乳房链球菌多位点序列分型和遗传进化特征分析

为探究中国不同地区来源的乳房链球菌（Streptococcus uberis，S.uberis）之间的多样性和遗传进化关系，本研究以部分地区分离到的38株乳房链球菌为研究对象，通过对其最小抑菌浓度（minimun inhibitory concentration，MIC）测定和全基因组框架测序，获取38株菌耐药性、耐药基因及毒力基因等相关信息；通过多位点序列分型技术（multilocus sequence typing，MLST）分析了7个管家基因的等位基因谱、序列型（sequence type，ST）和克隆复合体（clonal complexes，CCs），并构建系统进化树。MIC试验共采用15种抗生素，除头孢噻呋和氟苯尼考外，38株乳房链球菌对其中13种抗生素具有不同程度耐药，且江苏地区的耐药性明显强于新疆地区；耐药和毒力基因比对后发现，仅24株菌携带耐药基因，剩余14株则不携带耐药基因且属新疆地区分离株，与MIC试验结果相符；15种毒力基因中，除cfu、hasA/B和lbp外，余下12种毒力基因的携带率均高达100%。MLST结果表明，38株乳房链球菌共属于17个ST型，其中有15个ST型从未报道，仅supan01 1株菌属于ST-5 CCs。进化树结果揭示，38株分离株之间的亲缘性较远，分布呈地区依赖性，且中国部分地区当前流行的乳房链球菌与西方国家流行的菌株差异较大。本研究丰富了乳房链球菌的MLST分型数据库，为了解中国乳房链球菌遗传特性和分布特点提供了参考依据。     

The exploitation of the genome in the search for determinants of virulence in Streptococcus uberis

Despite much success in the control of mastitis in dairy cattle, intramammary infection with Streptococcus uberis remains a threat to herd health. This organism is a frequent cause of mastitis worldwide. Recent advances in the ability to genetically manipulate this bacterium, coupled to the determination of a representative genome sequence have already enabled the investigation of certain aspects of disease pathogenesis. Further use of such technology coupled to reliable models of disease and post-genomic analysis will permit the elucidation of further interactions between pathogen and host. This additional information can be usefully targeted at identification of candidates for inclusion in effective vaccines. This communication reviews the current, reported progress using this technology for S. uberis.     

Research Progress on the Staphylococcus aureus Immune Evasion in Bovine Mastitis

Staphylococcus aureus(SA)bovine mastitis is a chronic and subclinical infectious disease, which is hard to control. It brings unpredictable losses to dairy-farming industry. Immune escape effect of SA plays an important role in the occurrence and development of mastitis, and some virulence factors are mostly associated with the immune evasion of bacteria. In this paper, some important immune evasion mechanisms of SA in recent researches were reviewed in order to provide theoretical reference for further prevention and treatment of mastitis in dairy cows.     

奶牛乳腺炎金黄色葡萄球菌免疫逃避机制研究进展

金黄色葡萄球菌（Staphylococcus aureus,SA）型奶牛乳腺炎是一种较难控制的慢性、亚临床型传染病,给奶牛养殖业带来巨大的经济损失。多种SA免疫逃避机制在乳腺炎发生、发展过程中起重要作用,且SA的免疫逃避机制多与菌体毒力因子相关。本文对近年研究的奶牛乳腺炎SA的免疫逃避机制进行了综述,以期为SA型奶牛乳腺炎的防治研究提供参考。     

Prototheca zopfii genotype 2: the causative agent of bovine protothecal mastitis?

In order to clarify the epidemiology of bovine protothecal mastitis, 30 Prototheca zopfii mastitis isolates were genetically investigated. Based on the 18S rDNA, which allows a differentiation of the former species P. zopfii in two distinct P. zopfii genotypes and Prototheca blaschkeae sp. nov., newly developed genotype-specific PCR-assays as well as RFLP-assays were applied.

All mastitis isolates investigated could be assigned to P. zopfii genotype 2 suggesting that this genotype is the aetiological agent of bovine Prototheca mastitis.     

