奶牛乳房炎无乳链球菌的分离及PCR鉴定

利用THB(Todd-Hewitt Broth)固体培养基和色素试验培养基选择培养无乳链球菌,参照GenBank中无乳链球菌参考菌株(Accession:AF015927.1、JQ289582.1)16SrRNA和种属特异性基因cfb(CAMP因子)序列设计引物,对奶样中分离的12株疑似无乳链球菌进行鉴定。结果显示,经PCR扩增后,被检测的12株细菌均可扩增出预期大小的16S rRNA基因序列,而12株中有8株可以扩增出预期大小的cfb基因序列,条带单一,特异性好。序列BLAST显示,12株菌的16S rRNA基因序列与NCBI上报道的无乳链球菌相应序列高度同源(>99.0%),各分离菌株间的16S rRNA基因序列也高度同源(99.0%～100.0%);cfb基因序列与NCBI上已报道的无乳链球菌相应序列具有高度同源性(>99.0%),各菌株间cfb基因序列也高度同源(100.0%)。经选择培养与PCR鉴定结果可以确定12株疑似菌株中有8株为无乳链球菌。     

奶牛乳房炎相关链球菌PCR检测方法的建立

利用编码3-磷酸甘油醛脱氢酶的gapC基因具有高度特异性的特点,建立PCR方法鉴定与奶牛乳房炎相关的链球菌。根据已有gapC基因序列设计1对引物,以分离自患乳房炎奶牛乳样的10株革兰阳性球菌、10株革兰阳性杆菌、10株革兰阴性杆菌作为待检菌株,进行PCR扩增。结果表明,在10株革兰阳性球菌中,有8株球菌可以扩增出约1011bp的目的条带,而其他2株革兰阳性球菌及20株杆菌均无相应PCR产物出现。通过传统的生化鉴定与16S rDNA序列分析相结合证实,能扩出gapC基因的8株革兰阳性球菌分别为乳房链球菌(Streptococcus uberis)、牛链球菌(S.bovis)与猪链球菌(S.suis)。说明基于gapC基因的PCR方法,用于鉴定奶牛乳房炎相关链球菌具有较强的特异性。     

牛乳中无乳链球菌PCR快速检测方法的建立

根据GenBank公布的的SIP基因序列,设计一对特异性引物,通过对PCR方法的优化,建立了牛无乳链球菌性乳房炎的快速PCR检测方法。用建立的PCR检测方法对患牛乳中无乳链球菌总DNA进行扩增．获得大小为945bp的DNA片段。特异性试验表明,停乳链球菌、金黄色葡萄球菌、大肠杆菌DNA均未扩出条带;敏感性试验表明,该对引物能够检测到的最低DNA浓度为1．25ng;重复试验表明该方法具有良好的重复性和稳定性。     

Nisin抗菌肽治疗奶牛无乳链球菌性隐性乳房炎后主要乳成份的变化

将36头无乳链球菌引起的隐性乳房炎奶牛分成试验组和对照组,每组18个病例.试验组奶牛用Nisin抗菌肽经患病乳房内灌注,每天1次,连续治疗3 d,对照组奶牛不治疗.分别采集治疗前和治疗后1、3、5周的奶样进行细菌学检查,体细胞含量测定和乳成份分析.结果表明,经Nisin治疗后,细菌阳性乳区数和体细胞教(SCC)大于500 000/mL乳区数量均显著降低,牛奶中脂肪、蛋白质、乳糖和总固体含量有升高趋势,尤其是蛋白质和乳糖的含量显著高于治疗前.由此可见,Nisin乳房内注入对于奶牛无乳链球菌性隐性乳房炎具有良好的治疗作用.     

奶牛乳房炎中大肠杆菌PCR检测方法的初步建立

大肠杆菌是引起奶牛乳房炎的一种主要细菌.为了建立PCR直接检测奶牛乳房炎致病菌的方法,本试验以大肠杆菌为主要研究对象,根据已发表的致病性大肠杆菌16-23S rRNA特异性序列设计并合成一对引物,对昆明市的某发生奶牛乳房炎的病牛的牛奶进行大肠杆菌PCR检测,并对扩增片段进行序列分析,建立一种直接从牛奶中检测大肠杆菌的PCR快速诊断方法.结果表明,该方法能够检测到乳样中的大肠杆菌,而且具有快速、准确和特异的优点.     

