次数、剂量和途径的试验结果表明,重组质粒的治疗作用具有剂量和次数依赖性,3次注射450μg或2次注射400μg的治疗效果相当,乳房基部注射的治疗效果优于乳池注射。以CMT结果为主要指标的对比治疗试验结果表明,重组质粒对隐性奶牛乳房炎的治疗效果与青链霉素合剂接近,有效率分别为19／22(86．4％)和8／9(88．9％),优于中药乳炎消的治疗效果(9／13,69．2％)。3种药物治疗后,奶样中的细菌总数及葡萄球菌、链球菌和大肠杆菌等代表性致病菌的数量有明显下降,重组质粒和抗菌素治疗组的奶产量上升幅度高于中药治疗组。结果表明,表达人溶菌酶基因的重组质粒对奶牛乳房炎具有可靠、持久的治疗效果,可以代替抗菌素使用。     

人溶菌酶基因hLYZ在小鼠乳腺中的表达及其抗菌活性检测

从人胎盘组织中克隆获得人溶菌酶基因hLYZ,选用体内表达载体pcDNA3.1,构建表达载体pcDNA3.1hLYZ,并转染至小鼠体内,采集小鼠乳汁和乳腺组织,通过RT-PCR方法检测基因的表达水平,用免疫组化反应、Western blotting检测蛋白的表达,并通过体外抗菌活性检测重组人溶菌酶的抗菌活性。经酶切鉴定和核苷酸序列测定,表明重组质粒pcDNA3.1-hLYZ构建正确,小鼠乳清的SDS-PAGE电泳显示在约14700处有特异的蛋白条带出现,Western blotting检测表明成功表达了人溶菌酶,根据回归方程计算酶活性为4011U/mL,体外抗菌活性检测结果显示重组人溶菌酶对大肠杆菌有明显裂解作用。本试验成功地在小鼠乳腺中表达了重组人溶菌酶,表达的蛋白具有较高的酶活性,这些结果为哺乳动物乳腺生物反应器的研究奠定了基础。     

奶牛乳腺炎疫苗免疫试验报告

对奶牛乳腺炎疫苗进行免疫试验，结果显示：试验所用疫苗对奶牛临床乳腺炎的免疫效果较明显，并使奶牛乳中体细胞数大幅下降，避免了乳中体细胞数升高对奶牛产量的不良影响。在不考虑奶牛重复感染临床乳腺炎情况下，疫苗对奶牛临床乳腺炎的保护率可达到84．75％。     

利用乳汁体细胞研究共轭亚油酸钙对奶牛乳腺基因表达的作用

8头泌乳中期的奶牛被随机分为2组，分别是对照组（每头牛：基础日粮＋棕榈酸钙200g／d）和试验组（每头牛：基础日粮＋共轭亚油酸钙200g／d），试验期14d，检测了奶产量、乳成分，分析奶牛的血液变化，并采用荧光定量PCR对乳汁体细胞中脂蛋白脂酶（LPL）、乙酰辅酶A羧化酶（ACACA）的基因表达进行检测。结果显示，与对照组相比，试验组奶牛的乳产量、乳蛋白、乳糖和乳汁体细胞含量没有显著影响，但是显著降低了乳脂肪含量（P〈0．05），对照组和试验组奶牛乳脂肪含量分别为0．0326g／mL和0．0244g／mL。检测血液指标发现，共轭亚油酸钙显著升高了奶牛血液中高密度脂蛋白的含量（P〈0．05）。基因分析发现，试验组奶牛乳汁体细胞中LPL、ACA—CA的基因表达显著下调，表明共轭亚油酸钙抑制了奶牛乳腺细胞脂肪酸合成酶的基因表达。     