奶牛乳房炎主要病原菌感染流行规律研究进展

乳房炎作为全球性奶牛养殖业的重要疾病,因对社会经济、养殖行业和人类健康的巨大影响使人们从发病机理、疾病诊断、预防等方面对其进行了不断的研究与探讨。作者立足于前期尤其是近几年的研究文献,以引起奶牛乳房炎的重要原因——病原菌为中心展开,探讨其感染类型、奶牛与病原菌及病原菌相互之间的关系。奶牛乳房炎是由包括病原菌的侵袭及宿主奶牛自身抵抗力等多种因素共同作用导致,乳腺对病原菌的入侵应答能力,病原与病原之间或促进或抑制都对其发生产生影响。金黄色葡萄球菌是公认的奶牛乳房炎的主要传染性病原菌,其独特的生物学特性使得其在体内难以治疗,并且耐药性普遍存在;无乳链球菌是环境性还是传染性病原尚有争议,在中国感染率较高,对大部分抗生素仍旧敏感,相对于其他病原菌治愈率较高,畜群清除容易;大肠埃希氏菌被作为环境性病原体,乳房炎感染的分离率仍较高,抗生素治疗结果还有待商榷,耐药性普遍存在并增强;牛支原体近十年的感染率有所上升,在国内外都是严重的乳房炎致病因素,治疗效果不佳,且耐药性正在增强,疫苗开发困难。文章对上述几种主要病原菌的流行、危害与防治等研究进行综述,以期为奶牛生产管理和科学研究方面提供参考。     

奶牛乳腺炎无乳链球菌毒力相关因子

链球菌、金黄色葡萄球菌和大肠杆菌是引起奶牛乳腺炎的3大病原菌，在链球菌属中无乳链球菌是引起奶牛乳腺炎的重要病原菌之一，由无乳链球菌导致的乳腺炎约占隐性乳腺炎发病率的56.25%。无乳链球菌入侵奶牛乳腺的过程主要包括感染、黏附上皮细胞、侵入上皮细胞、损伤机体和免疫逃避等过程。无乳链球菌的毒力因子具有附着和侵袭机体细胞的作用，使菌体在奶牛乳腺表面形成生物被膜，进而干扰机体的正常免疫功能并引起疾病。本文主要阐述了无乳链球菌在入侵乳腺组织过程中发挥主要作用的毒力因子的种类、作用机制以及调控过程，旨在通过抑制其相关毒力因子的活性，从而阻断无乳链球菌在乳腺中感染和传播，进而为预防和治疗链球菌型乳腺炎提供新的思路。     

Advances on Virulence Factors of Streptococcus agalactiae of Dairy Cows Mastitis

Streptococcus, Staphylococcus aureus, and Escherichia coli are the three major pathogens causing mastitis in dairy cows, and Streptococcus agalactiae (S.agalactiae) in Streptoco-ccus is one of the important pathogens causing mastitis in dairy cows, which accounts for around 56.25% of the incidence of sub-clinic mastitis. The process of S.agalactiae invasion mammary glands of dairy cows mainly includes:infection, adhesion, invasion, and then damage the mammary tissue and entail the inflammation. The virulence factor of S. agalactiae has the function of attaching and invading the mammary gland epithelial cells so that the bacteria could form the biofilm on the surface. After infection, the irritation of the mammary tissues by infectious processes serves to the immune response. In this paper, we summarized the types, mechanism, and regulation process of S. agalactiae virulence factors which plays a major role in the invasion of mastitis, aimed to show the activity of S. agalactiae by inhibiting the activity of its related virulence factors in preventing dairy cow mastitis and provide new ideas for the treatment.     

Interpretation of California mastitis test scores using Staphylococcus aureus culture results for screening of subclinical mastitis in low yielding smallholder dairy cows in the Dar es Salaam region of Tanzania

