生产群奶牛金黄色葡萄球菌毒素基因检测及PFGE分型研究

隐性乳房炎患牛与健康牛外观无异,不易判断,对奶牛业造成的损失严重.金黄色葡萄球菌是引起隐性乳房炎的重要病原菌,准确判断金黄色葡萄球菌型隐性乳房炎是预防和控制乳房炎的重要途径.目前国际上普遍采用乳中体细胞数(SCC)作为检测隐性乳房炎的主要指标,但是约40％的金黄色葡萄球菌型乳房炎牛乳汁中SCC低于20万/mL.笔者依据相邻两月SCC差值高低,采集两个奶牛场荷斯坦牛生产群第一胎泌乳牛奶样65头份,从中检出有6头份含金黄色葡萄球菌.用PCR方法扩增金黄色葡萄球菌和耐甲氧西林金黄色葡萄球菌(MRSA)的特异基因nuc和mecA,结果发现,nuc基因检出率为100％(12/12),未检测到mecA基因.进一步用多重PCR方法检测金黄色葡萄球菌各种毒素基因,其中PVL基因在高SCC与低SCC奶样中的检出率分别为33.3％和100％,差异显著,这可能与PVL破坏白细胞有关.PFGE分型结果表明,所检测到的金黄色葡萄球菌可分为4个簇、5种类型,其中P2型(PVL)为流行株.结果提示,在荷斯坦牛生产群中,如果奶牛产奶量有所下降而SCC也不高时,应辅以分子检测手段来判断是否患有金黄色葡萄球菌型隐性乳房炎.     

奶牛乳房炎金黄色葡萄球菌8种毒力基因的PCR检测

为研究奶牛乳房炎金黄色葡萄球菌(Staphylococcus aureus,SA)主要毒力因子的分布情况,本试验利用PCR对70株临床型乳房炎、55株隐性乳房炎金黄色葡萄球菌的8种主要毒力基因进行检测。结果显示,nuc、ClfA、TSST-1、PVL、Hla、Hlb、FnBPA和FnBPB 8种毒力基因在临床型乳房炎SA菌株中的检出率分别为:100.0%(70株)、100.0%(70株)、0(0株)、5.7%(4株)、100.0%(70株)、11.4%(8株)、97.1%(68株)和100.0%(70株);在隐性乳房炎SA菌株中的检出率分别为:100.0%(55株)、100.0%(55株)、0(0株)、70.9%(39株)、98.2%(54株)、9.1%(5株)、100.0%(55株)和100.0%(55株)。结果表明,nuc、ClfA、Hla、Hlb和FnBPA 5种毒力基因是引起奶牛乳房炎的SA的最主要毒力基因;PVL基因可能是引起隐性乳房炎的重要致病基因。     

原料乳和临床乳房炎金黄色葡萄球菌毒力基因检测及药敏分析

对临床乳房炎（57株）和原料乳（44株）金黄色葡萄球菌菌株,用PCR方法检测mecA基因、PVL基因、ETs基因、SEs基因和TSST-1基因;采用CLSI指导说明执行琼脂稀释法药敏性试验。结果显示原料乳菌株中,84.09%携带有毒素基因,其中PVL的检出率为84.09%,肠毒素的检出率为52.27%,主要流行的肠毒素基因为sea（56.82%）,均未检测到携带mecA、ETs、TSST-1、sei和sej基因的菌株;同时得到10种毒素基因型,其主要流行的毒素基因型为PVL＋sea（29.55%）和PVL（27.27%）。临床菌株中,78.95%携带有毒素基因,其中PVL的检出率为28.07%,肠毒素的检出率为77.19%,主要流行的肠毒素基因为sea（47.37%）,没有检测到携带ETs、TSST-1和seh基因菌株;同时得到25种毒素基因型,其主要流行的毒素基因型为sea（19.30%）,其次是seb（7.02%）,sea＋sed＋sej（3.51%）和PVL＋sea＋seb＋sec＋seg＋sei（3.51%）。6株（10.53%）携带有mecA基因菌株均含有较多毒素基因。原料乳分离株对甲氧苄啶和头孢西丁的耐药率较高,分别为100%和86.36%,其次对氯霉素、红霉素、苯唑西林、头孢哌酮和庆大霉素的耐药率分别为11.36%、4.55%、2.27%、2.27%和6.82%,所有原料乳菌株均对环丙沙星敏感,同时得到8种耐药谱,多重耐药率达22%;临床乳房炎菌株对红霉素和甲氧苄啶的耐药率较高,分别为100%和71.93%,其次对氯霉素、庆大霉素、环丙沙星、头孢西丁和苯唑西林的耐药率分别为28.07%、26.07%、24.56%、19.30%和7.02%,临床乳房炎菌株对头孢哌酮和四环素的敏感率为100%,同时得到13种耐药谱,多重耐药率达77.19%。所有原料乳和临床乳房炎菌株均对万古霉素和阿米卡星敏感。临床乳房炎菌株携带的毒素基因和多重耐药率比原料奶菌株高,同时在临床乳房炎乳中检测到MRSA菌株,提示我们应加强乳及其乳制品的管理,并对奶牛乳房炎加以重视。     