重组禽腺联病毒介导的人溶菌酶在蛋清中的表达

为在鸡蛋清中表达人溶菌酶(hLYZ),将含有hLYZ输卵管特异表达盒的重组禽腺联病毒以每只鸡2×10~(11)经翅静脉注射正常产蛋母鸡,收集蛋清对重组蛋白的表达情况进行检测。RT-PCR结果显示,hLYZ只在输卵管部位表达,Western blot结果显示重组蛋白大小正确,抑菌活性表明注射重组病毒后的蛋清的抑菌活性明显优于正常蛋清的。动态表达水平检测结果显示,注射病毒后第1周即可以检测到重组hLYZ的表达,第5周时表达量最高,达68.3μg/mL,表达时间持续3个月之久。以上结果表明,重组禽腺联病毒能介导人溶菌酶在蛋清中的高效、长时表达,为人溶菌酶的开发应用提供了新途径。     

人溶菌酶cDNA的克隆及其在小鼠体内的表达

根据已发表的人溶菌酶 (human lysozyme,h L YZ) m RNA序列设计引物 ,以人乳腺第 1链 c DNA为模板 ,用 PCR扩增出 1.5 kb h L YZ双链 c DNA,其推导的氨基酸序列与国外发表的从胎盘、巨噬细胞和结肠中克隆的 h L YZ氨基酸序列同源性为 10 0 % ,与从人组织细胞中克隆的 h L YZ仅有 1个氨基酸差异 ,但与从中国人胎盘中克隆的 h L YZ具有6个氨基酸差异。将此 c DNA克隆入乳腺特异表达载体 ,用所获得的基因构件注射哺乳期小鼠 ,经 RT- PCR和微球菌溶解试验证明 ,上述基因构件能有效地驱动目的基因在小鼠乳腺中表达 ,表达量为 6 9.3mg/ L,表达具有较好的组织特异性。这些试验结果表明 ,本研究构建的表达 h L YZ c DNA的基因构件可以用于乳腺生物反应器的研制。     

The prevalence of mastitis in primiparous heifers in eleven Waikato dairy herds

The objective of the study was to determine the prevalence of mastitis among primiparous heifers at calving and at drying off in 11 Waikato dairy herds during the 1993-94 dairy production season. Duplicate quarter milk samples were collected aseptically from 458 heifers within 5 days after calving for bacteriological analysis. Mastitis was diagnosed in at least one quarter in 35.6% of these heifers. Coagulase-negative staphylococci were isolated from 21.8% of the heifers. The prevalence of coagulase-negative staphylococci varied between herds from 4.3% to 44.8%. Environmental streptococci caused mastitis in 12.2% of heifers, ranging from 5.6% to 24.1% between herds. Streptococcus uberus was the pathogen identified most frequently at calving and accounted for more than 90% of the streptococcal isolates. Staphylococcus aureus and coliforms were isolated from less than 1% of samples. Clinical mastitis was observed in 8.1% of heifers at calving; environmental streptococci were isolated from 67.6% of these clinical clinical cases. Only 2.8% of heifers developed clinical mastitis during lactation and environmental streptococci were isolated from 38.5% of these cases. The prevalence of mastitis among 428 of the heifers at drying off was 64.7%; a 1.8 fold increase during lactation. Corynebactetium bovis was isolated from 43% of heifers at drying off even though it was not isolated from any heifers at calving. During the season, the prevalence of Staphylococcus aureus mastitis increased to 2.8% while mastitis caused by environmental streptococci declined to 2.8%. The prevalence of environmental mastitis pathogens decreased during lactation while contagious pathogens increased in each of the 11 herds. Ineffective post-milking teat sanitation probably contributed to the increase in mastitis caused by contagious pathogens. Specific factors were not determined that affected the variation in prevalence between herds.     