Screening of subclinical mastitis under field conditions is done using the California mastitis test (CMT). CMT score of ≥1 corresponding to ≥500,000 somatic cells ml⁻¹ is commonly used as threshold of subclinical mastitis in temperate countries. However, given the innately high physiological level of somatic cells in low yielding dairy cows, this threshold may not apply to low yielding dairy cows. The current study was undertaken to investigate the clinical utility of CMT for screening of Staphylococcus aureus subclinical mastitis in low yielding smallholder dairy cows in Tanzania. A total of 1151 of quarter-milk samples were CMT tested, of these 914-originated from cows with a lactation period of 14–305 days. All samples were screened for subclinical mastitis by the CMT as well as microbiological culture of single, duplicate (two consecutive) and triplicate (three consecutive) samples as a gold standard. For the duplicate and triplicate quarter-samples, cows were considered positive for S. aureus subclinical mastitis if results of microbiologic culture for S. aureus were positive for two of two, and for at least two of the first three consecutive quarter-milk samples collected from that cow, respectively. Using a CMT score of ≥1 would classify 78.6% of the 940 quarter-samples as positive. Eighty-two percent of the samples in which S. aureus was isolated had CMT scores ≥2; this would classify 51.6% of the 940 quarter-samples as positive. For the single sample, this cut-off had sensitivity, specificity and likelihood ratio for S. aureus of 0.87, 0.83 and 4.24, respectively. For the duplicate quarter-milk samples this cut-off had sensitivity, specificity, and likelihood ratio of 0.94, 0.86, and 5.19. While, for the triplicate quarter-milk samples this cut-off had sensitivity, specificity and likelihood ratio of 0.97, 0.92 and 7.47, respectively. Based on these results and practical considerations, it is concluded that CMT score of ≥2 corresponding to ≥800,000 somatic cells Ml⁻¹ is the best cut-off to correctly identify S. aureus intramammary infections in low yielding dairy cows in Tanzania.     

新疆褐牛乳房炎抗性相关基因的表达及生物信息学分析

文章旨在分析新疆褐牛乳房炎抗性相关基因的分子生物学功能，为分子标记辅助育种工作在新疆褐牛乳房炎抗性性状中的研究奠定理论基础。试验采用DAVID 6.8软件对课题组前期研究及相关文献报道的乳房炎抗性相关基因（共20个基因）进行功能GO富集及KEGG信号通路分析。采用String 10.0数据库进行蛋白相互作用网络分析，结合生物信息学分析和相关文献的研究结果，挑选出JAK2和STAT5A基因，通过使用实时荧光定量PCR技术检测JAK2、STAT5A基因在不同乳房健康程度的新疆褐牛血液中的相对表达量。GO富集分析得到，20个基因主要涉及细胞膜、免疫应答和调节防御反应等生物过程；KEGG富集分析发现，这些基因主要参与JAK-STAT信号通路、趋化因子信号通路、ErbB信号通路等；PPI分析发现，20个基因中有8个基因之间存在直接或间接联系；实时荧光定量试验发现，JAK2、STAT5A基因在健康新疆褐牛血液的表达量极显著或显著高于患乳房炎新疆褐牛（P<0.01；P<0.05）。通过生物信息分析和实时荧光定量试验，进一步从分子水平探讨了不同乳房健康程度的新疆褐牛与JAK2、STAT5A基因表达之间的内在联系。     

奶牛乳房炎主要病原菌基因检测技术研究进展

奶牛乳房炎是奶牛三大疾病之一,给奶牛业带来严重经济损失的同时,也间接影响人类的健康。奶牛乳房炎主要病原菌快速、准确的诊断对奶牛乳房炎的治疗与预防具有重要的意义。文章全面综述了PCR、real-time PCR、基因芯片和环介导等温扩增(LAMP)技术在奶牛乳房炎主要病原菌诊断中的应用,旨在为奶牛乳房炎的综合防制提供参考。     

无乳链球菌毒力基因与耐药性的相关性研究进展

乳房炎是奶牛养殖业中最常见的一种疾病，会造成奶牛乳腺发炎肿胀，使产奶量大幅下降。作为一种多因素性疾病，奶牛乳房炎发病原因复杂，饲料污染、病原菌感染以及环境因素均可以导致乳房炎的发生。无乳链球菌（Streptococcus agalactiae）作为奶牛乳房炎的主要致病菌，常会造成乳腺组织的局部炎症，随着无乳链球菌分离率的增加以及抗菌药物的滥用，其对抗菌药物的耐药性逐步攀升，给临床治疗带来了一定的困难。此外，无乳链球菌能够定植于宿主体内，破坏宿主的免疫系统，而其自身毒力因子会引起宿主机体疾病的产生。随着相关研究的深入，发现毒力与耐药性本身互为关联，耐药性的增强以不同的方式影响毒力，表明了耐药性与毒力因子之间存在着一定的相关性。笔者主要回顾了国内外链球菌以及其他细菌毒力基因与耐药性之间相关性的研究进展，从无乳链球菌毒力因子及耐药性等多种角度入手，分析了无乳链球菌的毒力机制以及毒力基因，并对近5年国内不同地区无乳链球菌耐药性进行统计，从细菌种类、环境和相关机制对链球菌的毒力和耐药性之间的关系展开综述，以期为无乳链球菌所引起的临床症状筛选出合理的抗菌药物并对毒力基因与耐药性的相关性研究提供理论参考。     

