共表达NDVF和IBDV VP0基因重组鸡痘病毒遗传稳定性研究

将共表达NDVF和IBDVVP0基因重组鸡痘病毒（重组鸡痘病毒vFV282）接种10日龄～11日龄SPF鸡胚，连续传10代，分别对第5、8、10代传代毒进行病毒毒价测定和PCR鉴定；重组鸡痘病毒vFV282接种鸡胚成纤维细胞，分别对第1、5、10、15、20、25、30代传代毒进行病毒毒价测定和PCR鉴定；重组鸡痘病毒vFV282翅翼刺种于鸡体上，连续传8代，对第3、5、8代传代毒进行病毒毒价测定和PCR鉴定。结果表明，重组鸡痘病毒vFV282在SPF鸡胚上传10代、在SPF鸡胚成纤维细胞传30代、在鸡体传8代后，其毒力未返强，F基因和VP0基因未发生缺失和变异。     

新疆维吾尔自治区阿拉尔市某规模化养殖场猪乳源金黄色葡萄球菌的分离鉴定及其毒力、耐药性分析

[目的]分离鉴定新疆维吾尔自治区阿拉尔市猪乳源金黄色葡萄球菌,了解其致病性和耐药性。[方法]从阿拉尔市某规模化养殖场收集6份新鲜猪乳样本,采用甘露醇高盐琼脂（MSA）培养基分离乳样中的金黄色葡萄球菌,利用表型鉴定方法及细菌16S rDNA序列分析法对分离到的菌株进行鉴定,检测分离菌株的生物被膜形成能力,利用K-B纸片扩散法进行药物敏感性试验,利用PCR法检测毒力基因和耐药基因。[结果]经分离培养及分子生物学鉴定,从6份乳样中获得2株金黄色葡萄球菌,分离率为33.33%(2/6);2株金黄色葡萄球菌均具有强生物被膜形成能力（+++）;2株金黄色葡萄球菌均对利福平敏感,均对青霉素、头孢西丁、四环素、红霉素、替米考星、克林霉素和磺胺甲噁唑耐药;在检测的5种耐药基因中,2株金黄色葡萄球菌均携带ant(4)基因和mecA基因;在检测的20种毒力基因中,共检测到6种毒力基因,其中,2株金黄色葡萄球菌均携带seg、sei、hlb、fnbA、clfA基因,有1株携带hla基因。[结论]该养殖场猪乳源金黄色葡萄球菌生物被膜形成能力强,携带多种毒力基因和耐药基因,表现为多重耐药性。     

共表达NDV F和IBDV VPO基因重组鸡痘病毒遗传稳定性研究

将共表达NDV F和IBDV VP0基因重组鸡痘病毒(重组鸡痘病毒vFV282)接种10日龄～11日龄SPF鸡胚,连续传10代,分别对第5、8、10代传代毒进行病毒毒价测定和PCR鉴定;重组鸡痘病毒vFV282接种鸡胚成纤维细胞,分别对第1、5、10、15、20、25、30代传代毒进行病毒毒价测定和PCR鉴定;重组鸡痘病毒vFV282翅翼刺种于鸡体上,连续传8代,对第3、5、8代传代毒进行病毒毒价测定和PCR鉴定.结果表明,重组鸡痘病毒vFV282在SPF鸡胚上传10代、在SPF鸡胚成纤维细胞传30代、在鸡体传8代后,其毒力未返强,F基因和VP0基因未发生缺失和变异.     

共表达NDV F和IBDV VP0基因重组鸡痘病毒遗传稳定性研究

将共表达NDV F和IBDV VP0基因重组鸡痘病毒(重组鸡痘病毒vFV282)接种10日龄~11日龄SPF鸡胚,连续传10代,分别对第5、8、10代传代毒进行病毒毒价测定和PCR鉴定;重组鸡痘病毒vFV282接种鸡胚成纤维细胞,分别对第1、5、10、15、20、25、30代传代毒进行病毒毒价测定和PCR鉴定;重组鸡痘病毒vFV282翅翼刺种于鸡体上,连续传8代,对第3、5、8代传代毒进行病毒毒价测定和PCR鉴定。结果表明,重组鸡痘病毒vFV282在SPF鸡胚上传10代、在SPF鸡胚成纤维细胞传30代、在鸡体传8代后,其毒力未返强,F基因和VP0基因未发生缺失和变异。     

