清宫液2号质量标准的研究

为了建立清宫液2号的质量标准,试验采用薄层色谱法(TLC)对清宫液2号中丹参、红花进行定性鉴别;采用高效液相色谱法(HPLC)测定丹参中主要有效成分隐丹参酮和丹参酮ⅡA的含量。结果表明:供试品薄层色谱中,在与丹参、红花对照药材相应的位置上,分别显示相同颜色的斑点;隐丹参酮检测浓度在7.384~36.92μg/mL范围内与峰面积积分值线性关系良好(r=0.999 5),丹参酮ⅡA检测浓度在39.32~196.6μg/mL范围内与峰面积积分值线性关系良好(r=0.999 1)。说明建立的方法简便、快速、准确,可以用于清宫液2号的质量控制。     

高效液相色谱法测定苄星邻氯青霉素乳房注入剂中苄星邻氯青霉素的含量

用反相高效液相色谱法测定苄星邻氯青霉素乳房注入剂中苄星邻氯青霉素的含量。采用AgilentEclipseXDB-C185μm4.6×150mm色谱柱;流动相为0.1mol/L磷酸二氢钠-乙腈(3:1);检测波长为220nm;柱温为40℃。在该色谱条件下,苄星邻氯青霉素在20μg/ml～180μg/ml浓度范围内线性关系良好,相关系数r=0.99999;100%浓度时平均回收率为100.7%,RSD=0.04%。该方法快速灵敏,准确实用。     

高效液相色谱法测定癸氧喹酯含量试验方法的建立

本试验建立了高效液相色谱法测定癸氧喹酯干混悬剂含量的方法。采用XTerra C18柱(150 mm×4.6 mm,5μm)色谱柱;流动相:水+乙腈+甲醇(20:3:77);检测波长为265 nm。实验表明在10~60μg/m L的浓度范围内,峰面积与浓度呈良好线性关系,相关系数r=0.9999;平均回收率为99.8%;RSD=0.64%。该方法简便、快速,能准确、有效地测定癸氧喹酯干混悬剂的含量。     

AC4原料药含量的HPLC测定方法研究

为建立测定AC4原料药含量的高效液相色谱(HPLC)测定方法,色谱柱:Diamosil C18色谱柱(4.6 mm×250 mm,5μm),流动相为甲醇-0.2%磷酸水溶液,65∶35(v/v),流速为1.0 m L/min,检测波长251 nm。结果显示,AC4浓度在20~200μg/m L范围与峰面积呈现良好的线性关系,回归方程A=43743ρ+26 016,R2=0.9999(n=5),平均回收率100.3%,RSD为0.54%,样品至少在8 h内稳定。表明该法快速简捷、准确度高,适用于AC4原料药含量测定。     

中药复方子宫灌注液中盐酸小檗碱和丹酚酸B的质量控制

为了建立中药复方子宫灌注液的质量标准,采用薄层色谱法(TLC)对其中的黄连和丹参进行定性鉴别,并采用高效液相色谱(HPLC)法测定黄连中盐酸小檗碱和丹参中丹酚酸B的含量。结果:薄层色谱斑点清晰,阴性对照无干扰;盐酸小檗碱含量测定的线性方程为Y=38553X-89683(r=0.9998),进样量在4.1616~83.232μg/m L浓度范围内具有良好线性关系,平均回收率为98.59%,RSD为0.70%;丹酚酸B含量测定的线性方程为Y=1×107X-80303(r=0.9999),进样量在0.02952~1.476 mg/m L浓度范围内具有良好线性关系,平均回收率为98.60%,RSD为1.86%;结论:该方法简便易行,专属性强,重复性好,可用于该制剂的质量控制。     

高效液相色谱法测定恩诺沙星、盐酸小檗碱粉中两种有效成分含量

建立了恩诺沙星、盐酸小檗碱粉中两种有效成分含量的高效液相色谱测定法。采用C18色谱柱（4.6 mm×250 mm,5μm）,检测波长265 nm,流动相为乙腈-0.005 moL/L庚烷磺酸钠溶液（用磷酸调节pH值至3.0）（40∶60）,流速1.0 mL/min。结果显示,恩诺沙星在10.0～200.0μg/mL范围内含量与峰面积呈良好的线性关系,相关系数为0.9999（n=6）,平均回收率101.3%,RSD为2.0%;盐酸小檗碱在5.0～100.0μg/mL范围内含量与峰面积呈良好的线性关系,相关系数为1.0000（n=6）,平均回收率97.0%,RSD为2.6%。本方法简便、准确、快速、重现性好,可用于该制剂的含量测定。     