山东地区乳房炎牛奶中大肠杆菌的分离鉴定及耐药性分析

试验旨在了解山东地区乳房炎牛奶中大肠杆菌的污染状况及耐药情况。选择山东省3个地区的规模化奶牛场共采集227份牛奶样品，采用细菌学方法对大肠杆菌进行分离鉴定，用微量肉汤稀释法检测分离菌对11种常规抗菌药物的敏感性，采用PCR方法对常见的13种耐药基因、8种毒力基因和Ⅰ类整合子基因盒结构进行分析。结果显示，从227份牛奶样品中共分离出71株大肠杆菌；大肠杆菌对1种及1种以上抗菌药耐药的菌株达到77.5%，多重耐药率为15.5%，其中对多黏菌素耐药率为52.2%，对阿莫西林-克拉维酸耐药率为39.4%，而所有菌株均对新霉素表现为敏感。PCR检测耐药基因、毒力基因和Ⅰ类整合子结果显示，β-内酰胺类耐药基因中bla_TEM基因携带率为100%，其中全部为bla_TEM-1基因，bla_CTX-M基因携带率为32.4%，其中主要为bla_CTX-M-15基因，没有检测到bla_SHV、bla_OXA基因；多黏菌素的耐药基因mcr-1携带率为29.6%；喹诺酮类耐药基因中aac（6'）-Ⅰb-cr基因携带率为29.6%，qnrB基因携带率为20.8%，没有检测到qnrA和qnrC耐药基因；对8种毒力基因检测分析结果显示，仅Hly毒力基因没有被检出，Ecs3703、Irp2基因的检出率较高，分别为90.1%和63.4%，71株大肠杆菌中共有11株携带Ⅰ类整合子，检出率为15.5%，11株大肠杆菌携带6种耐药基因盒结构，最主要的耐药基因盒排列为dfr17-aadA5。本研究结果表明，山东地区乳房炎牛奶中大肠杆菌的耐药现象严重，携带毒力基因Ecs3703、Irp2的大肠杆菌可能是引起奶牛乳房炎的致病菌，Ⅰ类整合子的检测在细菌耐药性与基因携带率方面发挥着关键作用，可为临床预防和治疗奶牛乳房炎大肠杆菌病提供理论依据。     

内蒙古地区乳房炎奶样与环境中细菌的分离鉴定及耐药性分析

【目的】研究内蒙古地区部分奶牛养殖场乳房炎生鲜乳样本及环境样本致病菌种类和耐药性,为乳房炎的防控提供临床用药指导和参考。【方法】采用平板划线法对内蒙古地区4个规模化养殖场468份(乳房炎生鲜乳样本199份,乳房炎奶牛饲养环境样本269份)样本进行细菌分离培养,采用形态学观察、革兰氏染色镜检以及16S rDNA测序对分离出的细菌进行鉴定,且对引发乳房炎的主要致病菌进行药敏性试验。【结果】高盐甘露醇培养基上呈现出小、白且偏黄色的菌落,经16S rDNA测序比对,与金黄色葡萄球菌高度相似的菌株共56株,分离率为11.97%;在伊红-美蓝培养基上呈现出黑紫色泛有金属光泽的菌落,经16S rDNA测序比对,与大肠杆菌高度相似的菌株共44株,分离率为9.40%。在199份生鲜乳样本中,金黄色葡萄球菌和大肠杆菌的分离率最高,分别为21.11%和17.09%;在269份环境样本中,金黄色葡萄球菌和大肠杆菌的分离率分别为5.20%和3.72%。药敏试验结果显示,金黄色葡萄球菌对青霉素、磺胺异噁唑、氨苄西林、阿莫西林/克拉维酸的耐药性相对较高,耐药率分别为90.63%、78.13%、75.00%、68.75%;对庆大霉素、环丙沙星、复方新诺明的敏感性较高。大肠杆菌对氨苄西林、头孢噻吩、磺胺异噁唑存在明显耐药性,耐药率分别为100%、94.29%、45.71%;对链霉素、卡那霉素、庆大霉素、多粘菌素E、美罗培南、阿莫西林/克拉维酸以及利福昔明表现出较高敏感性。【结论】内蒙古地区引发奶牛乳房炎的主要致病菌为金黄色葡萄球菌、大肠杆菌;金黄色葡萄球菌对青霉素、磺胺异噁唑、氨苄西林、阿莫西林/克拉维酸的耐药性相对较高;大肠杆菌对氨苄西林、头孢噻吩、磺胺异噁唑存在明显的耐药性。     

