单测试片法定性快速检测乳中金黄色葡萄球菌的研究

目的 建立单测试片定性快速检测乳中金黄色葡萄球菌的方法。方法 选择7.5%氯化钠肉汤增菌液, 使用拍打式均质器, 匀浆时间1~1.5 min进行样品处置。采集不同基质样品, 设计多种方案对研究单测试片方法、国标方法与测试片方法同时进行验证。结果 该方法检测金黄色葡萄球菌特异性高, 特异性达到96.7%。最小检出量0.5 CFU/mL。阳性检出率高。结论 该方法准确性可靠, 特异性、灵敏度都高, 检出限低, 适用于乳中的金黄色葡萄球菌快速定性检测。     

Effect of segregation on prevention of intramammary infections by Staphylococcus aureus

Effectiveness of segregating cows with Staphylococcus aureus intramammary infections was studied over 1 yr. Nine herds were split into control (n = 5) or segregated (n = 4) groups. Cows with S. aureus intramammary infections were milked last in segregated herds. Monthly milk samples were collected aseptically for microbiologic analysis. Mean incidences of S. aureus intramammary infections were 3.7 and 4.3 cases/100 cow-mo in segregated and control herds. The mean prevalence of S. aureus intramammary infections decreased in both segregated and control herds during the study. Mean percentages of cows with S. aureus intramammary infections at the beginning and end of the study were 33.7 and 21.5 in segregated herds and 25.3 and 15.0 in control herds. Cows in all herds with S. aureus intramammary infections were preferentially culled. There were no significant differences in incidence and prevalence of S. aureus intramammary infections between groups, suggesting that S. aureus intramammary infections can be controlled without segregation.     

Study on the relationship between milk immune factors and Staphylococcus aureus intramammary infections in dairy cows

The distribution of Staphylococcus aureus within herds seems to be related to interactions among the shedding characteristics of the bacteria, their pathogenicity and mammary gland immune status. The aim of the present study was to investigate the relationships between selected mammary gland immune factors and intramammary infections associated with Staph. aureus. Overall, 70 cows from five commercial dairy herds were included in the study and quarter milk samples were assessed using bacteriological and cytological tests. We evaluated differential cell count, lysozyme concentration, N-acetyl-beta-glucosaminidase (NAGase) activity, cell viability and respiratory burst activity in randomly chosen quarter milk samples from each cow. Staph. aureus intramammary infection elicited different responses in the mammary gland immune defences investigated. Polymorphonuclear leucocytes (PMN) as a proportion of total somatic cells in milk, cell viability and NAGase activity were higher in infected quarters, while the proportions of macrophages and lymphocytes, respiratory burst activity and lysozyme levels were lower. Mean values differed among herds, but the differences were not significant. These changes were associated with Staph. aureus infection. The reduced respiratory burst activity together with the increase in the proportion of PMN suggests that both the number and activity of PMN could influence the susceptibility of the mammary gland to pathogens. Indeed, the logistic model adopted suggests that impairment of milk immune factors could be concurrent with the development of an infection.     

Efficacy of different Lysigin formulations in the prevention of Staphylococcus aureus intramammary infection in dairy heifers

The objective was to compare the efficacy of two experimental Staphylococcus aureus mastitis bacterins and a currently marketed five-isolate-based Staph. aureus bacterin (Lysigin, Boehringer Ingelheim Vetmedica, Inc.) with unvaccinated controls. Forty-seven Holstein-Friesian heifers were randomly assigned to one of four groups such that Group 1 (n=11) received a three-isolate experimental bacterin, Group 2 (n=11) received a five-isolate experimental bacterin, Group 3 (n=14) received Lysigin, and Group 4 (n=11) served as unvaccinated controls. Vaccinations were administered twice 28 d apart in late gestation. All groups were challenged with a heterologous strain of Staph. aureus (ATCC 29740) on days 6, 7, and 8 of lactation. Mastitis score, somatic cell count (SCC), milk culture yield, and total daily milk yield data were collected before and after challenge. All 47 cattle developed a Staph. aureus IMI post-challenge with three animals in Group 1 and one animal in Group 3 clearing their Staph. aureus IMI by the end of the study. However, there was no evidence of a difference between vaccinates and control with regard to Staph. aureus clearance rates post-challenge (P> or =0.214). Cattle vaccinated with Lysigin had a lower mean duration of clinical mastitis and lower total mastitis score post-challenge than controls (P=0.045 and P=0.046, respectively). Overall, there was no evidence that any of the vaccinated groups had a lower mean SCC than control (P> or =0.148) for the tested study days. Likewise there was no evidence that vaccinates had greater milk yield than controls post-challenge (P=0.617). Hence, there was no evidence that the vaccines reliably prevented Staph. aureus IMI, but Lysigin showed benefit in reducing the clinical severity and duration of clinical disease post-challenge. Neither of the experimental bacterins appeared to perform better than Lysigin.     

