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1.
Lautz S Kanbar T Alber J Lämmler C Weiss R Prenger-Berninghoff E Zschöck M 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2006,53(9):434-438
Phenotypic properties and species-specific PCR tests based on the nuc gene of Staphylococcus intermedius and S. aureus, and a conserved region of 16S rDNA were used to identify 45 S. intermedius and four S. aureus isolated from samples of dogs during routine diagnostics. Four S. pseudintermedius strains used for control purposes reacted positively with the S. intermedius nuc PCR showing the close relationship between both species. Investigating the 45 S. intermedius and four S. pseudintermedius strains for the prevalence of the exfoliative toxin SIET encoding gene yielded the presence of the gene for 21 of the S. intermedius and two of the S. pseudintermedius strains. Partial sequencing of the toxin gene of a single S. intermedius strain and comparing this sequence with that obtained from GenBank revealed an almost complete identity. The presence of the exfoliative toxin gene could mainly be found among S. intermedius isolated from skin and wound infections and from otitis externa possibly indicating a role of this toxin for the clinical symptoms. 相似文献
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Zubeir IE Kanbar T Alber J Lämmler C Akineden O Weiss R Zschöck M 《Veterinary microbiology》2007,121(1-2):170-176
Methicillin/oxacillin resistance of 10 S. intermedius strains was investigated by conventional and molecular methods. The strains tested had been isolated in Germany during routine veterinary microbiological examinations of specimens from a small animal clinic between May and September 2005. Epidemiological relationships of the strains were studied by macrorestriction analysis of their chromosomal DNA using pulsed field gel electrophoresis (PFGE). Species identity of the 10 S. intermedius strains was confirmed by conventional methods and by PCR mediated amplification of S. intermedius specific segments of thermonuclease encoding gene nuc. As controls, four methicillin/oxacillin resistant S. intermedius (MRSI) strains obtained from specimens sent by four veterinarians and three selected methicillin/oxacillin sensitive S. intermedius (MSSI), also obtained from the small animal clinic, were tested. The 10 strains, representing approximately 6% of all S. intermedius isolated from the clinic throughout the time period mentioned above, and the four MRSI obtained from veterinarians, were methicillin/oxacillin and penicillin resistant using disk diffusion tests and could be cultivated on oxacillin resistant screening agar base (ORSAB). Both resistances could be confirmed by multiplex PCR detecting the resistance genes mecA and blaZ. The three MSSI were methicillin/oxacillin sensitive in all tests. Epidemiological investigation by macrorestriction analysis of the chromosomal DNA of the strains by pulsed field gel electrophoresis revealed that all 10 MRSI strains obtained from the clinic and the four MRSI strains obtained from veterinarians, in contrast to the three MSSI strains, represent identical or closely related bacterial clones possibly indicating a cross-infection of the animals in the clinic and the distribution of a single MRSI clone in the pet population. 相似文献
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Terauchi R Sato H Hasegawa T Yamaguchi T Aizawa C Maehara N 《Veterinary microbiology》2003,94(1):19-29