中国北方奶牛金葡菌乳房炎感染现状及耐药性和流行类型研究进展

奶牛乳房炎,尤其是由金黄色葡萄球菌(金葡菌)感染引起的隐性乳房炎是最难以防控的奶牛常见病。近五年来,本课题组对中国北方六个省市九个荷斯坦牛场的生产群高体细胞数(SCC)和低SCC奶牛以及临床乳房炎奶牛进行了十一批次采样,共采集奶样1 112头份,分离出191株金葡菌,所检测牛的平均金葡菌感染率达11.7%,其中生产群奶牛金葡菌感染率为12.4%,临床乳房炎牛为10.8%。对这些金葡菌进行药敏试验以及毒素基因检测,发现氯霉素和阿米卡星对金葡菌有较强抑制性,毒素基因检出率最高者是杀白细胞素基因PVL(70.2%)。依据金葡菌PFGE分型结果绘制中国北部及华东地区奶牛乳源金葡菌的流行图谱。结合国内外相关研究结果,对比分析了国内外奶牛金葡菌乳房炎的感染情况以及金葡菌的耐药性、毒素基因和流行类型,并提出了降低奶牛感染金葡菌乳房炎的相关策略。     

奶牛乳房炎致病型耐甲氧西林金黄色葡萄球菌的分离鉴定

为探究引起奶牛乳房炎的耐药性致病菌,本研究针对西安地区显性乳房炎奶样中的耐甲氧西林金黄色葡萄球菌（MRSA）进行系统研究。采用常规细菌学鉴定法对34头（次）荷斯坦牛76个乳区152份奶样的金黄色葡萄球菌（Staphylococcus aureus,S.aureus）进行了分离培养,共检出92株阳性菌株,检出率为60.5%;对其进行S.aureus致病基因nuc的特异性PCR检测,共检出28株含nuc基因的阳性菌株,检出率为30.4%;进一步针对MRSA耐药基因mecA进行特异性PCR扩增,共检出5株MRSA,检出率为17.9%。研究结果为MRSA耐药菌的有效检测及奶牛乳房炎的治疗提供了指导。     

奶牛乳房炎病例中金黄色葡萄球菌毒素基因的检测

金黄色葡萄球菌是引起奶牛乳房炎的重要病原菌之一，其不仅给奶业生产造成严重损失，而且携带毒素基因的金黄色葡萄球菌在适当环境温度、pH、介质下可产生毒素，影响乳产品质量安全，危害人体健康。金黄色葡萄球菌常产生的、临床意义较大的毒素为肠毒素（SEs）、剥脱毒素（ETs）和中毒休克毒素-1（TSST-1）三类。国外有较多奶牛乳房炎金黄色葡萄球菌毒素基因流行情况的调查报道，国内少有这方面的研究。因此，本文就我国重要奶产地呼和浩特地区奶牛乳房炎金黄色葡萄球菌的sea、seb、sec、sed、see、eta、etb、tst等8种常见毒素基因的流行情况进行调查研究，以期为食源性疾病的监控和分子检测提供科学依据。     