Shedding pattern of Staphylococcus aureus from bovine intramammary infections

Twenty-one quarters of seven cows were experimentally infected with Staphylococcus aureus (ATCC 29740) to study the shedding pattern in quarter milk samples. Of 991 consecutive quarter milk samples collected from infected quarters during the trial, 745 were positive for S. aureus by bacteriological culture. The sensitivity of a single quarter milk sample to determine infection status of a quarter was 74.5% based on the mean of each gland's recovery pattern. Sensitivity of bacterial culture increased to 94% and 98% by including a second and a third consecutive sample. Because S. aureus is shed in a cyclical manner from mammary glands, consecutive samples would be advisable for accurate diagnosis of infected quarters.     

二重环介导等温扩增法快速检测乳粉中沙门氏菌和金黄色葡萄球菌

为建立能够同时检测乳粉中沙门氏菌和金黄色葡萄球菌的二重环介导等温扩增(loop-mediated isother-mal amplification,LAMP)方法.针对沙门氏菌invA基因、金黄色葡萄球菌nuc基因保守区域设计LAMP引物,优化引物浓度,并通过熔解曲线分析判断扩增靶标来源,建立同时检测沙门氏菌和金黄色...     

Efficacy of intramammary tilmicosin and risk factors for cure of Staphylococcus aureus infection in the dry period

The objective ofthis study was to evaluate the efficacy of intramammary tilmicosin, administered at drying-off, for eliminating Staphylococcus aureus infection, and to identify risk factors for S. aureus cure during the dry period. A total of 219 naturally infected cows, representing 308 quarters, were randomized to receive either one of two treatments at drying-off. Cows received either an intramammary infusion of 500 mg of benzathine cloxacillin, or a sterile solution containing 1,500 mg of tilmicosin. All cows had quarter milk samples taken aseptically three times before dry-off, and at wk 1, 2, and 4 of the subsequent lactation. Overall, 62% of cows and 67.5% of quarters infected with S. aureus cured during the dry period. The cure following administraton of tilmicosin was 67.3 and 72.5% for cows and quarters, respectively. By comparison, the cure achieved with cloxacillin was 56.9 and 62.9% of cows and quarters. Cows receiving tilmicosin were 2.1 times more likely to cure. The cure rate for cows decreased as the linear score on the last DHI test increased, and as the amount of S. aureus being shed increased. Quarters that cultured positive multiple times before drying-off were less likely to cure. Staphylococcus aureus infections located in front quarters of the udder were 2 times more likely to cure than those in hind quarters. Results of this study demonstrate that intramammary tilmicosin at drying-off is efficacious in curing existing S. aureus during the dry period. Risk factors associated with the cure of S. aureus were identified.     

Effect of vacuum fluctuation during milking on the development of intramammary infection from teat duct colonization by Staphylococcus aureus

Twelve of twenty-one udder quarters with colonized teat ducts became infected within 13 d when milked with a machine producing large cyclic and irregular vacuum fluctuations in the teatcup; of 23 quarters milked under the same conditions, but with metal shields fitted inside the liners to protect the teat apex (Thiel, 1974), only 2 quarters became infected in the same period. The introduction of a post-milking teat disinfectant teat dip resulted in the elimination of orifice colonization from 15 teats dipped in Na hypochlorite solution (40 g/l available chlorine) and from all but 2 of 15 teats dipped in an iodophor solution (5 g/l available iodine).     