Effect of teat dipping with a germicide barrier teat dip in late gestation on intramammary infection and clinical mastitis during the first 5 days post-partum in primiparous cows

The effect of teat dipping with a barrier teat dip prior to parturition on intramammary infection (IMI) and clinical mastitis during the first 5 days post-partum was investigated in a split udder trial in 149 Holstein-Frisian heifers. Their left front and right hind quarters were dipped three times weekly (i.e. Monday, Wednesday and Friday) with a barrier teat dip containing 0.1% polyvidon iodine from day 260 of gestation until parturition. The opposite quarters (right front and left hind quarter) served as untreated control. Bacteria were isolated from 52.2% of quarter milk samples collected immediately after parturition prior to first machine milking. Staphylococcus aureus and coagulase-negative staphylococci (CNS) were predominantly found in the samples (29.2 and 35.6% of the positive samples, respectively). At parturition 6.7% of the heifers showed signs of clinical mastitis and another 27.5% developed signs of clinical mastitis during the first five days of lactation. No significant differences were found between treated and control quarters regarding IMI and incidence of clinical mastitis. Teat dipping prior to parturition in primigravid dairy heifers did not improve udder health in this trial.     

Comparative efficacy of three dry-cow antibiotic formulations in spring-calving New Zealand dairy cows

AIM: To evaluate the efficacy of a dry-cow antibiotic preparation containing cloxacillin plus ampicillin in a formulation that gives a 10-week duration of action, in comparison to products containing cephalonium (10-week action) or cloxacillin alone (7-week action). METHODS: A total of 493 cows were selected from 6 spring-calving dairy herds in the Manawatu region of New Zealand, according to the criteria of the SAMM plan, to receive intramammary antibiotic therapy at the end of lactation (drying off). Cows were randomly allocated to receive 1 of the 3 dry-cow antibiotic products under investigation. Cows were examined twice during the dry period and twice daily during the first 10 days of their subsequent lactation for the presence of mastitis. Milk samples were collected from individual quarters at the time of drying off and at 7 and 28-35 days after calving, for determination of milk somatic cell counts (SCC). Bacteriology was carried out on milk samples taken from cows that developed mastitis during the first 10 days after calving. RESULTS: No cows developed mastitis during the dry period. Sixteen cows developed clinical mastitis within 10 days of calving; there was no difference in incidence between treatments. Streptococcus uberis was the most commonly isolated organism. Mean SCC on Day 7 were lower (p = 0.019) in cephalonium-treated quarters (189.9+/-28.4 x 10(3) cells/ml) than in cloxacillin-treated quarters (388.7+/-71.2 x 10(3) cells/ml); values in quarters receiving cloxacillin plus ampicillin were intermediate (252.0+/-47.0 x 10(3) cells/ml). SCC were similar between treatment groups on Day 28-35. CONCLUSIONS: The use of a combination of cloxacillin plus ampicillin was effective for the prevention of mastitis during the dry and peri-calving-periods in pastured dairy cattle.     

Management factors associated with the incidence of clinical mastitis over the non-lactation period and bulk tank somatic cell count during the subsequent lactation

