The Distribution of Soluble Copper- and Zinc-Binding Proteins in Liver and Kidney of Chronic Copper-Poisoned Goats

The distribution of copper and zinc among the soluble proteins in the liver and kidney from chronic copper-poisoned goats was examined after gel filtration of the proteins. The concentrations of copper in the liver and kidney cortex from five experimentally copper-poisoned goats were: 550–810 µg/g liver and 190–420 µg/g kidney cortex (wet weight). In general the copper-binding proteins from, both the liver and kidney samples were separated into two different fractions with approximate molecular weights (m.w.) of > 65,000 and 10,000, respectively. From the liver samples, varying amounts of copper were eluted in a fourth fraction with m.w. < 2,000. In the majority of kidney samples the dominating copper-binding protein fraction was the high molecular weight fraction. Absolute amounts of copper recovered in the metallothionein-like protein fraction were nearly the samt for all samples investigated. The distribution of zinc-binding proteins in both liver and kidney samples was nearly the same. The high molecular weight fraction dominated, and no zinc was bound to metallothionein-like proteins.     

Measurement of Staphylococcus Aureus Nuclease and Antinucleases: Applicability for The Assessment of Mastitic Milk

The concentration of the nuclease produced by Staphylococcus aureus and titres of antinucleases were determined in the udder secretions of 539 cows, most of them with clinical S. aureus mastitis. On average, the nuclease was detected in 85 % of the samples from mastitic quarters. In milk from most cows, antinucleases neutralize the activity of the nuclease, and a successful demonstration of this thermostable enzyme presupposed the inactivation of antibodies by heating the samples on boiling water-bath. The nuclease was demonstrated most frequently, and at highest concentration, in samples from cows with a severe mastitis and from cows which did not recover completely.The test for nuclease, performed out both non-heated and heated quarter samples, and the antimuclease test, may supplement conventional laboratory methods. The advantages and limitations of the methods in the diagnosis of mastitis are discussed.     

奶牛乳腺炎主要致病菌的分离与鉴定

对山东省济南、泰安和临沂等地区5个奶牛场的160份乳样进行了临床型乳腺炎和隐性乳腺炎的判定检测,并进行了主要致病菌的分离鉴定。结果表明,160份乳样中有127份呈乳腺炎阳性,其中临床型乳腺炎占36．22％,隐性乳腺炎占63．78％;从这些乳样中分离到了主要致病菌金黄色葡萄球菌、大肠杆菌和无乳链球菌,金黄色葡萄球菌依据溶血型的不同主要有α溶血、β溶血、α＋β溶血和不溶血四种;大肠杆菌依据O抗原诊断血清得出主要有02、021、076和088这四种型;无乳链球菌依据溶血型主要为β溶血型。     

PCR扩增Nuc基因检测奶牛乳腺炎致病性金黄色葡萄球菌

为建立奶牛乳腺炎金黄色葡萄球菌致病菌株的检测方法,依据金黄色葡萄球菌耐热核酸酶Nuc基因序列,设计合成特异性引物,通过聚合酶链式反应扩增Nuc基因,并将扩增基因克隆入T载体,进行序列测定,对所分离的奶牛乳腺炎病原菌进行鉴定.结果显示:金黄色葡萄球菌扩增出特异性条带,大小为694bp;其他菌株未出现条带.敏感性检测的最低浓度为1.25×103 cfu/mL.本试验所建立的方法具有特异性强、敏感度高的特点.     

PCR扩增Nuc基因检测奶牛乳腺炎致病性金黄色葡萄球菌

为建立奶牛乳腺炎金黄色葡萄球菌致病菌株的检测方法,依据金黄色葡萄球菌耐热核酸酶Nuc基因序列,设计合成特异性引物,通过聚合酶链式反应扩增Nuc基因,并将扩增基因克隆入T载体,进行序列测定,对所分离的奶牛乳腺炎病原菌进行鉴定。结果显示:金黄色葡萄球菌扩增出特异性条带,大小为694bp;其他菌株未出现条带。敏感性检测的最低浓度为1.25×103cfu/mL。本试验所建立的方法具有特异性强、敏感度高的特点。     