Effects of teat dipping on intramammary infections by staphylococci other than Staphylococcus aureus

Effects of intramammary infections in herds 1) either not teat dipping or postmilking teat dipping with either 2) linear dodecyl benzene sulfonic acid, 3) chlorhexidine, or 4) iodophor containing products were determined. Duplicate quarter foremilk samples were collected once from all lactating cows in 16 herds, four for each practice. Mean percentage of quarters infected with Staphylococcus species other than Staph. aureus was 11.0 in herds not teat dipping and 7.2 in herds teat dipping. Prevalence of Staphylococcus species intramammary infections in herds teat dipping with linear dodecyl benzene sulfonic acid was comparable to herds not teat dipping and greater than in herds using chlorhexidine and iodophor. The predominant Staphylococcus species in herds not teat dipping was Staph. epidermidis (37.1%). Staphylococcus hyicus constituted 48.5% of total Staphylococcus species intramammary infections in herds teat dipping and 12.4% in herds not teat dipping. Differences were not observed among SCC for quarters infected with different Staphylococcus species. Application of germicidal teat dips appeared to have selectively altered both prevalence and distribution of Staphylococcus species intramammary infections.     

Saltatory rolling circle amplification for sensitive visual detection of Staphylococcus aureus in milk

Monitoring Staphylococcus aureus with high sensitivity is very important for ensuring milk quality and food safety. In this study, we used a rapid nucleic acid isothermal amplification method, saltatory rolling circle amplification (SRCA), for the detection of Staph. aureus in milk. The results of the SRCA method can be assessed visually by the presence of white precipitate or by fluorescence measurement. Thirteen Staph. aureus strains and 31 non-Staph. aureus strains were used to evaluate the specificity of SRCA. The method exhibited excellent detection of Staph. aureus genomic DNA at a concentration of 7.8 × 10¹ fg/µL when assessed by visible precipitate, and at 7.8 × 10⁰ fg/µL when detected by fluorescence after addition of the fluorochrome SYBR Green I. In artificially inoculated milk, the detection limits of SRCA were 5.6 × 10² cfu/mL by precipitate and 5.6 × 10¹ cfu/mL by fluorescence, respectively. Compared with conventional PCR approaches, the SRCA assay achieved at least 100-fold higher sensitivity. Moreover, the sensitivity, specificity, and accuracy of the SRCA-based system were calculated to be 100.00, 97.73, and 97.78%, respectively. These results indicate that SRCA has potential application as a sensitive and visual technique for the detection of Staph. aureus in milk.     

Efficacy of a biological response modifier in preventing Staphylococcus aureus intramammary infections after calving.

A change in the epidemiology of mastitis in recent years has emphasized the role of the udder immune system in the pathogenesis of Staphylococcus aureus. Therefore, if the bovine or udder immune capability could be enhanced, susceptibility to Staph. aureus could be reduced and antibiotic efficacy could be increased. Immune system defense mechanisms could be enhanced by vaccination and by biological response modifiers. Within this latter group, a biological response modifier obtained from Parapox ovis that was attenuated over 200 tissue culture passages was recently developed and commercialized in some European countries. This study reports the results of a field trial on the efficacy of this biological response modifier in reducing Staph. aureus intramammary infection (IMI) after calving in primiparous and pluriparous cows. The trial included 106 cows sampled six times (55 cows from herd A and 51 from herd B) for a total of 2544 quarter milk samples. The analysis of IMI prevalence showed that 25.09% of samples were bacteriologically positive in the placebo group, and 23.17% of the positive samples were observed in the biological response modifier group. Staphylococcus aureus IMI had a frequency of 11.44% in the placebo group and 6.00% in the biological response modifier group. The dynamic of the hazards showed significantly lower rates in the biological response modifier group than in the placebo group (risk ratio = 0.47). Treatment with the parapox-containing biological response modifier showed significant reduction of Staph. aureus IMI around calving, and this reduction was attributed to an increase in immune defenses.     

