Characterization of a novel type of MLSB resistance plasmid from Staphylococcus saprophyticus carrying a constitutively expressed erm(C) gene

An erm(C)-carrying plasmid of unusual size and restriction map, designated pSES22, was identified in a Staphylococcus saprophyticus strain and sequenced completely. Constitutive expression of the erm(C) gene from pSES22 is based on a novel 22-bp tandem duplication in the erm(C) translational attenuator. Comparative analysis of the deduced Erm(C) amino acid sequence revealed that Erm(C) from pSES22 - together with an Erm(C) methylase from S. hyicus - represented a separate branch in the homology tree of Erm(C) methylases. Structural comparisons showed that plasmid pSES22 differed distinctly from all other completely sequenced erm(C)-carrying resistance plasmids. However, pSES22 was similar to several members of a diverse group of small plasmids, all of which carried closely related plasmid backbones consisting of the genes repU and pre/mob, but differed in their resistance genes.     

Antibiotic susceptibility and prevalence of erythromycin ribosomal methylase gene, erm(B) in Streptococcus spp

The aim of this study was to investigate drug resistance and the genetic relatedness of erythromycin-resistant Streptococcus spp. from different animals and humans in Taiwan. Cumulatively, 248 isolates were collected from 15 animal species and human patients and the susceptibilities of the isolates to six antimicrobial agents including azithromycin (AZI), clarithromycin (CLAR), erythromycin (ERY), spiramycin (SPIR), amoxicillin (AMO), and enrofloxacin (ENRO) were determined by the agar dilution method. The results indicated that resistance among the 248 strains was highest for SPIR, followed by ENRO, CLAR, ERY, AZI, and AMO. The most common resistotypes of the isolates from mammals and aquatic animals were AZI-CLAR-ERY-SPIR (27.5%) and SPIR (55.1%), respectively. The presence of ERY-resistant genes was confirmed by PCR. The erm gene was amplified from 28 isolates (20.6%) by PCR for further investigation. The predominant erm gene in the ERY-resistant isolates was the erm(B) gene. The phylogenetic analysis of the erm(B) gene results indicated that there was a close genetic relationship among all the strains but the genotypic clusters did not show clear segregation of the isolates according to the source or region.     

Staphylococci on the skin of pigs: isolates from two farms with different antibiotic policies.

Staphylococci isolated from pigs on two farms were identified and their antibiotic resistance and plasmid profiles were examined. A highly resistant Staphylococcus hyicus was epidemic on one of the farms which was also that at which antibiotic-containing feedstuffs were used most often. Staphylococci from this farm were more often resistant to two or more antibiotics than were the strains from the other farm. The many plasmids present in these staphylococci prevented the determination of the genetic nature of the antibiotic resistance.     

兽医临床凝固酶阴性葡萄球菌分离株的耐药性及耐药基因检测

采用微量肉汤稀释法和D-试验法检测64株凝固酶阴性葡萄球菌（CNs）对10种抗菌药物的耐药性,并用PCR方法分别检测头孢西丁耐药菌株和红霉素耐药菌株中mecA基因以及erinA、ermB、ermC和msrA基因的携带情况。结果表明,所有菌株均对万古霉素和甲氧苄啶／磺胺甲恶唑敏感;泰妙菌素为耐药率（65．6％）最高的抗菌药物,其次是红霉素、氧氟沙星和β-内酰胺类药物。28株（43．8％）CNS对青霉素耐药,其中26株对头孢西丁耐药（MRS）并且均携带mecA基因。30株（46．9％）CNS对红霉素耐药,其中24株为MLSB耐药表型,主要由ermB基因介导。总之,兽医临床CNs分离株对常用抗菌药物的耐药性不同,mecA基因和ermB基因的携带分别是兽医临床MRS和MLSB表型产生的主要原因。     