Mastitis control programme in the developing dairy industry of tropical lowland Bolivia

Mastitis is an important disease in developing dairy industries. This paper describes a commercial mastitis control programme in Santa Cruz, Bolivia, based upon the five-point mastitis control plan and bulk tank somatic cell count (BTSCC) monitoring. Twelve farms which participated on the programme for 6 years are considered. Mean annual BTSCC fell steadily from 1200000 cells/ml to 461000 cells/ml over this time. This progressive improvement was probably a result of increasing use of and attention to detail in mastitis control practices. The control of subclinical mastitis improved in both hand-milked and machine-milked herds, though hand-milked herds consistently produced milk of higher BTSCC. The mastitis programme did not appear to influence the incidence of clinical mastitis. The paper concludes that a simple mastitis programme based on the five-point plan and BTSCC monitoring can be commercially implemented and effectively control subclinical mastitis in a tropical, developing country.     

不同地区3个奶牛场临床型乳房炎细菌病原种类分析

本试验通过对不同地区3个奶牛场临床型乳房炎细菌病原的分离鉴定,分析其种类和感染情况,并结合牧场环境状况和管理方式,提出控制乳房炎的建议.从中国福建、新疆、重庆3个不同规模化奶牛场采集61份临床型乳房炎乳样,采用传统微生物鉴定方法与PCR技术进行细菌分离鉴定.结果表明,61份临床型乳房炎乳样中单一感染44份(72.13%),混合感染15份(24.59%);共分离到细菌80株,经传统微生物鉴定方法与PCR技术鉴定主要为大肠杆菌、葡萄球菌和链球菌,其中传染性微生物13株(16.25%)、环境性微生物46株(57.50%)、机会性微生物11株(13.75%)、其他微生物10株(12.50%);3个奶牛场临床型乳房炎仍以单一感染为主,混合感染占有一定的比例,传染性致病菌和环境性致病菌为主要致病菌.在实际生产中,规模化牧场可以通过对乳房炎病原菌的分离鉴定来摸清牧场奶牛乳房炎的感染情况和致病菌种类,结合生产实际有助于了解牛群管理情况,发现问题并加以改善,更好的控制乳房炎的发生.     

The descriptive epidemiology of udder lesions in Northern Iraqi ewes

Pathological lesions of the mammary glands were studied in 3693 randomly selected ewes in Northern Iraq between July 1988 and June 1989. The lesions were encountered in 3.9% (143/3693) of the ewes. Prevalences of mastitis, neoplasms and miscellaneous lesions were 3.5% (131/3693), 0.2% (6/3693) and 0.2% (6/3693), respectively. Five patterns of mastitis were encountered: acute suppurative, subacute suppurative, subacute non-suppurative, chronic suppurative and chronic non-suppurative. Mammary neoplasms encountered in the present study included adenocarcinoma, papilloma of the skin of the teat and haemangiopapilloma of the cistern. The miscellaneous lesions of the mammary gland included subepidermal cysts; hyperplasia, metaplasia and dysplasia of the ductal epithelium; thelitis; the presence of corpora amylacia; and blind teats. The highest prevalence of mastitis was during May and June. Significant differences were not encountered in the prevalence of mastitis in various age groups and breeds. Bacteria that were isolated from mastitic udders included Staphylococcus spp., Corynebacterium pyogenes, Pasteurella hemolytica, Pasteurella multocida, and a mixture of Escherichia coli and Staphylococcus spp. The staphylococci were isolated from almost all types of mastitis, C. pyogenes from chronic supparative mastitis, and the pasteurellae from the acute and subacute mastitis. Fungi were not isolated.     