贵州地区牛源金黄色葡萄球菌分离株16S rRNA分子鉴定及RAPD分型研究

利用16S rRNA保守序列对引起贵州地区奶牛乳房炎的金黄色葡萄球菌进行分离鉴定,建立随机多态性扩增体系对金黄色葡萄球菌分离株进行基因分型研究。结果表明,69份奶样中共分离出金黄色葡萄球菌37株,DNA随机多态性扩增（random amplified polymorphic DNA,RAPD）结果显示这37株金黄色葡萄球菌扩增产物在1～7条带之间,产物大小为400～4500 bp。菌株可分为7个基因型,其中Ⅰ型7株（18.9%）、Ⅱ型2株（5.4%）、Ⅲ型2株（5.4%）、Ⅳ型1株（2.7%）、Ⅴ型19株（51.4%）、Ⅵ型1株（2.7%）、Ⅶ型5株（13.5%）,Ⅴ型为该地区的流行优势菌群。地理和气候环境对病原菌流行传播的影响是病原菌基因型分布差异的重要原因。     

奶牛乳房炎金黄色葡萄球菌黏附素研究进展

金黄色葡萄球菌的黏附素是其引起奶牛乳房炎的关键因素之一,与金黄色葡萄球菌在奶牛乳房中定植有关的的黏附素主要是ClfA、FnBPA和FnBPB3种。曾有对金黄色葡萄球菌黏附素表达调控的研究发现,在金黄色葡萄球菌感染的早期主要表达黏附素,之后才表达毒素和荚膜,所以人们认为,对金黄色葡萄球菌的黏附进行干预,可以减少或阻止毒素和荚膜的表达,而以黏附素作为靶位进行疫苗研制可能是预防奶牛金黄色葡萄球菌性乳房炎的有效途径。国外对金黄色葡萄球菌的黏附素的研究历史相对较久,并且也取得了许多可喜成绩,而国内对金黄色葡萄球菌的研究相对滞后。因此,研究金黄色葡萄球菌黏附素对预防由金黄色葡萄球菌引起的奶牛乳房炎具有广阔的前景。     

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.     

奶牛乳腺炎源金黄色葡萄球菌噬菌体P82的分离及特性研究

【目的】 为开发噬菌体"鸡尾酒"制剂防治奶牛乳腺炎提供生物学材料。【方法】 以实验室分离的奶牛乳腺炎源金黄色葡萄球菌82为宿主菌,通过点滴法和双层平板法在奶牛场的污水、弃奶和粪便混合物中分离并纯化其噬菌体,通过噬菌斑及透射电子显微镜对该株噬菌体的形态特征进行观察。利用核酸酶处理分析该噬菌体核酸类型,并对其裂解谱、最佳感染复数、一步生长曲线、热稳定性、pH稳定性进行研究。【结果】 分离筛选出金黄色葡萄球菌82的裂解性噬菌体P82,电镜下观察其头部为二十面体,大小约为120 nm×83 nm,有一条带可伸缩尾鞘的长尾,长约126 nm,属于有尾噬菌体目,肌尾噬菌体科。通过琼脂糖凝胶电泳鉴定其核酸类型为双链DNA （dsDNA）。噬菌体P82的最佳感染复数为0.001,对奶牛乳腺炎源金黄色葡萄球菌裂解率为36.51%（23/63）,宿主谱较广;其生长潜伏期约为25 min,爆发期约为45 min,裂解量约为74 PFU/cell;在pH 4.0~12.0时活性稳定,在pH 2.0和13.0时则完全失去活性。噬菌体P82热稳定性较高,在70 ℃作用60 min后仍有裂解能力,在80 ℃作用40 min时则失去裂解能力。【结论】 本研究分离的噬菌体P82效价较高、裂解能力强、增殖速度快、噬菌谱广,对不同温度和pH均有较好的耐受度,能作为专一性裂解奶牛乳腺炎源金黄色葡萄球菌的噬菌体候选株,可与其他噬菌体混合制成噬菌体"鸡尾酒"制剂用于防治奶牛乳腺炎,为奶牛乳腺炎的噬菌体疗法提供材料。     

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.     

金黄色葡萄球菌毒力及其免疫原性评价方法研究

为建立评价金黄色葡萄球菌毒力和用于筛选免疫保护性菌株的技术方法,选取小鼠腹腔攻毒方法确定的强毒力和弱毒力菌株各3株,经小鼠后腿肌肉注射不同剂量,比较20 d的临床病变差异,确定小鼠后腿内侧肌肉注射0.25 mL、OD600=0.6的剂量可以评价不同金黄色葡萄球菌的毒力。利用该方法比较了6株强毒力菌株的毒力差异,并用于2株免疫保护性菌株的筛选。结果证明建立的金黄色葡萄球菌毒力评价方法可以精确、客观比较不同菌株毒力差异,并可用于免疫保护性菌株的筛选。     