HPLC法测定盐酸小檗碱预混剂的含量

采用HPLC法测定盐酸小檗碱预混剂中盐酸小檗碱含量。色谱柱为Agilent ZORBAXSB-C18柱(3.9mm×150mm,4μm),流动相为0.02mol/L磷酸二氢钾溶液(用磷酸调pH为2.5)-乙腈(70∶30,V/V),检测波长346nm,流速1.0mL/min,柱温为30℃,保留时间约6.2min。盐酸小檗碱浓度在5~80μg/mL范围内,峰面积与浓度呈良好的线性关系(r2=0.9999)。平均回收率99.5%,RSD为0.4%。该方法适用于检测盐酸小檗碱预混剂。     

高效液相色谱法同时测定复合有机酸化剂中5种成分的含量

建立高效液相色谱法同时分离测定复合有机酸化剂中的烟酸、乳酸、柠檬酸、苯甲酸、水杨酸含量.采用Agela-ASB(r)C18柱(250 mm×4.6 mm,5μm);盐酸溶液(0.02%,V/V,A)和乙腈(B)为流动相,梯度洗脱;流速1.0mL/min;检测波长210 mn;柱温:(35±2)℃.在此色谱条件下,5种有机酸分离良好,它们的峰面积响应与浓度分别为烟酸1～20 μg/mL;乳酸100～2000 μg/mL;柠檬酸100～2000μg/mL;苯甲酸2.5～50μg/mL;水杨酸2.5～50μg/mL,线性关系良好(r>0.9995).平均加样回收率均分别为99.1%,98.2%,97.2%,96.3%,96.0%;RSD 小于1.0%.该方法操作简便易行,系统适用性良好,方法准确可靠.     

HPLC法测定黄山草灌注剂中绿原酸的含量

为完善黄山草灌注剂的标准,试验采用HPLC法测定绿原酸的含量,色谱柱为Agilent C18柱(250mm×4.6mm,5μm),流动相为甲醇∶1%冰醋酸(20∶80),检测波长为324nm,流速为1.0m L/min,进样量10μL。结果表明绿原酸含量在20~120μg/m L范围内线性关系良好(r=0.9999),平均加样回收率为99.45%,RSD为0.45%。此方法操作简便、快捷、结果准确,可用于黄山草灌注剂的质量控制     

HPLC法同步测定维生素预混合饲料中5种B族维生素

本实验建立同步测定维生素预混合饲料中烟酸、烟酰胺、维生素B6、维生素B2和维生素B1等5种B族维生素的高效液相色谱(HPLC)法。采用Hypersil C18色谱柱(250 mm×4.0 mm,5μm),流动相为p H 3.0的磷酸盐缓冲液(0.01 mol/L KH2PO4-0.005 mol/L辛烷磺酸钠-0.5%三乙胺)-甲醇(V/V,75∶25)等度洗脱,柱温为35℃,流速为1.0 m L/min,紫外检测器在波长275 nm进行检测,外标法定量。结果表明,烟酸和烟酰胺的线性范围为75~1500μg/m L,维生素B6、维生素B2和维生素B1的线性范围为25~500μg/m L,线性相关系数为0.9996~0.9999,平均回收率为98.5%~100.6%,相对标准偏差(RSD)为0.21%~0.61%。此方法具有简单、快速和准确的特点,适用于维生素预混合饲料中5种B族维生素含量的同步测定。     

奶牛乳房炎抗生素防治失败原因探讨

奶牛乳房炎主要由病原微生物引起,抗生素的应用在奶牛乳房炎控制中起了重要作用。本文着重从病原微生物—宿主—抗生素三个方面及相互作用关系中论述了奶牛乳房炎抗生素治疗失败的机制,包括:抗生素的选择、吸收、分布及抗生素的细胞内分布;细菌的被动选择、耐药性和细胞内寄生;细菌对吞噬细胞功能的影响,抗生素对吞噬细胞功能的影响及乳腺的重新感染。     

A framework for evaluation of on-farm mastitis diagnostics in Australia

Numerous culture-based diagnostics are available on the Australian and international markets for on-farm detection of bacterial pathogens in milk. Use of such diagnostics may provide an opportunity to improve the prudent use of antimicrobials in udder health management. Farms are low-resource settings in terms of diagnostic microbiology capacity. The World Health Organisation has identified criteria for the evaluation of diagnostic tests in low resource settings based on Accuracy, Sensitivity, Specificity, User-friendliness, being Rapid or Robust, Equipment-free and being Deliverable (ASSURED). Here, we review how those criteria can be interpreted in the context of microbiological diagnosis of mastitis pathogens, and how on-farm diagnostics that are currently available in Australia perform relative to ASSURED criteria. This evaluation identifies multiple trade-offs, both with regard to scientific criteria and with regards to convenience criteria. More importantly, the purpose of testing may differ between farms, and test performance should be evaluated relative to its intended use. The ability of on-farm mastitis diagnostics to inform mastitis treatment decision-making in a timely and cost-effective manner depends not just on test characteristics but also on farm-specific pathogen prevalence, and on the farm enterprise's priorities and the farm manager's potential courses of action. With most assay evaluations to date conducted in professional laboratories, there is a surprising dearth of information on how well any of the diagnostic tests perform on-farm and, indeed, of the on-farm decision-making processes that they aim to inform.     