奶牛乳房炎无乳链球菌的分离与鉴定

利用THB固体培养基和色素培养基初步筛选出奶牛乳房炎中无乳链球菌,以分离的12株疑似菌的基因组DNA为模板进行PCR扩增,对扩增产物进行分析,结合选择培养的生理生化特性对分离菌进行鉴定。结果表明,12株疑似菌中有8株为无乳链球菌.     

我国奶牛乳房炎无乳链球菌抗生素耐药性研究

为了查明我国奶牛乳房炎无乳链球菌对抗生素耐药情况,指导临床合理用药,从我国部分地区奶牛场采集的临床型奶牛乳房炎病乳中分离鉴定出无乳链球菌115株,采用K-B纸片法测定了这些菌株对抗生素的耐药情况。结果表明,无乳链球菌对目前临床上使用的大部分抗生素,如头孢唑啉、头孢噻肟、丁胺卡那霉素、卡那霉素、庆大霉素、四环素、强力霉素、麦迪霉素、林可霉素、氟苯尼考、多黏菌素B、环丙沙星、氟哌酸、氨苄青霉素/舒巴坦、头孢噻肟/棒酸和头孢他啶/棒酸均比较敏感;但对青霉素G、氨苄青霉素、链霉素、恩诺沙星、阿莫西林/棒酸和复方新诺明等有一定耐药性,其耐药率达50%～100%。     

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。     

通辽地区奶牛乳房炎无乳链球菌的分离与鉴定

为研究通辽地区奶牛乳房炎优势性病原菌,试验对30份奶牛临床型乳房炎奶样进行细菌分离,经血液琼脂平板分离培养、生化试验及药敏试验共分离到14个菌株,最终鉴定为无乳链球菌。药敏试验结果表明,分离菌株对临床上常用的链霉素及磺胺类药物均已产生不同程度的耐药性,而对卡那霉素、庆大霉素及麦迪霉素表现出较好的敏感性。     

Milk protein profiles in response to Streptococcus agalactiae subclinical mastitis in dairy cows

The objective of this study was to investigate the milk protein profiles of normal milk and those of milk during the course of subclinical mastitis, caused by natural Streptococcus agalactiae infection. Two‐dimensional gel electrophoresis and liquid chromatography mass spectrometry were used to assess protein profiles and to identify the proteins. The results showed that S. agalactiae subclinical mastitis altered the protein profiles of milk. Following Mascot database matching, 11 and 12 protein types were identified in the milk collected from healthy and S. agalactiae subclinical mastitic udders, respectively. The distinct presence of the antibacterial protein cathelicidin‐1 was detected in infected milk samples, which in turn was highly correlated to the severity of subclinical mastitis as represented by the milk somatic cell count (r = 0.616), but not the bacterial count. The protein profile of milk reveals changes in the host response to S. agalactiae intramammary infection; cathelicidin‐1 could therefore serve as a biomarker for the detection of subclinical mastitis in dairy cows.     

The economic benefit of treating subclinical Streptococcus agalactiae mastitis in lactating cows

The economic benefits of treating lactating cows for Streptococcus agalactiae mastitis were studied at a large (689 milking cows) central California dairy. Postcure milk production of case cows (infected, treated, and cured) was compared with production of paired control cows (uninfected) and was matched for yield, days in milk, days in gestation, and parity. A simulation was used to plot expected lactation curves for mastitic cows (infected, not treated) with characteristics similar to those of each control cow, and these curves were compared with actual case-cow lactation curves. The difference in actual and expected production was used to calculate net economic benefits of treatment. Comparison of expected with actual production indicated a net benefit from treatment of $396/cow for cows treated in early lactation and $237 for cows treated in midlactation, but a net loss of $55 for cows treated in late lactation. Lactation number did not have a significant impact on economic benefits of treatment. In contrast to other studies indicating no economic benefit from treating mastitis during lactation, this study's positive results may have been attributable to the high cure rate (98%) and the subclinical form of mastitis being treated. Streptococcus agalactiae mastitis treatment during early and midlactation would appear to be an economically justifiable option for dairy managers.     