牛源耐甲氧西林金黄色葡萄球菌的分离鉴定

从奶牛分离得到耐甲氧西林金黄色葡萄球菌（methieillin—resistant Staphylococcus aureus,MRSA）。按常规方法复苏菌株,用血浆凝固酶试验鉴定SA。用KB法、琼脂筛选法、生化鉴定法（VITEK32）、聚合酶链反应等鉴定MRSA。常规分离鏊定的葡萄球菌468株,其中凝固酶阴性葡萄球菌（coagulase negative Staphylococcus,CNS）361株,检出率为77．149,6,金黄色葡萄球菌（Staphylococcus aureus,SA）105株,检出率为22．44％,MRSA36株检出率为34．29％,2株未定型,占0．42％。而MRSA在乳房炎奶样中检出率高达34．29％,表明新疆兵团部分垦区牛场MRSA已经呈蔓延趋势。     

上海地区奶牛的乳房炎金黄色葡萄球菌耐药性

为调查上海地区奶牛乳房炎金黄色葡萄球菌的耐药性,采集上海地区乳房炎患病乳样60 份,进行金黄色葡萄球菌的分离鉴定及药敏实验。结果表明：经分离鉴定,共检出8 株金黄色葡萄球菌,分离率为13.33%;药敏实验结果表明,分离得到的金黄色葡萄球菌对青霉素G和氨苄西林的耐药率最高,均为100.00%,对磺胺甲恶唑的耐药率次之,为50.00%,对头孢克洛、头孢唑肟、洛美沙星、诺氟沙星、四环素、庆大霉素、红霉素、克林霉素和氯霉素的耐药率均为37.50%,对环丙沙星和氧氟沙星的耐药率均为25.00%,对头孢噻肟、头孢他啶     

奶牛乳房炎金黄色葡萄球菌疫苗研究进展

奶牛乳房炎是影响乳品工业经济效益的重要疾病之一,引起奶牛乳房炎的致病菌约有150多种,但金黄色葡萄球菌是引起奶牛乳房炎的主要病原。由它引起的乳房炎很难治愈,而且对抗生素产生耐药性的菌株已经相当普遍,因此使用预防或治疗性疫苗防治乳房炎就成为首选。然而金黄色葡萄球菌的致病力由许多不同的因子(如黏附素、荚膜等)决定,这使得制备非常有效的疫苗变得很困难。本文就这些致病因子在疫苗研制方面的进展做一简要介绍。     

奶牛乳房炎金黄色葡萄球菌的分离鉴定

为检测江苏省某奶牛场中乳房炎是否为金黄色葡萄球菌所致,采取4份患病乳汁样本进行试验分离和鉴定,并针对金黄色葡萄球菌采用分离后鉴定,结果显示,分离到的2株致病菌为金黄色葡萄球菌,其他菌株还包括大肠杆菌、肠球菌等,为多种致病病原菌同时引起,主要为金黄色葡萄球菌感染。     

金黄色葡萄球菌感染小鼠乳房炎模型的建立

取25只分娩8～10d的昆明系小鼠随机分成5组(n=5),分别为阴性对照、生理盐水(pss)组和不同剂量金黄色葡萄球菌(1.0×102cfu/50μL、1.0×103cfu/50μL、5.0×103cfu/50μL)试验组,经乳头导管对第4对乳腺50μL/只注射,24h后观察结果,一侧乳腺甲醛固定制作切片观察组织病理变化,另一侧冰上研磨后取匀浆用高盐甘露醇鉴别培养基进行细菌计数,检测组织匀浆上清中的TNF-α含量。结果显示,试验组随着注射细菌数量的增加,病理变化加重,分离到的金黄色葡萄球菌数量显著增加,乳腺匀浆中TNF-α含量明显升高。结果表明,注射1.0×103cfu组可以引起较典型的小鼠乳房炎,且病理变化适中,适合用来建立金黄色葡萄球菌小鼠乳房炎模型。     

Field trial of a staphylococcal mastitis vaccine in dairy herds: clinical, subclinical and microbiological assessments