奶牛乳房炎金黄色葡萄球菌的分离鉴定及毒力基因检测

为研究奶牛乳房炎金黄色葡萄球菌毒力基因的分布情况,本试验通过MALDI Biotyper系统对分离自3个奶牛场的25株金黄色葡萄球菌进行鉴定及聚类分析,并采用PCR方法对25株金黄色葡萄球菌的nuc、clfA、fnbA、fn-bB、tsst-1、sea、seb和sec等14种毒力基因进行检测。结果显示,25株金黄色葡萄球菌被分为3个类群,各个类群之间及类群内菌株遗传距离较远。14种毒力基因中有7种被检出,检出率分别是nuc(100%)、fnbA (100%)、seg(64%)、clfA(52%)、fnbB(32%)、tsst-1(16%)和sea(4%),其余未检出。结果表明,不同奶牛场甚至同一奶牛场内存在多种金黄色葡萄球菌的流行株,且金黄色葡萄球菌携带毒力基因的数量和种类不同,并存在多种毒力基因组合,为奶牛金黄色葡萄球菌性乳房炎的防治提供基础研究。     

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。     

鸢尾黄素作为MgrA抑制剂对小鼠金黄色葡萄球菌肺炎的治疗作用

本研究旨在从中药小分子库中筛选出金黄色葡萄球菌（Staphylococcus aureus）转录调控因子MgrA的单体抑制剂,并探讨其对金黄色葡萄球菌主要毒力因子的影响及对小鼠金黄色葡萄球菌肺炎的治疗作用。使用荧光各向异性分析法进行抑制剂的筛选,通过实时荧光定量PCR、溶血试验及纤维蛋白原黏附试验考察药物对MrgA调控的相关毒力基因转录表达及对溶血和黏附的抑制作用,采用热稳定迁移试验对其抑制机制进行初步探讨,最后通过建立小鼠肺炎模型评估鸢尾黄素对金黄色葡萄球菌诱导的小鼠肺炎的治疗作用。结果显示,鸢尾黄素作为MgrA的抑制剂,在不影响细菌生长的低浓度下（IC₅₀=20.35 μg/mL）就能显著抑制MgrA的活性（P<0.05）;实时荧光定量PCR结果显示,鸢尾黄素可显著降低fnba基因的转录水平（P<0.05）,极显著降低hla和RNAⅢ基因的转录水平（P<0.01）,同时极显著上调ebh、srap和spa基因的转录水平（P<0.01）;溶血试验显示,8 μg/mL鸢尾黄素可极显著抑制USA300菌株的溶血作用（P<0.01）;纤维蛋白原黏附试验证实32 μg/mL鸢尾黄素可极显著抑制USA300菌株的黏附活性（P<0.01）;热稳定迁移试验揭示了鸢尾黄素是通过与MgrA结合从而抑制其活性。小鼠肺炎试验结果显示,鸢尾黄素能极显著提高金黄色葡萄球菌感染小鼠的存活率（P<0.01）,极显著减少其肺脏载菌量（P<0.01）,并减轻小鼠肺脏组织的病理损伤和炎症反应。以上结果表明,鸢尾黄素能通过抑制转录调控因子MgrA的活性对金黄色葡萄球菌感染引起的小鼠肺炎起到治疗作用,可作为开发治疗金黄色葡萄球菌感染的先导化合物,并为以毒力因子MgrA为靶标的药物研发提供了理论依据。     

乳牛衣原体性流产的病原鉴定及免疫原性研究

对采自甘肃、陕西和宁夏奶牛场的发生不明原因流产乳牛病料进行了间接血凝试验（IHA）、PCR、病原分离鉴定和MOMP基因检测，证实病原为鹦鹉热衣原体（Chlamydia psittaci）。对分离株Sx5和Nx进行毒力稳定性和免疫原性测定，用鸡胚连传20代，SX5和NX株的毒力比较稳定，毒力效价分别为10^-11ELD50和10^-10ELD50；用这2株菌对小鼠和牛做免疫效力试验，结果显示，免疫鼠5／5和5／5保护，对照鼠0／5保护；免疫牛3／3和3／3保护，对照牛0／3保护。表明，SX5和NX株具有良好的免疫原性，可以作为疫苗研制的候选菌株。     