Unique features of bovine lymphocytes exposed to a staphylococcal enterotoxin

We previously demonstrated that stimulation of bovine peripheral blood mononuclear cells (PBMCs) with staphylococcal enterotoxin C (SEC), led to an inversion of the CD4⁺:CD8⁺ T cell ratio and generation of an atypical CD8⁺ T cell subpopulation expressing CD26. In the present study, we examined T cell apoptosis and proliferation profiles of PBMC subpopulations in cultures stimulated with SEC. Unlike when stimulated with concanavalin A, nucleic acid synthesis in bovine PBMC cultures stimulated with SEC was low during the first four days but increased greatly on day 5. In contrast, nucleic acid synthesis in human PBMC cultures stimulated with SEC increased continuously. To investigate the mechanism of delayed bovine T cell proliferation, various cell phenotypes were monitored. The inversion of the bovine CD4⁺:CD8⁺ T cell ratio in PBMC cultures stimulated by SEC was associated with higher proliferation and lower apoptosis of CD8⁺ T cells compared to CD4⁺ T cells. The mRNA levels for interleukin (IL)-4 and IL-13 were sustained over 4 days but IL-12 mRNA levels dropped to background on day 2. These data suggest that SEC induces a prolonged Th-2-biased microenvironment, and together with the inversion of the bovine CD4⁺:CD8⁺ T cell ratios in bovine PBMC cultures with SEC, may in part explain the inability of the mammary immune system to establish an effective response to Staphylococcus aureus infections.     

我国部分地区奶牛乳房炎病例中金黄色葡萄球菌血清型及毒力调查

为了解我国奶牛乳房炎病例中金黄色葡萄球菌(S.aureus)流行血清型和不同血清型菌株的毒力情况,本研究采用玻板凝集和双重PCR方法分别对北京、山西、内蒙古、山东、浙江和新疆奶牛乳房炎乳样中分离的191株S.aureus进行血清分型并从各血清型中随机选取100株菌以小白鼠进行毒力测定.结果表明:这些地区牛源S.aureus中,336PS型为流行血清型,占60.2％(115/191),CP8型占19.4％(37/191),CP5型占11.5％(22/191),未分型菌株占8.9％(17/191).毒力测定显示:336PS、CP8和CP5型菌株中强毒力菌株分别占65.1％、62.9％及61.9％,未分型菌株中强毒力菌株为22.2％.该调查为我国奶牛乳房炎S.aureus疫苗菌株的筛选提供了依据.     

Production of lipid mediators in mastitic milk of cow

Bovine mastitis is one of the most prevalent and costly diseases in the dairy industry. Lipid mediators are signaling molecules which coordinately and intricately modulate inflammation. They are produced from polyunsaturated fatty acids (PUFAs) in the cellular membrane via several enzymes including cyclooxygenase (COX) and lipoxygenase (LOX). In the present study, we performed comprehensive analysis of lipid production in milk obtained from clinical or subclinical mastitic cows using liquid chromatography/mass spectrometry. We detected 26, 24, and 40 kinds of lipid constantly in healthy, subclinical, and clinical mastitic milk, respectively. In clinical mastitic milk, the amount of a major n‐6 PUFA, arachidonic acid (AA), tended to increase, whereas amounts of major n‐3 PUFAs, eicosapentaenoic acid and docosahexaenoic acid, tended to decrease. The amounts of several AA‐derived lipids including COX‐catalyzed prostaglandin (PG) D₂ and PGE₂, and LOX‐catalyzed leukotriene (LT) B₄ were increased in clinical mastitic milk. Although subclinical mastitic milk represented similar trend of lipid production to healthy milk, amounts of several lipids such as LTD₄, 14,15‐dihydroxyeicosatrienoic acid, and 14‐epoxyeicosatrienoic acid changed. These findings would be helpful for better understanding of mastitis pathology and give us some insights to develop a new diagnostic and therapeutic strategy.     

A Working Model for the Evaluation of Different Immunological Traits as Indicators of Resistance to Infection in Dairy Cattle

In order to determine the relative value of different immunological traits as indicators of resistance to infection, an analysis was performed based on records of immunological traits in young bulls and “health card” records of clinical mastitis in their half sisters. The correlation analysis suggested that young bulls with high levels of serum immunoglobulin have half sisters with low mastitis frequency. A significant positive correlation was found between the antibody response peak against human serum albumin and susceptibility to mastitis. The results of this analysis should not be considered conclusive as the data were limited. Further work is required on a larger body of material. The model described here may be considered for future use as it enables immediate selection of animals.     