甘肃部分地区奶牛乳房炎金黄色葡萄球菌的细菌多位点序列分型与毒力检测

为研究甘肃地区牛源金黄色葡萄球菌(Staphylococcus aureus)的耐药情况、基因型总体结构特征及毒力因子的分布情况,从甘肃省东、中和西部3个地区的3个县市采集41头罹患临床型乳房炎奶牛的93份乳样,采用鉴别培养基分离纯化细菌。采用K-B法对分离株进行耐药性分析,常规PCR方法检测mecA抗性基因和14种常见毒力基因以及多位点序列分型(multilocus sequence typing, MLST)。结果显示,93份乳样中分离出28株金黄色葡萄球菌;分离株有不同程度的耐药且多为多重耐药(16株),多重耐药菌株占57.14%,未发现耐万古霉素菌株(VRSA),万古霉素敏感率为100%;28株分离菌中共检出mecA阳性11株,检出率为39.3%;共获得9个已知ST型;有12种毒力基因不同程度被检出,其中nuc、spa和coa检出率为100%,hlb、set1、clfA、FnBPA和seb阳性检出率均大于50%,sak、seg、PVL和sea的检出率依次为28.6%、7.9%、14.3%和21.4%,而FnBPB、hla基因未检出。研究结果可为甘肃省牛源金黄色葡萄球菌的耐药性评价、溯源分析和治疗提供参考依据。     

我国部分省区牛源金黄色葡萄球菌抗菌药物敏感性调查

为调查我国奶牛乳房炎奶样中金黄色葡萄球菌的药物敏感性情况,以便为该病的临床用药提供参考,本试验采用纸片扩散法对从北京、山西、内蒙古、新疆、浙江5省区奶牛乳房炎奶样中分离的148株金黄色葡萄球菌进行了抗菌药物敏感性检测。北京、山西、内蒙古分离株的临床药敏试验显示,所有菌株对头孢西丁、多西环素、四环素敏感或中介,对红霉素、克林霉素、青霉素、复方新诺明严重耐药,对氯霉素、环丙沙星和庆大霉素的敏感性有地域性差异,66%～100%的菌株同时对3种以上的抗菌药物耐药;新疆86株分离株和浙江20株分离株的临床药敏试验显示,所有菌株除对红霉素、克林霉素、青霉素、复方新诺明耐药外,对多西环素和四环素完全耐药,86%以上的分离株对5种以上抗菌药物耐药,同时还发现头孢西丁耐药株。调查结果显示,5省区乳房炎奶样中的金黄色葡萄球菌除对头孢类(头孢西丁)敏感外,对其他类型抗菌药物均存在一定程度的耐药,尤其对大环内酯类(红霉素)、林可胺类(克林霉素)、青霉素类(青霉素)、磺胺类(复方新诺明)抗菌药物严重耐药,对3～5种以上不同类型抗菌药物的多重耐药也较为普遍。调查表明,牛源性金黄色葡萄球菌耐药性的广泛存在将对金黄色葡萄球菌性奶牛乳房炎的抗菌药物治疗形成严重挑战。     

内蒙古地区致奶牛子宫内膜炎葡萄球菌超抗原基因分布及耐药性研究

为了解内蒙古地区致奶牛子宫内膜炎葡萄球菌超抗原基因分布及其耐药情况,采用PCR对分离到的葡萄球菌进行鉴定并检测超抗原基因在分离菌中的分布,应用微量肉汤稀释法开展分离菌的药敏试验和PCR检测β-内酰胺类耐药基因BlaZ和mecA的流行情况.结果表明,共分离到金黄色葡萄球菌28株(52.8％),凝固酶阴性葡萄球菌(CNS) 25株(47.2％);超抗原基因SEJ在分离菌中的携带率最高(70.0％),超抗原基因SEA、SEB及SEE携带率最低(1.9％),其他超抗原基因携带率在9.4％～43.4％之间;分离到的葡萄球菌对青霉素耐药率最高(96.2％),对万古霉素最敏感,有多重耐药菌株检出;分离菌中BlaZ携带率达98％,金黄色葡萄球菌中未检出mecA,但CNS中mecA携带率为24.5％.内蒙古地区奶牛子宫内膜炎致病葡萄球菌有12种肠毒素及eta、etb、TSST-1检出,其中SEJ基因的携带率最高;葡萄球菌对β-内酰胺类耐药率较高,并且发现了多重耐药菌株的流行.     