江苏地区奶牛乳房炎凝固酶阴性葡萄球菌耐药基因分析

对江苏地区多个奶牛场采样,并对奶牛乳房炎(Bovine mastitis,BMT)检测阳性奶样进行病原菌分离、鉴定。对分离出的凝固酶阴性葡萄球菌(Coagulase-negative Staphylococci,CNS)分别进行20种药物的敏感性试验和相关耐药基因检测。结果显示,BMT检测阳性的奶样有610份,528份检出细菌,共检出细菌802株,鉴定为CNS的217株;大部分CNS对万古霉素、左氟沙星、氯霉素、磷霉素、呋喃妥因敏感,对青霉素、大观霉素具有较强耐药性;CNS对β-内酰胺类(blaZ)、大环内酯类(ermC)和四环素类(tetK、tetM、tetL、tetO)耐药基因的检出率分别为29.95%、2.30%、24.88%、1.84%、1.84%和0.00%。     

薄层色谱法应用中的注意环节

薄层色谱法(thinlayerchromatography简写TLC)是一种物理化学的分离技术,即将吸附剂或载体(支持剂)均匀的铺在玻璃板上或塑料板上使成薄层,在此薄层上进行色谱分离的方法,又称薄层色谱。目前,饲料及药物市场不断地开发和研究新产品,各个品种大量涌现市场,由于有些饲料添加剂和药物结构相似,不易分离和鉴别,而且有些饲料添加剂和药物虽然鉴别和含量都符合国家标准规定要求,但是却含有大量杂质,而运用薄层色谱法恰好能解决这些问题。由于吸附剂对不同化合物的吸附力大小不同,对极性大的化合物吸附力强,对极性小的物质吸附力相应地较弱。因此…     

不同方法从小鼠胚胎干细胞分化拟胚体

建立简便有效的胚胎干细胞体外分化研究中制备拟胚体的方法。本实验以3种方法形成拟胚体：滋养层过生长法、悬浮法和胶原酶处理法,并与传统的悬滴法进行了比较。在不同的分化培养体系中以最终分化出搏动的心肌细胞评价形成有分化发育能力的拟胚体的效率。以RT—PCR检测了拟胚体和心肌细胞形成中基因的表达。结果表明,4种方法分化出搏动心肌细胞的比例不同。滋养层过生长法形成拟胚体的比例接近悬滴法．而悬浮法和胶原酶处理形成拟胚体的比例显著低于滋养层过生长法和悬滴法。形态学研究显示,拟胚体发育经历了简单拟胚体阶段到囊状拟胚体或成熟拟胚体。在拟胚体成熟过程中特异性胚层分化发育分子标记HNF-4、Bmp-4与Otx-2表达上调,与心肌细胞分化相关的基因α—MHC、β-MHC、GATA-4及Nkx2．5同样表达上调。表明不同途径可获得不同比例的有发育能力的拟胚体,在体外分化研究中滋养层过生长法则是简便有效的获得正常发育拟胚体的方法。     

Characterization of strains of Staphylococci from infections in dogs and cats

Forty strains of Staphylococci from infections of dogs and cats were characterized with respect to their coagulase and thermonuclease activities, and 19 strains for their fermentation of mannitol anaerobically. Thermonuclease correlated well with tube coagulase activity but there was a poor correlation between these two characters, the ability to ferment mannitol anaerobically, and the presence of bound coagulase. Fifty percent of the organisms were resistant to penicillin due to the presence of β-lactamase (penicillinase). There was a strict correlation between detection of β-lactamase by the disc diffusion and the slide penicillinase tests. Antibiotics to which organisms were resistant also were bacitracin (52·5%), lincomycin (20%), streptomycin (17·5%), tetracyclines (12·5%) and chloramphenicol (7·5%).     