Aim: To evaluate associations between management decisions related to the control of mastitis, including the infusion of antibiotics at the end of lactation (dry-cow therapy; DCT), on the incidence of clinical mastitis over the non-lactating period and the bulk tank somatic cell count (BTSCC) in the subsequent lactation. Methods: Dairy herd owners (n=158) provided information via a retrospective survey about (a) the proportion of their herds treated with DCT; (b) DCT management, including: number of occasions on which cows were dried off; manipulation of feed and water intake around drying off; infusion technique (partial vs full depth insertion of cannula); and hygiene before and after DCT infusion; (c) occurrence of mastitis and frequency of occurrence following drying off and in the subsequent lactation; (d) number of cows culled for mastitis-related conditions; (e) reasons for culling; (f) incidence of clinical mastitis; and (g) stock purchase policy with regard to mastitis. The BTSCC for each vat of milk supplied for the 1999/2000 and 2000/2001 seasons, and records of antibiotic purchases were collated for each herd. The probability that >2% of cows within a herd were diagnosed with clinical mastitis over the dry period was initially examined using univariate analysis (i.e. chi2 or logistic regression) and associated factors (p<0.2) were offered to a reverse stepwise logistic regression model. Factors hypothesised as being associated with the average lactation log10 BTSCC for the 2000/2001 season were initially examined using univariate analysis (i.e. ANOVA or linear regression analysis) and associated factors (p<0.2) were then tested using a forward manual model-building approach. Results: Increasing the percentage of the herd treated with DCT at the end of lactation was associated with reduced probability that >2% of a herd would be diagnosed with clinical mastitis over the non-lactating period and with a lower BTSCC in the subsequent lactation (p<0.01). A lower BTSCC was associated with small herds (<150 cows; p<0.05), not reducing feed intake around drying off (p<0.05), checking for clinical mastitis over the dry period in the milking parlour rather than at pasture (p<0.05), partial insertion of the DCT cannula (p<0.01), and use of 'change in udder shape' during lactation as a diagnostic criterion for mastitis (p<0.05). The incidence of clinical mastitis over the dry period was positively associated with reduced feeding around drying off (p=0.05) and the estimated volume of milk being produced at the time of drying off (p=0.014). Conclusions: Use of dry cow therapy was associated with fewer cases of clinical mastitis over the non-lactating period and reduced BTSCC over the subsequent lactation. Reduced BTSCC was also associated with smaller herds, use of partial (compared with full depth) insertion of the DCT cannula, not reducing feed intake at the time of drying off, checking for clinical mastitis over the dry (non-lactation) period in the milking parlour, and use of udder shape for diagnosis during lactation. Control of clinical mastitis and BTSCC involves a range of management practices that need to be used in conjunction with DCT. Keywords: Dairy cows, mastitis, dry-cow therapy, somatic cell count, management practices.     

Teat canal closure in non-lactating heifers and its association with udder health in the consecutive lactation

Prevention of heifer mastitis is a key element in preventing mastitis. Since pathogenesis of heifer mastitis is not fully understood yet, combating it is arduous. The goal of this study was to examine whether the premature loss of the keratin plug of the teat canal represents an important risk factor for the development of heifer mastitis. 84 dairy heifers (German Holstein) from six high-yielding dairy herds in Northern Germany were examined between March 2005 and June 2006. Each quarter was examined clinically at least three times before calving. If the teat canal of a quarter was open, secretion samples were collected for bacteriological analysis. After calving, four quarter samples were collected during the first lactation. A rapid increase of the proportion of open teat canals was detected in the preparturition period. No teat canals were open till 80 days before parturition, while 60% of the teat canals were open at 60 days before parturition. A time-scheduled infection pattern was observed: during the days 90-61, 60-31, and 30-0 before calving, skin inhabitants (coagulase-negative staphylococci, coryneform bacteria), cow-related, and environment-related microorganisms prevailed, respectively. A total of 77% of intramammary infections encountered at parturition had established previously. No significant association between the duration of open teat canals before calving and the udder health post partum was found. However, the incidence of clinical mastitis during first lactation was influenced largely by the duration of infection ante partum and the mastitis pathogen involved. This study shows that a high proportion of teat canals already opens several months before calving and that opening of the teat canal before calving is an important factor in the aetiology of heifer mastitis.     

Development of cell content and shedding of Prototheca spp. in milk from infected udder quarters of cows