Optimization of the PCR for detection of Staphylococcus aureus nuc gene in bovine milk

Staphylococcus aureus is an economically important and a major mastitis-causing pathogen that also poses food safety and antimicrobial resistance threats. Substances in mastitic milk inhibit the Taq DNA polymerase reaction (Taq PCR) making it of limited use for detecting S. aureus mastitis. In the study reported here, a set of oligonucleotide primers of 21 and 24 bases was used in Taq-PCR to amplify DNA from S. aureus (isolates from bovine mastitis). A specific amplicon of 270 bp was generated as predicted. Replacing Taq DNA polymerase with Thermus thermophilus (Tth) DNA polymerase alone (Tth-PCR) raised the sensitivity of S. aureus detection in milk from experimentally infected cows from 65 to 80%. Combining the use of Tth DNA polymerase and the purification of crude DNA extract using Chelex-100 before PCR raised the sensitivity to 100%. In a random survey involving 100 milk samples from cattle not infected with S. aureus, the test was 100% specific. With milk samples from clinical cases of bovine mastitis, 100% sensitivity and specificity were also observed. It is concluded that Tth-PCR on milk samples with the purification of crude DNA extracts using Chelex-100 is as sensitive as but faster than conventional milk bacteriological culture techniques and is highly specific. The modified PCR correlates with elevated somatic cell counts, detects evidence of chronic and resolving infection based on S. aureus-specific DNA and circumvents the endogenous inhibitory effects of milk.     

Evaluation of breed-dependent differences in the innate immune responses of Holstein and Jersey cows to Staphylococcus aureus intramammary infection

Mastitis is one of the most prevalent diseases of cattle. Various studies have reported breed-dependent differences in the risk for developing this disease. Among two major breeds, Jersey cows have been identified as having a lower prevalence of mastitis than Holstein cows. It is well established that the nature of the initial innate immune response to infection influences the ability of the host to clear harmful bacterial pathogens. Whether differences in the innate immune response to intramammary infections explain, in part, the differential prevalence of mastitis in Holstein and Jersey cows remains unknown. The objective of the current study was to evaluate several parameters of the innate immune response of Holstein and Jersey cows to intramammary infection with Staphylococcus aureus, a common mastitis-inducing pathogen. To control for non-breed related factors that could influence these parameters, all cows were of the same parity, in similar stages of milk production, housed and managed under identical conditions, and experimentally infected and sampled in parallel. The following parameters of the innate immune response were evaluated: acute phase protein synthesis of serum amyloid A and lipopolysaccharide-binding protein; total and differential circulating white blood cell counts; milk somatic cell counts; mammary vascular permeability; milk N-acetyl-beta-d-glucosaminidase (NAGase) activity; and production of the cytokines, interferon (IFN)-gamma, interleukin (IL)-12, tumour growth factor(TGF)-alpha, and TGF-beta1. The temporal response of all of these parameters following infection was similar between Holstein and Jersey cows. Further, with the exception of changes in circulating neutrophils and NAGase activity, the overall magnitude of these parameters were also comparable. Together, these data demonstrate that the innate immune response of Holstein and Jersey cows to Staph. aureus intramammary infection remains highly conserved despite previously reported differences in mastitis prevalence, as well as genotypic and phenotypic traits, that exist between the two breeds.     

Short communication: influence of Staphylococcus aureus intramammary infection on serum copper, zinc, and iron concentrations

The goal of the present study was to characterize changes in serum trace mineral concentrations in cattle with experimentally induced Staphylococcus aureus mastitis. Nine primiparous Holstein-Friesian cattle were challenged with approximately 150 cfu of Staph. aureus ATCC29740 by intramammary infusion on d 6, 7, and 8 of lactation. Serum Cu, Zn, and Fe concentrations were determined immediately before and at 24, 48, and 72 h after the final intramammary infusion of Staph. aureus. Infection status (cfu/mL of Staph. aureus), milk somatic cell count, and mastitis score were also determined at these times. Infection resulted in a decrease in mean serum Cu, Zn, and Fe concentrations to 89, 83, and 81% of preinfection concentrations at 24 h postchallenge. One-way analysis of variance for repeated measures demonstrated a significant change in serum zinc concentration. The reductions in trace mineral concentrations were of less magnitude than observed following experimental E. coli mastitis.     

