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.     

Determination of antibodies to Streptococcus agalactiae in the milk of dairy cows using the ELISA test

A sensitive 4-layers ELISA test for determination of antibodies against the pathogens of Streptococcus agalactiae in cows' milk was used for diagnosis of mastitis, with the aim to broaden these methods. Antigen was linked on the solid phase in the form of the whole bacteria, and milk was tested, diluted in the ratio of 1:10. Antigen bound-specific antibodies were labelled with pig antibodies against bovine immunoglobulins and in the next layer with rabbit antibody conjugated with peroxidases against pig immunoglobulins. After test visualisation and reading on the photometre, the results were given in the positivity per cent as a 100-multiple of the proportion of absorbance of the unknown sample and the positive control after subtraction of the negative control. Milk was examined in 36 dairy cows from three various breeding herds by that method. The samples were parallelly examined bacteriologically and cytologically. In the milk of dairy cows with positive S. agalactiae finding, the main level of antibodies expressed a positivity per cent, was 15.0%, while in bacteriologically negative animals it was only 6.2%. The dairy cows were divided into 8 groups, characterizing various stages of mastitis, according to the results of the individual treatments.     

木糖醇对奶牛无乳链球菌的抑菌作用及其生物被膜干预

通过电镜观察木糖醇对引起奶牛乳房炎的无乳链球菌生物被膜形成的影响并探索其抑菌机理。首先采用卡尔加里生物被膜发生装置与结晶紫染色法定量分析和评价无乳链球菌生物被膜形成能力,再选取生物被膜形成能力强的典型菌株,应用内置载体片法构建生物被膜,并进行扫描电镜观察;最后,分别通过透射及扫描电镜分析不同浓度的木糖醇对无乳链球菌及其生物被膜的影响。内置载体片法试验结果显示,无乳链球菌的生物被膜完全成熟需要连续培养4 d,此时细菌黏附于接触表面,分泌多糖基质等并逐渐将其自身包绕其中;透射电镜观察结果显示,木糖醇可通过破坏无乳链球菌结构来抑制其生长,且随浓度增加抑菌效果亦显著增加。此外,不同质量浓度木糖醇溶液(0.05,0.15,0.30和0.50 kg/L)处理的载体片上生物被膜细菌数量均有不同程度的减少,其中以0.50 kg/L最为显著。结果表明,木糖醇可改变无乳链球菌超微结构,也可通过破坏其生物被膜来抑制细菌生长。     

Managerial risk factors of intramammary infection with Streptococcus agalactiae in dairy herds in Ohio.

Dairy herds in Ohio were selected by stratified random sampling for participation in a disease-monitoring study to relate Streptococcus agalactiae intramammary prevalence to herd management and environmental conditions. Of 48 herds studied, 27 herds had at least 1 cow infected with this pathogen. Management and environmental conditions were assessed by direct observation as well as by an interview with the dairy producers. One-way ANOVA or chi 2 analysis, with presence or absence of Streptococcus agalactiae as the dependent variable, was used to test each of 70 independent variables. Variables found significant at P less than 0.20 were further evaluated by use of logistic regression. Our sample size permitted only 4 independent variables to be simultaneously evaluated by logistic regression. The most predictive risk factors were identified as poor teat and udder hygiene, poor environmental sanitation, large herd population, and use of a shared washcloth for premilking cleaning of teats and udders.     

Relationship between morphological characteristics of the teat duct and prevalence of intramammary infections with Streptococcus agalactiae in dairy cows

The objective of the study was to determine the relationship between morphological findings of the surface of teat duct particularly the level of ceratosis and the prevalence of intramammary infections (IMI). The study was conducted on a commercial dairy herd housing about 3000 lactating dairy cows. We examined 891 quarters in the middle of lactation. Duplicate samples of quarter foremilk were collected monthly. The bacteriological status of quarters was determined according to the recommendations of IDF. At the same time teats were evaluated by clinical examinations. The appearance of teat skin lesions and the status of the teat duct especially the existence of hyperceratosis (HC) was documented. Four classes of teat duct hyperceratosis were defined: without, slight, medium and severe HC. The rate of IMI in different classes of hyperceratosis of teat duct was compared by Chi-square analysis. Prevalences of intramammary infections were determined three times (P1, P2 and P3) during the study period. Prevalence of infection was high for S. aureus (P1: 5.6% vs. P2: 4.5% vs. P3: 4.3%), Sc. agalactiae (P1: 2.7% vs. P2: 2.6% vs. P3: 2.8%) and CNS (P1: 10.7% vs. P2: 8.8% vs. P3: 9.6%). Furthermore we detected IMI caused by other streptococci, yeast, E. coli and mixed infections. A positive correlation between status of HC and prevalence of IMI for Sc. agalactiae was found. At the second and third sampling time the rate of intramammary infection with Sc. agalactiae in quarters with medium HC (P2: 9.21% and P3: 13.73%) differed significantly (p < 0.05) compared to groups without (P2: 1.56% and P3: 1.91%) and slight hyperceratosis (P2: 2.33% and P3: 2.56%). The results of our study indicate a correlation between morphology of teat duct surface, especially regarding to Sc. agalactiae. On one hand HC can cause high intramammary infection rate with Sc. agalactiae. On the other hand it is possible that HC is the consequence of a quarter infection with Sc. agalactiae. Further research is required.     