It was the objective of this study to analyse shedding patterns and somatic cell counts in cows and quarters infected with Prototheca spp. and to evaluate two approaches to identify infected animals by somatic cell count (SCC) or by bacteriological analysis of pooled milk samples. Five lactating dairy cows, chronically infected with Prototheca spp. in at least one quarter were studied over 11 weeks to 13 months. Quarter milk samples and a pooled milk sample from 4 quarters were collected aseptically from all quarters of the cows on a weekly basis. Culture results of quarter milk and pooled samples were compared using cross tabulation. SCC of quarter milk samples and of pooled samples were related to the probability of detection in the infected quarters and cows, respectively. Shedding of Prototheca spp. was continuous in 2 of 8 quarters. In the other quarters negative samples were obtained sporadically or over a longer period (1 quarter). Overall, Prototheca spp. were isolated from 83.6% of quarter milk samples and 77.0% of pooled milk samples of infected quarters and cows. Somatic cell counts were higher in those samples from infected quarters that contained the algae than in negative samples (p < 0.0001). The same applied for composite samples from infected cows. Positive samples had higher SCC than negative samples. However, Prototheca spp. were also isolated from quarter milk and pooled samples with physiological SCC (i.e. < 10(5)/ml). Infected quarters that were dried off did not develop acute mastitis. However, drying off had no effect on the infection, i.e. samples collected at calving or 8 weeks after dry off still contained Prototheca spp. Results indicate that pre-selection of cows to be sampled for Prototheca spp. by SCC and the use of composite samples are probably inadequate in attempts to eradicate the disease. However, due to intermittent shedding of the algae in some cows, single herd sampling using quarter milk samples probably also fails to detect all infected cases. Therefore, continuous monitoring of problem cows with clinical mastitis or increased SCC in herds during eradication programs is recommended.     

Posology and Field Efficacy Study with Novobiocin for Intramammary Infusion in Nonlactating Dairy Cows

Four dose levels of novobiocin (50, 200, 400, 600 mg) were compared with no drug for the intramammary treatment of Staphylococcus aureus, Streptococcus agalactiae and other streptococcal infections present in the udder of dairy cows at the initiation of the dry period. Treatment success was evaluated by comparing the microbiological status of duplicate pretreatment quarter milk samples collected at drying off with the microbiological status of duplicate quarter milk samples collected four to ten days postcalving. Infection status of 1318 cows in 75 herds in five geographic locations was determined. Treatment effects on infected cows were evaluated by least squares analysis of variance with treatment, herd, lactation number, days dry and milk production at drying off considered as variables. The dose of 400 mg novobiocin per quarter was demonstrated to be significantly more effective (P < 0.05) than no drug and significantly better than (P < 0.05) or equal to the other doses for curing infections caused by S. aureus, S. agalactiae and other streptococci. A significant reduction (P < 0.05) in the overall rate of new udder infections acquired during the dry period was observed in cows treated with ≥ 200 mg novobiocin at drying off. The data supported the conclusion that the cow rather than the quarter is the appropriate experimental unit in the evaluation of intramammary mastitis treatments. Herd and lactation number were the most significant variables affecting cures.     

The prophylactic effect of a dry-cow antibiotic against Streptococcus uberis

The prophylactic use of a dry-cow antibiotic for reducing the incidence of mastitis due to Streptococcus uberis was studied in four seasonally calving dairy herds involving 378 cows. The treatment was a long-acting dry-cow antibiotic preparation administered immediately after the last milking of lactation. New intramammary infections were identified by comparing the bacteriological status of quarters at drying off with that after calving, or through manual udder palpation during the dry period. The administration of dry-cow antibiotic to uninfected quarters at drying off reduced the overall incidence of new infections with Streptococcus uberis from 12.3% for untreated quarters to 1.2% of quarters (p<0.01). The reduction was significant (p<0.01) for both dry-period and post-calving infections. The susceptibility of uninfected quarters to new infection by Streptococcus uberis appeared to be unrelated to the infection status of a cow at drying off. Clinical infections during the dry period were most prevalent (97%) in quarters identified as having open teat canals. Fewer open teat canals (p<0.05) were observed among antibiotic treated quarters over the first 4 weeks of the dry period. Treated quarters had a lower (p<0.05) incidence of new clinical infection during the ensuing lactation and lower somatic cell counts. This did not affect production levels of milk, milk fat or protein. The results clearly indicated a prophylactic benefit for the dry cow antibiotic treatment against new Streptococcus uberis infections during the dry period.