A rounding effect was demonstrated in cultured cells inoculated with the culture filtrates (CFs) of 60 strains of Staphylococcus intermedius derived from dogs affected with pyoderma. Exfoliative toxin (ET)-like toxin (ETLT) was isolated from the CF of S. intermedius strain D-52, which exhibited strong rounding activity and then was purified by gel filtration on a Sephadex G-75 column, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The ETLT caused exfoliation in 1-day-old chickens, suckling Syrian hamsters, and dogs, but not in suckling mice. The ETLT was serologically different from exfoliative toxin A (ETA), exfoliative toxin B (ETB), exfoliative toxin C (ETC), S. hyicus exfoliative toxin A (SHETA), and SHETB, as shown by Western blot analysis. The molecular weight of the ETLT was estimated at 30 kDa by SDS-PAGE. In the present study, we propose the ETLT was a novel type of ET, S. intermedius exfoliative toxin (SIET). 相似文献
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Wakita Y Shimizu A Hájek V Kawano J Yamashita K 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2002,64(3):237-243
Staphylococcus intermedius from pigeons, dogs, foxes, mink, and horses, was characterized by pulsed-field gel electrophoresis (PFGE) to evaluate the use of this typing method for discriminating among strains. SmaI cut the chromosomal DNA into 7-13 fragments ranging from approximately 48 kb to 655 kb, with most of the detectable fragments being smaller than 172 kb. S. intermedius from various animals had a high degree of restriction fragment length polymorphism. Pigeon strains have a similar genotype, despite the difference in their isolation area. Phage typing indicated that the dog, fox, and mink strains belong to the canine I or canine II type. The PFGE method further differentiated the mink strains from the dog and fox strains with regard to three fragments between 256 kb and 570 kb. As such, genomic DNA fingerprinting by PFGE appears to be an effective technique for discriminating S. intermedius strains from various animals. A combination of PFGE typing and phage typing would provide more detailed information than the single method for ecological investigations of S. intermedius. 相似文献
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Wakita Y Kawano J Shimizu A Hajek V Tomisaka E Yasuda R Matsuo E 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2002,64(7):603-605
The development of a PCR assay based on the 16S ribosomal RNA gene (rDNA) sequence was carried out for the identification of Staphylococcus intermedius. Sixty-six strains of S. intermedius, 70 of Staphylococcus aureus and 2 of Staphylococcus hyicus were examined for the assay. The 16S rDNA, of which the PCR target fragment makes up 901 bp corresponding to the sequence data of the gene, was detected in all strains of S. intermedius, but it was not detected in any strains of either S. aureus or S. hyicus. These results suggest that the PCR allows a simple and precise identification of S. intermedius. 相似文献
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利用辐射杂种克隆板对猪NDUFS2(NADH dehydrogenase(ubiquinone)Fe-S protein 2)基因进行了染色体定位,克隆了该基因的CDS序列,用相关软件进行了基因结构和功能预测,并利用半定量RT-PCR对基因的组织表达谱进行了分析。研究结果表明:NDUFS2基因定位于猪的4号染色体SSC4q,与标记SW589紧密连锁。获得NDUFS2基因的CDS为1 392 nt,编码463个氨基酸。物种间系统进化树分析表明:猪与牛的NDUFS2基因序列同源性更高。结构预测发现猪NDUFS2基因具有一个NuoD保守结构域,与能量的产生和转化功能有关。根据组织表达谱分析结果,NDUFS2在猪的11种组织中的表达量有差异。 相似文献
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奶牛朊病毒基因克隆与序列分析 总被引：8，自引：0，他引：8
根据已报道正常牛朊蛋白(PrP^c)基因(PRNP)序列设计引物，采用PCR法扩增了6头荷斯坦奶牛的PRNP基因，将其克隆到T-Vector。序列测定及分析表明所克隆的奶牛PRNP基因片段为795bp，该基因内无内含子，包含了牛PRNP完整编码区序列，编码264个氨基酸的前体蛋白，推测其分子量约34ku。其中2头共同含有未曾报道的牛PRNP多态性位点M120I，无义突变G234A，但未引起酶切位点变异，未发现插入或缺失变异；与已报道牛PRNP序列(GenBank收录号为DI0613)相比，两者核苷酸序列同源性为99％，其编码的氨基酸同源性为99％。 相似文献
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致绵羊脑炎型肠球菌溶血素CylA基因的克隆及序列测定 总被引：3，自引：0，他引：3