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.     

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.     

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.     

双重PCR检测奶牛隐性乳房炎大肠杆菌与无乳链球菌方法的建立及应用

为快速检测奶牛隐性乳房炎主要致病菌大肠杆菌和无乳链球菌,分别针对2种致病菌16S-23S rRNA和16S rRNA基因设计2对特异性引物,优化并确立双重PCR反应体系。特异性检测表明,对其他对照菌株未扩增出目的条带;敏感性试验表明,该双重PCR对大肠杆菌、无乳链球菌的最小检测浓度分别为10~2、10~3cfu/mL。同时采用双重PCR与细菌学检查法对送检的96份奶样进行检测,结果双重PCR检出63份大肠杆菌阳性、54份链球菌阳性;细菌学方法检出29份大肠杆菌阳性、37份链球菌阳性。说明本研究建立的双重PCR方法敏感、快速,可用于检测大肠杆菌和无乳链球菌引起的奶牛隐性乳房炎。     

无乳链球菌体外感染奶牛乳腺上皮细胞

用分离到的无乳链球菌(Streptococcus agalactiae,GBS)及标准菌株侵袭原代奶牛乳腺上皮细胞建立体外感染细胞模型,并进行细胞凋亡检测。同时,采用qRT-PCR方法检测细胞受到侵袭后白细胞介素6(IL-6)、白细胞介素8(IL-8)、肿瘤坏死因子(TNF-α)等细胞炎性因子mRNA的转录水平。结果显示,标准菌株组(WL组)与空白组相比凋亡率差异极显著(P<0.01),分离菌株组(FL组)与空白组相比凋亡率差异极显著(P<0.01),分离菌株组与其标准菌株组的凋亡率差异也极显著(P<0.01)。qRT-PCR结果分析显示,标准株组TNF-α、IL-6及IL-8 mRNA转录水平与对照组相比,差异极显著(P<0.01);而分离菌株组TNF-αmRNA转录水平与对照组相比差异显著(P<0.05),IL-6及IL-8 mRNA转录水平差异不显著(P>0.05)。结果表明,与标准菌株相比,无乳链球菌分离株对奶牛乳腺上皮细胞侵袭和损伤能力较低,乳腺细胞的凋亡率也较低,当奶牛乳腺上皮细胞受到细菌侵袭的时候,细胞中IL-6、IL-8及TNF-α等炎性因子mRNA的转录水平都显著性提高,但在相同浓度下GBS标准株诱导细胞炎性因子的表达量显著高于GBS分离株。本试验结果为以后预防和控制GBS引起的隐性乳房炎提供试验依据。     

Treatment of persistent intramammary infections with Streptococcus uberis in dairy cows

A survey was conducted of the prevalence of environmental pathogens, especially Streptococcus uberis, as causes of clinical mastitis in dairy cows. The response of intramammary infections with S uberis to conventional treatment was monitored by taking milk samples for bacteriology and somatic cell counting seven, 14 and 21 days after the treatment. The results showed that 51 per cent of the infections failed to respond, and the odds of cases failing to respond was significantly increased when the individual quarter somatic cell count seven days after the treatment was greater than 201,000 cells/ml. Ninety-six per cent of the suspected S uberis isolates identified by culture were confirmed as S uberis by using the api 20 Strep system. Restriction endonuclease fingerprinting was used to type the strains of S uberis isolated from 75 milk samples from 32 cows. Analysis showed that 96 per cent of the cases of S uberis that failed to respond to conventional treatment were persistent infections with one strain rather than reinfections with different strains. The persistent cases of S uberis were treated further with an extended course of intramammary preparations containing either procaine penicillin with dihydrostreptomycin or cefquinome. There was no significant difference between the cure rates achieved by the two preparations, and 55 per cent of the cases that had failed to respond to conventional treatment responded to the additional treatment.     