奶牛乳房炎金黄色葡萄球菌的分离鉴定与耐药分析

为了解本地区规模化奶牛场奶牛乳房炎金黄色葡萄球菌的流行与耐药情况，本研究采集了300份奶牛乳房炎奶样，进行金黄色葡萄球菌的分离、培养，利用荧光PCR方法鉴定，同时对分离出的阳性菌株采用纸片扩散法进行耐药性分析。结果表明：从奶牛乳房炎样品中分离出42株金黄色葡萄球菌，分离率为14%；耐药性分析表明分离的金黄色葡萄球菌对青霉素、磺胺异噁唑耐药率超过70%，对庆大霉素、氧氟沙星、头孢哌酮敏感性大于90%。本研究为本地区奶牛乳房炎的防控与临床用药提供了依据。     

Genotypic characterization by polymerase chain reaction of Staphylococcus aureus isolates associated with bovine mastitis

Staphylococcus aureus is recognized worldwide as a pathogen causing many serious diseases in humans and animals, and is the most common aetiological agent of clinical and subclinical bovine mastitis. The importance of evaluating the combination of S. aureus virulence factors has been emphasized both in human and veterinary medicine, and knowledge about the genetic variability within different S. aureus populations would help in the design of efficient treatments. The aim of the present study was to determine the genetic profiles of S. aureus strains isolated from milk of cows suffering from clinical and subclinical mastitis in Belgium. The presence of about forty virulence-associated genes was investigated by specific polymerase chain reaction (PCR) amplification. A high number of genotypic subtypes were observed, demonstrating further the large variation in the presence of virulence genes in S. aureus isolates and the considerable diversity of strains populations that are able to cause mastitis in cows. In accordance with other studies, we showed that some genes are associated with mastitis-causing S. aureus isolates, whereas others are absent or rarely present. We also further highlighted the presence of conserved gene combinations, namely the enterotoxigenic egc-cluster and the bovine pathogenicity island SaPIbov. Importantly, the presence of isolates carrying genes coding for toxins involved in important human infections makes the milk of cows with mastitis a potential reservoir for these toxins, and therefore a potential danger in human health, which strengthens the importance to consider raw milk consumption and its processing very carefully.     

奶牛乳腺炎的细菌学研究

在对山东7个地区14个奶牛场临床型和隐性乳腺炎调查的基础上采集234头临床乳腺炎病牛乳样、241个隐性乳腺炎乳样并分别做了细菌学检查,结果表明:泌乳期临床型乳腺炎病原菌以凝固酶阴性葡萄球菌、金黄色葡萄球菌、链球菌、酵母菌和棒状杆菌为主;干奶期临床型乳腺炎病原菌以大肠杆菌、链球菌、金黄色葡萄球菌、凝固酶阴性葡萄球菌和酵母菌为主;隐性乳腺炎病原菌以凝固酶阴性葡萄球菌、金黄色葡萄球菌链球菌、酵母菌、假单胞菌和棒状杆菌为主;厌氧菌在隐性乳腺炎、干奶期乳腺炎和干奶期乳腺炎乳样的捡出率分别为 5．82％,4．17%,10．16％;隐性乳腺炎、干奶期乳腺炎细菌的共感染率较高,与泌乳期乳腺炎病原菌的差异极显著(P<0．01),隐性乳腺炎与干奶期乳腺炎病原菌共感染率差异不显著(P >0．05)。     

Haemolytic and proteolytic activity of coagulase-negative staphylococci isolated from mastitis cows

The aim of the present study was to assess the haemolytic and proteolytic activity of coagulase-negative staphylococci (CNS) isolated from cows with mastitis. The study was conducted on 100 CNS strains: S. xylosus (n=28), S. chromogenes (n=26), S. haemolyticus (n=25), S. sciuri (n=14), S. warneri (n=4), S. hominis (n=2), S. saprophyticus (n=1); 22 CNS were isolated from cows with clinical mastitis and 78 from those with subclinical mastitis. The CNS studied showed the ability to produce only alpha-haemolysin and belonged to one strain - S. haemolyticus (21.0% of isolated CNS strains). Haemolysin-positive CNS were responsible for both clinical and subclinical mastitis (22.7% and 20.5%, respectively). The ability to produce protease was found in 31.0% of CNS belonging to two strains: S. chromogenes and S. sciuri. Protease-positive CNS were the etiological factor of both clinical and subclinical mastitis (31.8% and 30.8%, respectively). All S. xylosus, S. warneri, S. hominis, and S. saprophyticus strains were found protease-negative and haemolysin-negative, irrespective of whether they caused clinical or subclinical mastitis in cows.     