宁夏地区牛源金黄色葡萄球菌毒力基因和大环内酯类抗生素耐药基因的检测及诱导耐药

采用PCR技术对231株牛源金黄色葡萄球菌进行7种毒力基因及2种大环内酯类耐药基因的检测,同时运用双纸片法即D试验对31株耐药表型为红霉素耐药而克林霉素敏感或中介菌株进行诱导耐药试验。结果显示:在231株金黄色葡萄球菌中,99.1%(229/231)的菌株检测到毒力基因hla,97.4%(225/231)的菌株检测到毒力基因hlb,98.3%(227/231)的菌株检测到毒力基因clfa,且同时携带hla、hlb和clfa等3种毒力基因的菌株占45.9%;毒力基因pvl、sea、seb和sec的检出率分别为1.73%(4/231),27.7%(64/231),29.0%(67/231)和5.6%(13/231)。介导大环内酯类抗生素耐药的耐药基因msrA、ermC的检出率分别为55.3%和67.5%。红霉素诱导克林霉素耐药的阳性率为90.3%(28/31)。结果表明:宁夏地区牛源金黄色葡萄球菌毒力基因检出率较高的是hla、hlb和clfa,毒力基因的组合较复杂,耐药基因msrA、ermC的检出率也较高,并且诱导耐药阳性率所占比例较高。     

红嘴鸥源致病性大肠杆菌毒力基因检测及致病性分析

以10株红嘴鸥源大肠杆菌云南省分离株为研究对象,采用PCR菌株进行毒力基因测定并进行小鼠致病性试验。PCR性率依次为astA(60%6株)、fuyA(60%6株)、Iss(50%5株)、iucD(50%5株)、tsh(40%4株)、Vat(30%3株)、papC(30%3株)、kpsⅡ(30%3株)、hlyE(20%2株)、irp2(20%2株)、fimH(10%1株),小鼠致病性试验结果显示携带毒力基因数量多的菌株致病力越强,小鼠发病时间死亡时间越短。结果说明:10株分离菌株均携带不同的毒力基因,不同毒力基因的组合与小鼠的致病力密切相关。不同毒力基因的组合对小鼠相关致病机理有一定关系,有待进一步研究。     

猪流行性腹泻病毒JX16株的分离鉴定及体外传代对其毒力的影响

2010年中国新出现了以新生仔猪高发病率、高死亡率为主要特征的变异株。为了明确山东地区猪流行性腹泻病毒（porcine epidemic diarrhea virus，PEDV）变异株特征，本试验利用Vero细胞对莒县某猪场的腹泻病料进行了病毒分离和体外传代培养。通过光学显微镜和细胞超薄切片观察、RT-PCR检测、间接免疫荧光试验、S基因序列进化分析、滴度测定和致病力试验进行病毒序列及特征分析，并探究接毒后细胞脱落时间的变化。光学显微镜观察发现，该病毒感染Vero细胞后能引起典型的合胞体病变；细胞超薄切片观察发现，病毒粒子多呈致密核芯，能引起宿主细胞线粒体肿胀，溶解；RT-PCR检测和间接免疫荧光试验结果证实其为PEDV，最终通过测序证实该毒株为变异毒株。体外传代培养至100代过程中，随着病毒代次升高，其对细胞适应性不断增强。接毒后细胞脱落时间由48 h逐渐缩短至17 h，病毒滴度由6代时的10^5.3TCID₅₀/mL逐渐升高至100代的10^7.5TCID₅₀/mL。致病力试验结果显示，病毒毒力随代次升高逐渐下降，40代时对仔猪已无致病力。本试验结果为PEDV突变株致病基因的确定及疫苗的研发奠定了基础。     

The effect of auto-vaccination therapy on the phenotypic variation of one clonal type of Staphylococcus aureus isolated from cows with mastitis