Differentially expressed genes associated with Staphylococcus aureus mastitis of Canadian Holstein cows

To study pathway specific gene expression within the immune-endocrine axis of dairy cows with Staphylococcus aureus mastitis, mRNA was collected from blood mononuclear cells (BMCs) and milk somatic cells (MSCs) of cows (n = 7) identified as culture positive for S. aureus and their matched negative control cows (n = 7) with no evidence of S. aureus mastitis. Labeled cDNA probes derived from BMCs and MSCs of infected and healthy cows were applied to a bovine immune-endocrine cDNA array containing 167 genes. Genes with a log₂ ratio ≥ 0.5 were considered to be up-regulated and genes with a log₂ ratio ≤ −0.5 to be down-regulated. In total, 22 genes were differentially displayed in BMCs and 16 genes in MSCs of case versus controls. Expression of selected genes in BMCs and MSCs were confirmed by real-time PCR. The RT-PCR results were highly correlated with microarray measurements. Some of these genes, such as interleukin (IL)-8 have been previously implicated in other bacterial diseases, and are known to regulate immune responses; whereas, others may reflect novel pathways or genes involved in progressive mammary gland disease. For example, IL-18 was up-regulated in BMCs but not MSCs of mastitic quarters, while IL-17 was more highly expressed in MSCs compared to BMCs. This study identified a number of differentially expressed genes associated with bovine S. aureus mastitis and demonstrates the intricacy of the patterns of gene expression that influence host response to a complex pathogen of significant relevance to both human and veterinary medicine.     

Analysis of Clinical Mastitis Pathogenic Bacterial Species in Three Dairy Farms of Different Areas

In order to control mastitis,clinical mastitis pathogenic bacterial species of three dairy farms in different areas were isolated and identified.The types and infection condition of pathogenic bacteria,environmental conditions and management methods of the farms were analyzed.61 clinical mastitis milk samples from three different large-scale dairy farms in Fujian,Xinjiang,and Chongqing were collected for bacterial isolation and identification using traditional microbial identification methods and PCR technology.Among clinical mastitis milk samples collected in this trial,single infection sample was 44 (72.13%)and mixed infections sample was 15 (24.59%).80 bacteria were isolated,mainly E.coli,Staphylococcus and Streptococcus,including 13 strains of infectious microorganisms (16.25%),46 strains of environmental microorganisms (57.50%),11 strains of chance of microorganisms (13.75%),10 strains of other microorganisms (12.50%).Clinical mastitis in three dairy farms were still given priority to single infection,and mixed infection occupies certain proportion,with infectious pathogens and environmental pathogens as the main pathogenic.In the actual production the dairy cow mastitis infection and pathogen species could be carried out through the isolation and identification of mastitis pathogens in large-scale dairy farms.It was helpful to identify the herd management problems and make improvements to better control the occurrence of mastitis combining with the actual production conditions.     

甘肃省乳房炎奶牛乳源肠球菌的分离、药敏试验及毒力基因原核表达载体的构建

为分离临床型奶牛乳房炎中的肠球菌,检测其耐药性和携带毒力基因的情况,本试验采集了甘肃省东、中和西部3个地区41头临床型乳房炎奶牛的奶样93份,并构建了肠球菌毒力基因的原核表达载体。试验使用选择性培养基分离纯化肠球菌,16S rRNA和生化试验结合的方法鉴定所分离菌株的种;选取16种抗菌药进行药敏试验;常规PCR方法检测11种毒力基因的携带情况,最后对具有免疫原性的毒力基因进行原核表达载体的构建。鉴定结果显示,93份乳样中18株为肠球菌,并分为9种。药敏结果显示,分离株多数为多重耐药菌（multiple resistant bacteria,MDR）,占88.89%,未发现耐万古霉素菌株（vancomycin-resistant enterococcus,VRE）,万古霉素敏感率94.12%,所有分离株至少对一种抗菌药耐药。PCR检测结果表明,11种毒力基因均有检出,cob基因检出率最高（44.44%）,efaA、hyl、ccf和esp基因检出率分别为33.33%、27.78%、27.78%和22.22%,Asa1、cylA、EF3314和gelE基因检出率均为16.67%,Ace和cylM基因检出最低（11.11%）;毒力基因组合因菌种不同而存在差异,选择具有免疫原性的Ace和gelE基因成功构建出原核表达载体pET32a-Ace和pET32a-gelE。本试验为后续绘制3个地区奶牛乳房炎流行病学区域谱以及制备相应抗体和亚单位疫苗提供了基础数据及生物材料。