Effects of adjuvants on the immune response of staphylococcal alpha toxin and capsular polysaccharide (CPS) in rabbit

This study was performed to isolate a vaccine strain of S. aureus from clinical or subclinical mastitis and to choose the most optimal adjuvant for immune response of alpha toxin and capsular polysaccharide (CPS) of field strain. Of thirty strains of S. aureus isolated from milk of clinical or subclinical mastitis, V112 strain isolated from milk of gangrenous mastitis was used in this vaccine. Twenty one of rabbits were allocated into 5 groups based on adjuvants and immunized twice every 2 weeks for 8 weeks. This vaccine was composed of alpha toxin (10 hemolytic units) and formalinized whole cells (1 x 10(11) cells/ml. Five rabbits received PBS solution as a control group. The highest antibody titers against alpha toxin and CPS were observed in dextran sulfate- and aluminium hydroxide-adjuvant group at 8 weeks after immunization, respectively. These results of the study showed that one adjuvant could not induce strong and long-term immune response of alpha toxin and CPS antigens. Therefore, the use of combined adjuvants in subunit vaccine may be useful and feasible.     

凉州区奶牛乳房炎主要病原分离鉴定及药敏试验

乳房炎是奶牛最常见的疾病,随着奶牛产业的蓬勃发展,乳房炎已经成为制约该产业的瓶颈之一。从甘肃省武威市凉州区5个奶牛养殖场采集了1 000头荷斯坦奶牛共计3 991份奶样,对临床型和非临床型(隐性)乳房炎分别通过临床症状和兰州隐性乳房炎诊断液(LMT)进行诊断。结果显示:该地区奶牛临床型乳房炎发病率18.20%,亚临床型乳房炎发病率36.50%,总阳性率54.70%,总阳性乳区率54.72%。从阳性奶样中分离纯化得到的主要病原菌为链球菌属、金黄色葡萄球菌和大肠杆菌,且在不同类型乳房炎中的分布情况不同,临床型中链球菌属最多(54.37%),亚临床型中金黄色葡萄球菌最多(48.56%)。对主要病原菌进行药敏试验,结果表明治疗凉州区奶牛乳房炎的最佳药物应为喹诺酮类和庆大霉素类。     

Antipenicillinase in the serum of dairy cows

Serum from dairy cows was tested for inhibitory effect on penicillinase from a penicillin-resistant Staphylococcus aureus strain of mastitic origin. Among cows with subclinical mastitis caused by penicillin-resistant S. aureus there was a significantly higher frequency of individuals with penicillinajse-inhibiting serum than among healthy cows. Among the subclinical cases, a foregoing penicillin treatment of clinical mastitis appeared to increase the serum antipenicillinase activity.     

Isolation and Identification of Pathogenic Bacteria Causing Bovine Subclinical Mastitis in Eastern Hebei Province

In this study, the milk samples of 1 021 cows in eight dairy farms in Eastern Hebei Province were collected and detected with LMT reagent and somatic cell count for subclinical mastitis. Pathogenic bacteria in subclinical mastitis positive milk samples were isolated and identified.The results showed that 60.63%(619/1 021) of the sampled cows were diagnosed with subclinical mastitis, and mixed infections accounted for 88.21%(546/619) of the cases. In addition, 82 strains of 14 species were isolated from the subclinical mastitis positive milk samples, including 36 strains of Staphylococcus(43.90%), 33 strains of Streptococcus(40.24%), 8 strains of Enterobacteriaceae(9.76%) and 5 strains of Corynebacterium(6.10%), respectively. The results proved that Staphylococcus aureus and Streptococcus agalactiae are the main pathogenic bacteria causing bovine subclinical mastitis in Eastern Hebei Province.     

Evaluation of levamisole for use in control of bovine Staphylococcus aureus mastitis

Levamisole was injected into cows six times at weekly intervals in the dry period and its effect on existing subclinical Staphylococcus aureus mastitis monitored through the following milking season. Levamisole therapy failed to eliminate or modify subclinical S. aureus mastitis. In a second experiment, a single levamisole administration at drying off did not prevent new experimentally-induced S. aureus infections in the first two weeks of the dry period and there were similar numbers of cases of clinical and subclinical mastitis in treated and control groups. However, levamisole may have reduced the severe clinical manifestations of the bacterial challenge, since gangrenous mastitis and abortion were only observed in the control cows.