以致绵羊脑炎型肠球菌（Enterococcus）染色体DNA为模板，PCR扩增溶血素CylA的结构基因，将PCR产物克隆于pMD19-T载体，对PCR产物进行测序鉴定。与已发表的肠球茵溶血素CylA基因序列比较，同源性达99．3％。 相似文献
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Iyori K Futagawa-Saito K Hisatsune J Yamamoto M Sekiguchi M Ide K Son WG Olivry T Sugai M Fukuyasu T Iwasaki T Nishifuji K 《Veterinary dermatology》2011,22(4):319-326
Staphylococcal exfoliative toxins are known to digest desmoglein (Dsg) 1, a desmosomal cell–cell adhesion molecule, thus causing intraepidermal splitting in human bullous impetigo, staphylococcal scalded skin syndrome and swine exudative epidermitis. Recently, a novel exfoliative toxin gene (exi), whose sequence shares significant homology with previously identified exfoliative toxins, was isolated from Staphylococcus pseudintermedius. Little is known about the pathogenic involvement of this toxin in canine pustular diseases such as impetigo. The aim of this study was to determine whether EXI, the product of the exi gene, digests canine Dsg1 and causes intraepidermal splitting in canine skin. An exi gene was isolated from chromosomal DNA of an S. pseudintermedius strain obtained from a pustule of a dog with impetigo, and was used to produce a recombinant EXI by Escherichia coli expression. When purified recombinant EXI was injected intradermally into normal dogs, it caused the development of vesicles or erosions with superficial epidermal splitting. In addition, the EXI abolished immunofluorescence for Dsg1, but not for Dsg3, at the injection sites. Moreover, the EXI directly degraded baculovirus‐secreted recombinant extracellular domains of canine Dsg1, but not that of canine Dsg3, in vitro. The EXI also degraded mouse Dsg1α and swine Dsg1, but not human Dsg1, mouse Dsg1β and Dsg1γ. Conversely, recombinant SIET, previously designated as S. intermedius exfoliative toxin, did not cause intraepidermal splitting or degradation of any Dsgs. These findings indicate that EXI has a proteolytic activity that digests canine Dsg1, and this characteristic might be involved in the pathogenesis of intraepidermal splitting in canine impetigo. 相似文献
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为了克隆从患肺炎犊牛和健康犊牛上分离的多杀性巴氏杆菌血红素结合受体(heme acquisition system receptor,HasR)基因并进行序列分析,分别以致犊牛肺炎和健康犊牛携带的多杀性巴氏杆菌DNA为模板,通过PCR反应扩增出血红素结合受体基因的全部序列,PCR产物纯化后克隆到pMD19-T载体上,经菌液PCR和酶切鉴定后进行序列测定。各菌株HasR基因序列比对结果发现,Pm-BS-a、Pm142-x-4、Pm142-x-3、Pm149-x、Pm-BS-d的序列之间亲源关系较近,而Pm149-xby与参考菌株Pm70的同源性较高。健康牛源的多杀性巴氏杆菌与致犊牛肺炎多杀性巴氏杆菌之间HasR基因的同源性非常高,说明致犊牛肺炎多杀性巴氏杆菌是一种条件性致病菌,其引起的犊牛肺炎可能属内源性感染。 相似文献
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人脑源性神经营养因子基因(hBDNF)在转基因家蚕丝腺中的特异表达 总被引：3，自引：1，他引：2
家蚕丝腺具有强大合成与分泌蛋白质的能力,利用其作为生物反应器生产高附加值外源蛋白有着广阔的市场前景。以人血白细胞基因组DNA为模板,扩增并克隆了人脑源性神经营养因子基因(hBDNF)核苷酸序列。序列分析表明,克隆的hBDNF核苷酸序列与已发表序列(GenBank登录号:NM_170735)的同源性为100%。随后采用家蚕丝胶基因(Ser1)启动子,以增强型绿色荧光蛋白(EGFP)为筛选标记,将hBDNF构建到piggyBac转座表达载体并注射入家蚕早期胚胎,在G1代筛选获得了54头转基因阳性个体。经分子检测证实,hBDNF已整合到家蚕基因组并在丝腺有较高水平的特异表达。 相似文献
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In this study, bacterial cultures were collected from five sites on each of 50 healthy cats and 48 cats with inflammatory skin disease (ISD), to determine prevalence of carriage and relative frequency of methicillin resistance in coagulase-positive staphylococci and Staphylococcus schleiferi ssp. schleiferi. Latex agglutination testing for penicillin-binding protein 2a (PBP2a) and pulsed field gel electrophoresis (PFGE) were performed on all methicillin-resistant (MR) isolates. Polymerase chain reaction (PCR) for the mecA gene was performed on MR S. intermedius and S. schleiferi isolates. Staphylococcal chromosomal cassette (SCCmec) typing was performed on all MR S. aureus isolates. Coagulase-positive staphylococci and S. schleiferi ssp. schleiferi