Development of a highly sensitive and specific assay to detect Staphylococcus aureus in bovine mastitic milk

Diagnosis of udder infections with Staphylococcus aureus by bacteriological milk testing of quarter milk samples is often not satisfactory. To get reliable results, repeated sampling is necessary, which is normally too expensive. Therefore, we developed a test that allows the highly specific detection of Staph. aureus in bovine milk samples at very low concentrations. It is based on a fast procedure to prepare bacteria from milk, followed by DNA extraction and quantitative PCR. The whole analysis is done within 5 h. For clinical milk samples, the analytical sensitivity of the assay was 50.7 times and 507 times higher than conventional bacteriology with 100 and 10 μL, respectively. The diagnostic specificity was 100%. The test is further characterized by a low intra- and interassay variability as well as by a good recovery of Staph. aureus from raw milk. Furthermore, a high correlation (R = 0.925) between the agar plate counts and the quantitative PCR methodology over the whole range of measurement was found. In addition, our test revealed considerably more positive results than bacteriology. Due to its favorable properties, the assay might become an important diagnostic tool in the context of bovine mastitis caused by Staph. aureus.     

Staphylococcus aureus strains in primiparous and multiparous cows in six herds with a high prevalence of Staph. aureus intramammary infections

The proportion of different strains of Staphylococcus aureus was tested in four groups of lactating dairy cows in six herds with a high overall prevalence of Staph. aureus using random amplified polymorphic DNA PCR. Group 1 included primiparous cows in early lactation (<50 days in milk, DIM). Group 2 consisted of primiparous cows in late lactation (>250 days in milk). Groups 3 and 4 were multiparous cows in the respective stages of lactation. Eight cows from each group on each farm were tested. Overall quarter prevalence of Staph. aureus ranged from 23.4 to 32.0% in the herds. Of the 130 isolates included in the analysis 86.9% were high prevalence strains (more than three isolates per herd), while 13.1% were strains that were only identified in one or two samples. Low prevalence strains were found in all six herds. The proportion of low prevalence strains was higher in multiparous than in primiparous cows (odds ratio, OR 4.4, 1.2-16.6). It is concluded that low prevalence Staph. aureus strains are common even in herds with a high prevalence of Staph. aureus and that their frequency is lower in primiparous cows than in older cows.     

免疫磁球捕获-PCR检测牛奶中金黄色葡萄球菌的研究

对免疫磁球捕获-PCR(IMS-PCR)检测牛奶中金黄色葡萄球菌进行了初步研究。优化了免疫磁球制备参数,确定了金黄色葡萄球菌免疫磁球对目标菌的结合时间。研究结果显示,当纯菌浓度为101~104CFU/m L水平时,金黄色葡萄球菌免疫磁球对目标菌的捕获率大于80%。通过对目标菌和非目标菌的检测,IMS-PCR检测方法显示了很强的特异性;在纯培养、无需增菌情况,IMS-PCR检测方法检测限为104CFU/m L;牛奶中的金黄色葡萄球菌经磁分离后增菌2h用PCR检测,可检测出104CFU/m L的金黄色葡萄球菌。     

免疫磁球捕获-PCR检测牛奶中金黄色葡萄球菌的研究

对免疫磁球捕获-PCR（IMS-PCR）检测牛奶中金黄色葡萄球菌进行了初步研究。优化了免疫磁球制备参数,确定了金黄色葡萄球菌免疫磁球对目标菌的结合时间。研究结果显示,当纯菌浓度为101¹04CFU/m L水平时,金黄色葡萄球菌免疫磁球对目标菌的捕获率大于80%。通过对目标菌和非目标菌的检测,IMS-PCR检测方法显示了很强的特异性;在纯培养、无需增菌情况,IMS-PCR检测方法检测限为104CFU/m L;牛奶中的金黄色葡萄球菌经磁分离后增菌2h用PCR检测,可检测出104CFU/m L的金黄色葡萄球菌。