Annual incidence, prevalence and transmission characteristics of Streptococcus agalactiae in Danish dairy herds

Contagious mastitis pathogens continue to pose an economic threat to the dairy industry. An understanding of their frequency and transmission dynamics is central to evaluating the effectiveness of control programmes. The objectives of this study were twofold: (1) to estimate the annual herd-level incidence rates and apparent prevalences of Streptococcus agalactiae (S. agalactiae) in the population of Danish dairy cattle herds over a 10-year period from 2000 to 2009 inclusive and (2) to estimate the herd-level entry and exit rates (demographic parameters), the transmission parameter, β, and recovery rate for S. agalactiae infection. Data covering the specified period, on bacteriological culture of all bulk tank milk samples collected annually as part of the mandatory Danish S. agalactiae surveillance scheme, were extracted from the Danish Cattle Database and subsequently analysed. There was an increasing trend in both the incidence and prevalence of S. agalactiae over the study period. Per 100 herd-years the value of β was 54.1 (95% confidence interval [CI] 46.0-63.7); entry rate 0.3 (95% CI 0.2-0.4); infection-related exit rate 7.1 (95% CI 5.6-8.9); non-infection related exit rate 9.2 (95% CI 7.4-11.5) and recovery rate 40.0 (95% CI 36.8-43.5). This study demonstrates a need to tighten the current controls against S. agalactiae in order to lower its incidence.     

鱼类无乳链球菌病

无乳链球菌亦称为B组链球菌,是一种在自然界广泛存在的革兰阳性菌,是人类的重要病原之一,也是鱼类的重要病原菌。近年来,无乳链球菌成为鱼类链球菌病的主要病原之一,鱼类感染无乳链球菌的常见临床症状包括眼球突出、腹部肿胀、脊骨弯曲、鳍条基部出血等,发病率和死亡率高,常造成严重的经济损失,危害着水产养殖业的健康发展。论文对鱼类无乳链球菌病的研究进行了总结,对鱼类无乳链球菌病病原、致病机理进行综述,介绍了鱼类无乳链球菌病常见的诊断方法,以及现阶段针对鱼类无乳链球菌病的疫苗开发情况,旨在进一步丰富、完善鱼类无乳链球菌病的研究资料,为更好的防治鱼类无乳链球菌病提供参考。     

In vitro antimicrobial activity of Combretum molle (Combretaceae) against Staphylococcus aureus and Streptococcus agalactiae isolated from crossbred dairy cows with clinical mastitis

Following the rapidly expanding dairy enterprise, mastitis has remained the most economically damaging disease. The objective of this study was mainly to investigate the in vitro antibacterial activities of ethanol extracts of Combretum molle (R.Br.Ex.G.Don) Engl & Diels (Combretaceae) against antibiotic-resistant and susceptible Staphylococcus aureus and Streptococcus agalactiae isolated from clinical cases of bovine mastitis using agar disc diffusion method. The leaf and bark extracts showed antibacterial activity against S. aureus at concentrations of 3 mg/ml while the stem and seed extract did not show any bioactivity. Although both leaf and bark extracts were handled in the same manner, the antibacterial activity of the bark extract against the bacterial strains had declined gradually to a lower level as time advanced after extraction. The leaf extract had sustained bioactivity for longer duration. The susceptibility of the bacteria to the leaf extract is not obviously different between S. aureus and S. agalactiae. Also, there was no difference in susceptibility to the leaf extract between the antibiotic-resistant and antibiotic-sensitive bacteria. Further phytochemical and in vivo efficacy and safety studies are required to evaluate the therapeutic value of the plant against bovine mastitis.     

Immunogenic properties of Staphylococcus aureus and Streptococcus agalactiae administered separately and in combination to lactating cows

Composite bacterins of Staphylococcus aureus and Streptococcus agalactiae, or either bacterin alone, were administered systemically to groups of lactating cows. The response to each bacterin was unaffected by the simultaneous administration of the antigens when assessed by comparing the antibody levels in milk by enzyme-linked immunosorbent assay. There was no evidence of cross reactivity of the antigens studied, nor immunopotentiation by either bacteria.