were isolated from 24 of 48 cats with ISD: Staphylococcus aureus (14 of 24, 58%), Staphylococcus intermedius (11 of 24, 46%), Staphylococcus schleiferi ssp. schleiferi (1 of 24, 4%), and Staphylococcus hyicus (1 of 24, 4%). Prevalence of MR was 7% for S. aureus, 0% for S. intermedius, 100% for S. schleiferi ssp. schleiferi, and 0% for S. hyicus. Coagulase-positive staphylococci were isolated from 17 of 50 healthy cats: S. aureus (10 of 17, 59%), S. intermedius (11 of 17, 65%), and S. schleiferi ssp. coagulans (1 of 17, 6%). Prevalence of MR was 20% for S. aureus, 18% for S. intermedius, and 0% for S. schleiferi ssp. coagulans. All MR isolates were positive for PBP2a via latex agglutination. Methicillin-resistant S. intermedius and S. schleiferi ssp. schleiferi isolates were also positive for the mecA gene via PCR. Methicillin-resistant S. aureus isolates were identified as SCCmec type II. Results of PFGE indicated heterogeneity among isolates. There was no significant difference in staphylococcal isolation or methicillin resistance between study groups. While present, MR coagulase-positive staphylococci are significantly less common in these study populations. 相似文献
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Kim TJ Na YR Lee JI 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2005,52(3):119-124
A total of 160 Staphylococcus intermedius isolates were recovered from cases of pyoderma in 2002 and were examined for susceptibility to 13 different antimicrobial agents. Ninety per cent (144) of the isolates were resistant to tetracycline, derivatives of which have been used until recently, and 18% (29) were resistant to chloramphenicol which was banned from use 13 years ago. The presence of genes encoding chloramphenicol acetyltransferase (CAT) and tetracycline resistance (tet); tet(K), (L), (M), and (O) were determined by PCR in the 29 chloramphenicol and tetracycline resistant isolates. Seventeen (59%) isolates contained the cat gene while 12 (41%) isolates did not carry the cat gene, implying there may be other genes for chloramphenicol resistance that were not detected by the primers (primer set 1) used in this study. The tet(M) gene was found in 28 (97%) of the resistant S. intermedius isolates, but none contained the tet(O) gene. All 29 isolates carried one or two tet genes; tet(K), (L), and (M), with four different distribution patterns. New PCR products, a 1.1 kb product using primer set 1 and a 0.2 kb product using primer set 2, were cloned and sequenced. A 904 bp fragment of S. aureus plamid pS194, including sequence from the streptomycin adenyltransferase gene (804 bp), was found inserted into the terminal region of the cat gene (GenBank accession no. AY604739), whilst the sequence of 0.2 kb was previously unpublished. 相似文献
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牛GM-CSF与金黄色葡萄球菌黏附素FnBPB共表达质粒的构建与原核表达 总被引：1，自引：0，他引：1
采用PCR方法对牛粒细胞-巨噬细胞集落刺激因子(GM-CSF)和金黄色葡萄球菌FnBPB的D区进行特异性的扩增,并通过重叠延伸PCR扩增GM-CSF-FnBPB串联基因,构建了克隆质粒pMD19-GM-CSF-FnBPB。利用表达载体pET-32a(+)对该融合基因片段进行原核表达,SDS-PAGE分析表明,在1mmol/L IPTG诱导浓度下,在39ku处出现了与目的蛋白一致的外源蛋白带,Western blot分析表明,该蛋白具有反应原性,进而证明该融合基因成功在原核细胞中表达。 相似文献
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中国牛亚科家畜GH基因编码区序列的遗传变异研究 总被引：2，自引：0，他引：2
采用PCR产物直接双向测序法,分段扩增普通牛、瘤牛、牦牛、大额牛和亚洲水牛共5个牛种的GH基因,并拼接成编码区全序列,分析中国牛亚科家畜不同牛种GH基因编码区序列变异及其分子进化特征。结果表明,牛GH基因编码区序列全长654bp,种间核苷酸突变率在0．1％～1．84％。5个牛种编码区序列定义了10种单倍型,瘤牛的单倍型多样性最高,大额牛和水牛均无单倍型多样性。GH基因编码区序列的密码子使用存在偏倚性,共发现了25个偏好性密码子。核苷酸的替代以转换为主,转换明显高于颠换,转换／颠换比为3．0。非同义突变位点远远少于同义突变位点,同义与非同义替代发生的速率比都小于或等于1,表明GH基因编码区序列不受达尔文正选择的影响。以GH基因单倍型序列为基础的分子进化树表明,水牛与普通牛、瘤牛、牦牛、大额牛间分化很明显;普通牛、瘤牛、牦牛、大额牛间序列分化并不明显,并且它们共同拥有一条相同的祖先核苷酸序列。说明中国牛亚科家畜GH基因编码区序列的变异相当贫乏,并且由于功能的约束表现得相当保守,进化速率相当缓慢。 相似文献