The aim of this study was to demonstrate the effect of auto-vaccine therapy on selected properties of Staphylococcus aureus strains, isolated from milk of cows with subclinical mastitis. The experiment was based on auto-vaccines which were prepared from S. aureus strains isolated from 16 cows. S. aureus strains isolated from cows on the 7th, 21st and 35th day following auto-vaccination, were analyzed phenotypically and genotypically. The isolated strains represented 17 biotypes all belonging to one clonal type. Increases of new biotypes of S. aureus were detected on the 35th day of therapy. Among 48 re-isolated strains, 18.75% (9/48) revealed single and 12.50% (6/48) multiple phenotypical changes. The present study demonstrated that during auto-vaccine therapy, S. aureus strains can change phenotypically, pointing out the necessity for using precise diagnostic methods, that would make possible a better assessment of the used therapy.     

Pattern of enterotoxin genes seg, seh, sei and sej positive Staphylococcus aureus isolated from bovine mastitis

PCR detection of the genes encoding the newly described staphylococcal enterotoxins (SE) SEG, SEH, SEI and SEJ was carried out for 104 randomly selected Staphylococcus aureus field strains isolated from cases of bovine mastitis. Sixty-one (58.7%) isolates were positive for one or more of these novel enterotoxin genes. Thirty-six field strains were classified as carrier of seg, 22 of sei gene and 23 were positive for sej gene. None of the 104 investigated ruminant S. aureus strains carried the seh gene. Thirty-seven of these S. aureus strains showed a combination of genes encoding enterotoxin types SEA to SEE or toxic shock syndrome toxin 1 (TSST 1). Thirteen cultures harboured only one, 28 two, 12 three and 8 four enterotoxin genes. Among the 61 S. aureus field strains 14 (23.0%) were positive for the genes encoding SEJ and SED and 10 (16.4%) isolates for those encoding SEG and SEI. Isolates harbouring the sed/sej genes were further characterized by macrorestriction analysis and pulsed-field-gelelectrophoresis (Pfge). Macrorestriction analysis revealed six patterns. Nine of these14 S. aureus isolates (64.3%) exhibited two patterns with a high degree of relationship (>80%).     

PCR-based detection of genes encoding virulence determinants in Staphylococcus aureus from birds

The present study was designed to comparatively investigate 19 Staphylococcus aureus strains isolated from specimens of 19 different birds during routine microbiological diagnostics. The S. aureus strains were characterized genotypically by polymerase chain reaction (PCR) amplification using 62 different oligonucleotide primers amplifying genes encoding staphylococcal cell surface proteins, exoproteins and two classes of the accessory gene regulator agr. All 19 investigated S. aureus were positive for the gene segment encoding a S. aureus-specific part of the 23S rRNA, the genes encoding thermostable nuclease (nuc), clumping factor (clfA) and coagulase (coa) and the gene segments encoding the Xr-repetitive region and the immunoglobulin G (IgG)-binding region of protein A (spa). In addition, all tested strains were positive for the genes hla and fnbA and negative for the genes seb, sec, sed, see, sej, tst, eta and etb. The remaining genes, including sbi, hlb, fnbB, ebpS, cna (domains A and B), cap5, cap8, set1, agr class I, agr class II, sea, seg, seh and sei were detected in a variable number of isolates. The presented data give an overview on the distribution of virulence determinants of S. aureus strains isolated from birds. This might be useful to understand the role of these virulence determinants in bird infections.     

Occurrence of enterotoxigenic strains of Staphylococcus aureus in raw poultry meat

The aim of this work was to determine the contamination of raw poultry meat with enterotoxigenic strains of S. aureus, using the PCR method. PCR is a rapid and sensitive method, which can show the presence in food of enterotoxigenic strains of S. aureus on the basis of specific gene sequences and detect the potential source of contamination before enterotoxins are produced. No coagulase-positive staphylococci strains were found in 65 samples of chicken parts, but these bacteria were present in 11 of 23 examined samples of minced turkey meat (48%). Using the primers for enterotoxin genes A to C, 4 of the 11 isolated S. aureus strains showed a positive result in the PCR. Three of the isolates represented the SEB gene and remaining one the SEC gene. The results obtained showed that PCR is sensitive and rapid method which may be used for detection and identification of enterotoxigenic S. aureus.     