Objective To assess the efficacy of a new staphylococcal mastitis vaccine under commercial dairying conditions.
Design A field trial involving 1819 cows and heifers conducted on seven dairy herds in Victoria. The trial was done 'blind'; approximately half the animals were vaccinated and the remainder were untreated controls.
Procedure The vaccine was given twice during the last 10 weeks of pregnancy. Effects of vaccination were assessed, during the ensuing lactation, on the basis of clinical and sub-clinical mastitis and microbiological investigations of the milk.
Results A total of 273 cases of clinical mastitis were recorded. Staphylococcus aureus was isolated from 112 of these, 45 cases in vaccinates and 67 cases in controls; the difference was not statistically significant. One herd was notable in having a high incidence of clinical staphylococcal mastitis. This herd accounted for 15.8% of the animals in the field trial but 54.5% of cases of clinical staphylococcal mastitis. For this herd, vaccinated animals had significantly lower incidence of clinical staphylococcal mastitis and prevalence of subclinical mastitis, relative to controls. An unexpected feature of the trial as a whole was the low incidence of clinical mastitis from which S aureus was isolated in pure culture (26.3% of cases) and the high incidence of clinical Streptococcus uberis mastitis (22.7% of cases).
Conclusions The trial showed that the vaccine was efficacious in reducing the incidence of clinical mastitis and prevalence of subclinical mastitis in a herd that had a serious staphylococcal mastitis problem.     

新疆石河子奶牛隐性乳房炎源金黄色葡萄球菌的分离鉴定及药物敏感性分析

为了解石河子地区部分规模化奶牛场奶牛隐性乳房炎源金黄色葡萄球菌的生物学特性及药物敏感性,为科学防治该病的发生提供参考依据,本试验选择了石河子周边地区不同规模的11个奶牛场,通过采用LMT法检测其隐性乳房炎的感染率,并从采集到的313份隐性乳房炎奶样中分离鉴定金黄色葡萄球菌,同时采用纸片扩散法（K－B法）对分离株的药物敏感性进行了分析。结果表明,试验区奶牛隐性乳房炎的总阳性率为44．56％（869／1950）,金黄色葡萄球菌的分离率为44．71％（93／208）;药敏试验结果表明,大部分菌株对阿莫西林、红霉素、四环素表现为高度耐药,耐药率分别为92．30％（24／26）、80．77％（21／26）、80．77％（21／26）;对庆大霉素、氧氟沙星、头孢唑啉表现为敏感,敏感率分别为57．70％（15／26）、76．92％（20／26）、76．92％（20／26）;部分菌株对克林霉素中度敏感,敏感率为46．15％（12／26）。建议在选用抗生素治疗奶牛隐性乳房炎时,以流行菌株的药物敏感性为依据。     

牛源金黄色葡萄球菌凝固酶分型与致病因子检测

为了解青海和甘肃地区奶牛养殖场的金黄色葡萄球菌(S.aureus)基因型分布状况,本研究对来自该两个地区的210份奶样进行了S.aureus的分离与鉴定,利用凝固酶基因(Coa)扩增和限制性内切酶AluⅠ酶切方法对所有分离株进行了凝固酶多态性分型。并对不同凝固酶基因型的S.aureus进行了白细胞毒素F (LukF)、白细胞毒素S (LukS)、α溶血素(α-hemolysin)、β溶血素(β-hemolysin)等主要致病因子检测。结果显示,共分离鉴定出35株S.aureus (青海13株,甘肃22株),所有分离株均能够扩增出Coa基因片段,并有5种PCR分型结果(本文简称PCR1～PCR5型),其中PCR 1、4和5型只有1种亚型,而PCR 2型有3种亚型,PCR 3型有2种亚型。PCR3型是青海(6/13)和甘肃地区(8/22)主要流行的基因型。致病因子检测结果显示,所有分离株均携带致病因子LukS和α溶血素;致病因子LukF和β溶血素检出率也很高,分别是30株(85.7%)和31株(88.6%)。β溶血素基因在甘肃地区检出率为95.5%(21/22),青海地区检出率为76.9%(10/13),致病因子LukF在甘肃地区检出率为91.0%(20/22),青海地区检出率为76.9%(10/13),且在各基因型中均有分布,甘肃地区S.aureus致病因子LukF和β溶血素检出率高于青海地区。本研究首次对青海和甘肃地区致奶牛乳房炎S.aureus进行了鉴定分析,为这两个地区奶牛乳房炎的防治提供了参考依据。     

金黄色葡萄球菌诱发建立乳房炎小鼠病理模型

为了建立小鼠乳房炎模型,将30只昆明小鼠随机分成3组,分别经第4对乳腺注入50μL的生理盐水、1.0×105CFU/mL和2.0×105CFU/mL的金黄色葡萄球菌,观察乳腺组织的外形和组织切片的变化;结果表明,注菌后24 h,小鼠出现明显的临床症状,乳腺出现不同程度的炎性症状,组织病理学显示腺泡腔内有大量嗜中性粒细胞浸润,泡腔间隔增宽。说明金黄色葡萄球菌能够诱发试验性乳房炎。