Automated detection of estrus and mastitis in dairy cows

The development and test of detection models for oestrus and mastitis in dairy cows is described in a PhD thesis that was defended in Wageningen on June 5, 2000. These models were based on sensors for milk yield, milk temperature, electrical conductivity of milk, and cow activity and concentrate intake, and on combined processing of the sensor data. The models alert farmers to cows that need attention, because of possible oestrus or mastitis. A first detection model for cows, milked twice a day, was based on time series models for the sensor variables. A time series model describes the dependence between successive observations. The parameters of the time series models were fitted on-line for each cow after each milking by means of a Kalman filter, a mathematical method to estimate the state of a system on-line. The Kalman filter gives the best estimate of the current state of a system based on all preceding observations. This model was tested for 2 years on two experimental farms, and under field conditions on four farms over several years. A second detection model, for cow milked in an automatic milking system (AMS), was based on a generalization of the first model. Two data sets (one small, one large) were used for testing. The results for oestrus detection were good for both models. The results for mastitis detection were varying (in some cases good, in other cases moderate). Fuzzy logic was used to classify mastitis and oestrus alerts with both detection models, to reduce the number of false positive alerts. Fuzzy logic makes approximate reasoning possible, where statements can be partly true or false. Input for the fuzzy logic model were alerts from the detection models and additional information. The number of false positive alerts decreased considerably, while the number of detected cases remained at the same level. These models make automated detection possible in practice.     

Relationship between teat morphological traits and subclinical mastitis in Frieswal dairy cows

The present study was aimed to investigate the relationship between selected morphological traits of teat and subclinical mastitis (SCM) in Frieswal crossbred dairy cows. A total of 1040 quarters from 261 lactating cows were evaluated for teat shape (bottle/fleshy/collapsed/conical/normal/pencil and short), teat-end shape (dished/flat/funnel/pocketed/pointed and rounded), teat orientation (aligned/misaligned) and teat position (front and rear; left-sided and right-sided). Each udder quarter was screened with California Mastitis Test (CMT) for the purpose of defining quarter health status. Data were analysed using Chi-square test and multivariable logistic regression procedure. An overall prevalence of SCM (CMT positive) at quarter level was 30.6%. Most of the teats had normal or cylindrical shapes (48%), dished teat-ends (40.7%), and aligned (central or squared) in orientation (65%). At bivariable level, significant association of SCM with teat shape, teat position, teat orientation, parity, and stage of lactation was observed (P < 0.05 to P < 0.001). Teat-end shapes showed some association with SCM (P = 0.07). Results of multivariable analysis showed that pencil-shaped teats were least associated with SCM (P < 0.05) as compared to other teat shapes. Prevalence of SCM was also higher in rear teats (P = 0.015), misaligned teats (P = 0.01), and cows in second or higher parities (P < 0.01) and late stage of lactation (P < 0.001). The results of the present study indicate that selected morphological traits of teat are associated with SCM in Frieswal crossbred cows; therefore, selection towards desirable morphological traits could help reduce mastitis in this breed.     

应用体细胞计数监测奶牛隐性乳房炎

奶牛乳汁中体细胞数的变化受多方面因素的影响。本文主要论述用体细胞计数检测奶牛隐性乳房炎技术、以及将体细胞数作为标记性状、选育具有乳房炎抗性奶牛的研究。     

Treatment of Streptococcus agalactiae mastitis in dairy cows: comparative efficacies of two antibiotic preparations and factors associated with successful treatment

A commercially available, penicillin-novobiocin, intramammary infusion product and a solution of procaine penicillin G (1.2 X 10(6) IU) in 10 ml of sterile saline solution were evaluated for their comparative efficacies against Streptococcus agalactiae mastitis in 3 California dairy herds. After composite milk samples from each cow in each herd were bacteriologically cultured, cows infected with S agalactiae (n = 228) were assigned randomly to 2 treatment groups. Milk samples were reevaluated bacteriologically 21 to 25 days after treatment. Both preparations were highly effective against S agalactiae in first-lactation cows and in cows scored negative or trace by use of the California Mastitis Test. Efficacy was significantly decreased in cows with California Mastitis Test scores of 1, 2, or 3. Herd and treatment were associated significantly with treatment success or failure. Most treatment failures were in one herd in cows that were given procaine penicillin G in sterile saline solution. Milk production and lactation stage were not associated with success or failure of treatment.     

Progressive pathology of severe Escherichia coli mastitis in dairy cows

An investigation was made using light and electron microscopy of the progressive pathological changes in nine experimental and two natural cases of severe Escherichia coli mastitis in dairy cows. The duration of infection varied from 18 hours to 13 days. Epithelial lesions were not found in glands which had been infected for more than 24 hours. However, the epithelia of the sinuses and large ducts became hyperplastic after 60 hours of infection and by six days hyperplasia was extensive on the crests of folds. The leucocyte response in the lumina of the glands and subepithelial tissue showed a progressive change from an acute neutrophil reaction to a chronic mononuclear cell infiltration within the first 36 hours of infection. The only changes affecting the secretory tissue occurred after six days of infection and were typical of mammary gland involution which was probably a direct consequence of anorexia.