Successful treatment of recurrent subclinical mastitis in cows caused by enrofloxacin resistant bacteria by means of the sequential intramammary infusion of enrofloxacin HCl-2H2O and ceftiofur HCl: a clinical trial

BackgroundRecurrent subclinical mastitis (RScM) due to resistant bacteria has low clinical and bacteriological cure rates, often requiring the culling of cows. The sequential intra-mammary administration of enrofloxacin hydrochloride-dihydrate (enro-C) followed by ceftiofur HCl may be useful for treating these cases.ObjectivesThis study assessed the bacteriological and clinical cure-efficacies of the sequentially intramammary administration of enro-C, followed by ceftiofur HCl to treat RScM in Holstein/Friesian cows.MethodsThis trial was conducted in a herd with a high prevalence of RScM, and 20 Holstein/Friesian cows were included: 45% suffering subclinical mastitis and 38.9% of the mammary quarters affected. Twenty-nine bacterial isolates in vitro resistant to enro-C were obtained (coagulase-negative Staphylococcus spp, 55.2%; Staphylococcus aureus, 27.6%; Escherichia coli, 6.9%; Streptococcus uberis, 6.9%; Corynebacterium bovis, 3.4%). Polymerase chain reaction-isolated the following genes linked to enro-C resistance: chromosomal (gyrA) and plasmid (aac(6'')-lb-cr). The treatments were as follows: twice-daily intramammary infusions of enro-C (300 mg/10 mL) for 5 days. Cows clinically considered treatment failures were also treated with intramammary ceftiofur (125 mg/10 mL, twice daily for 5 days. The clinical and bacteriological cure rates were carried out when completing each treatment phase and at 14 and 21 days, aided by a California mastitis test, somatic cell count, and failure to identify the initially causative bacteria.ResultsEnro-C achieved 65% clinical and bacteriological cure rates, and 100% cure rates were obtained after the rescue treatment with ceftiofur HCl.ConclusionsOutstanding clinical and bacteriological cure rates in cows affected by RScM were achieved with the consecutive intramammary infusions of enro-C, followed by ceftiofur HCl.     

Mastitis, polyarthritis and abortion caused by Mycoplasma species bovine group 7 in dairy cattle

OBJECTIVE: To report an outbreak of mastitis, polyarthritis and abortion caused by Mycoplasma sp bovine group 7 in three large, centrally-managed dairies and to review the relevant literature. DESIGN: Epidemiological, clinical and laboratory data were analysed, collated and reported. Multidisciplinary procedures were employed. These included clinical assessment and comprehensive laboratory investigations of affected calves, aborted foetuses and milk samples. Mycoplasma cultures and genetic analyses of isolates were undertaken to identify the aetiological agent. RESULTS: About 30% of 240 calves usually kept in a calf rearing facility developed severe polyarthritis as a result of Mycoplasma sp bovine group 7 infection between 2 and 3 weeks of age. Multiple abortions occurred on these farms. Mycoplasma sp bovine group 7 was recovered from the fibrinopurulent synovial exudates of four 14-day-old calves, from the stomach contents and lungs of two aborted foetuses, from 14 of 21 bulk milk and four of 10 mastitic quarters. Three bulk colostrum samples cultured during the outbreak were negative for mycoplasma. CONCLUSION: Mycoplasma sp bovine group 7 caused significant economic losses as a result of polyarthritis, abortion and mastitis. The disease probably originated from udder infections with spread being facilitated by the decreased use of tetracycline in the treatment of mastitis. Neonatal calves were most likely infected by the consumption of milk contaminated with the organism. Abortions presumably resulted from mycoplasmaemia. This appears to be the first report in Australia of bovine abortion resulting from Mycoplasma sp infection.     

Staphylococcal Nuclease in Udder Secretions of Cows with Acute Mastitis

High concentrations of nuclease produced by Staphylococcus aureus were demonstrated within a few hours by direct examination of udder secretions from cows with a severe staphylococcal mastitis. A positive correlation between the nuclease concentration and the severity of the mastitis was found. The cows with high nuclease concentrations were generally young individuals and/or in the first 2 weeks of the lactation period. Most had a low titre of antibodies against staphylococcal nuclease. Two-thirds of the cows in which high nuclease concentrations were demonstrated were culled or died because of the mastitis attack.The role which nuclease plays for staphylococcal virulence is discussed, and it is concluded that nuclease contributes to the pathogenicity of S. aureus.