Detection of the enterotoxins A, B, and C genes in Staphylococcus aureus from goat and bovine mastitis in Brazilian dairy herds

To determine the distribution of genes that encode enterotoxins A, B and C, 36 strains of Staphylococcus aureus isolated from goat mastitis and 64 isolated from bovine mastitis were analyzed by Multiplex PCR. Of the total strains studied, 37 (37%) were detected to have some of the SEs genes. From the bovine mastitis strains, 4 (6.3%) co-amplified the sea and seb genes and 2 (3.1%) were positive for the sec gene. From the goat mastitis strains, 31 (86%) tested positive to the Multiplex, and the sec gene was detected in all of them. The production of SE was detected in all strains harboring the corresponding gene. The results demonstrated that S. aureus isolated from goat mastitis had a higher enterotoxigenic potential than those isolated from bovine mastitis. Additionally, the presence of the sec gene in the majority of goat mastitis strains suggests a possible involvement of SEC in goat mastitis pathogenesis.     

Induced staphylococcal infections in the bovine mammary gland

In a study to develop and define a practical model of bovine mastitis caused by Staphylococcus aureus, induced infections were attempted in 203 bovine mammary glands of 41 cows, using 12 strains of S aureus. Approximately 100 colony-forming units of S aureus in saline solution were injected after milking, and milk samples were collected daily from test glands for 14 days to monitor the progress of infections and inflammatory responses. Relationships were examined for cow-related factors and for various characteristics of the strains of S aureus used to the development of a persistent intramammary infection. A dairy cow that was useful in this model was defined as follows: (1) the 2nd to 7th month in the 1st to 5th lactation; (2) producing milk from all mammary glands that contained less than 6 x 10(5) somatic cells/ml; and (3) having mammary glands that were free of any primary mastitis pathogen, as well as micrococci and Corynebacterium bovis. From the present study, it was not possible to define clearly a strain of S aureus which would be useful in the model, but 5 strains of S aureus were identified as being capable of producing persistent subacute infections with a high degree of repeatability.     

Evaluation of the presence of the bap gene in Staphylococcus aureus isolates recovered from human and animals species

The implication of biofilm in chronic bacterial infection in many species has triggered an increasing interest in the characterization of genes involved in biofilm formation. The bap gene is a newly identified gene that encodes the biofilm-associated protein, BAP, which is involved in biofilm formation in Staphylococcus aureus. So far the bap gene has only been found in a small proportion of S. aureus strains from bovine mastitis in Spain. In order to study the presence of the bap gene in S. aureus isolates obtained from other species and various locations, a collection of 262 isolates was tested by PCR, using published primers and dot-blot. The results indicated that none of the isolates carried the bap gene suggesting that the prevalence of this gene among S. aureus isolates should be very low.     

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。     

A real-time PCR assay to detect the Panton Valentine Leukocidin toxin in staphylococci: screening Staphylococcus schleiferi subspecies coagulans strains from companion animals

Recent reports suggest that methicillin-resistant strains of Staphylococcus schleiferi subspecies coagulans are now commonly isolated from dogs. Given the association of a potentially mobile SCCmec type IV element with lysogenic phage-encoded Panton Valentine Leukocidin (PVL) toxin genes in community-acquired methicillin-resistant Staphylococcus aureus strains we hypothesized that methicillin-resistant S. schleiferi ssp. coagulans strains may also encode PVL toxin genes. Forty S. schleiferi ssp. coagulans strains isolated from companion animals were studied. Susceptibility to oxacillin was determined by broth microdilution and all isolates were screened by PCR for the presence of the mecA gene. SCCmec typing was performed on 14 isolates. A real-time PCR assay was developed for the detection of the PVL genes using a SmartCycler. Pulsed-field gel electrophoresis (PFGE) was performed to determine whether S. schleiferi ssp. coagulans strains were homogeneous. Twenty-eight of the 40 isolates (70%) were resistant to oxacillin and 26/28 possessed the mecA gene by PCR. SCCmec IV was identified in seven strains; the other seven isolates were not typable by this technique. All 40 strains were negative for the PVL toxin gene. PFGE showed a heterogeneous population and 13 different profiles were determined. In conclusion, this study showed that PVL toxin genes were not detected in a heterogeneous population of methicillin-resistant S. schleiferi ssp. coagulans strains isolated from companion animals.