密码子优化的PRRSV GP5基因DNA疫苗在鼠体内免疫效力的评价

为了提高表达含有猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus,PRRSV）GP5基因DNA疫苗的死疫效应,本研究通过人工合成的方法将GP5基因的密码子改造为猪体嗜好的密码子,并将其作为免疫原基因,连同野生型GP5基因,分别插入表达载体pIRES1neo中,构建重组质粒pIR-optiGP5和pIR-GP5.将这两种质粒分别转染293T细胞,转染后48 h,采用间接免疫荧光和Western blot方法检测GP5基因的体外表达情况,结果两种检测方法显示重组质粒pIR-optiGP5的蛋白表达量明显多于pIR-GP5。为了评价DNA免疫质粒pIR-optiGP5的免疫效果,选取30只6～8周龄雌性BALB/c小鼠,将pIRES1neo、pIR-GP5、pIR-optiGP5分别以100μg/只剂量,进行肌肉多点注射,共免疫3次,每次间隔2周,同时,利用间接免疫荧光技术、流式细胞技术、淋巴细胞特异性增殖试验等方法检测其体液免疫和细胞免疫水平。结果显示：DNA免疫质粒pIR-optiGP5在二免后即可检测到荧光抗体,而pIR-GP5在三免后才能检测到;且发现用pIR-optiGP5质粒免疫小鼠,其CD4^＋、CD8^＋ T淋巴细胞百分数及特异性刺激指数均高于pIR-GP5免疫小鼠,推测经过密码子优化的GP5基因其蛋白在小鼠体内得到了较高水平的表达,并诱导了较强的免疫应答,这为进一步研究和设计有效的PRRSV DNA疫苗提供了新的思路。     

东方山羊豆脱水蛋白基因的克隆及初步分析

 采用ＳＭＡＲＴ ＲＡＣＥ 方法,从东方山羊豆盐诱导抑制性差减杂交ｃＤＮＡ 文库中分离到了一个脱水蛋白（GoDHN）基因。该基因ｃＤＮＡ 全长１１６９ｂｐ,开放阅读框８４３ｂｐ,编码２８１ 个氨基酸,编码的蛋白质分子量为２８．７１ｋＤａ。经实时荧光定量ＰＣＲ 分析,GoDHN 基因在东方山羊豆的茎和叶中表达量明显高于根中表达量,并且基因表达受ＡＢＡ、ＮａＣｌ和ＰＥＧ 的诱导,随着诱导时间的增加,表达量呈持续增长趋势。这些结果表明,DHN 基因在东方山羊豆的抗逆性中可能起到重要的调控作用。本研究成功构建了ｐＣＡＭＢＩＡ１３０２-ＤＨＮ 植物表达载体,为下一步转基因研究奠定了基础。     

维生素C的生理作用及其在饲料中的应用

维生素Ｃ又称抗坏血酸 (ＶｉｔａｍｉｎＣ) ,在自然界中以两种形式存在 ,即还原形式Ｌ -抗坏血酸和氧化形式Ｌ -脱氢抗坏血酸 ,大部分维生素Ｃ以还原形式存在 ,这种还原形式的维生素Ｃ在动物体内具有重要的生理作用。到目前 ,国内外许多科学家和学者就维生素Ｃ对畜、禽、鱼类的生理作用及其在饲料中的应用作了大量的研究。笔者对其作一综述。1　维生素Ｃ的生理作用维生素Ｃ作为强还原剂具有很多反应功能。目前了解最清楚的是它参与胶原蛋白的合成和“修复” ,维生素Ｃ是脯氨酸羟基化酶的辅酶 ,有助于脯氨酸羟基化形成羟脯氨酸。羟脯氨酸是胶…     

Investigation of domestic animals and pets as a reservoir for intimin- (eae) gene positive Escherichia coli types

Domestic animals belonging to seven different species (cattle, sheep, dogs, cats, pigs, chicken and goats) were investigated as natural reservoirs for attaching and effacing Escherichia coli (AEEC). For this, 2165 E. coli strains from faeces of 803 animals were examined for the presence of the intimin -(eae) gene as a characteristic of AEEC strains. Ten percent of the animals were found to excrete AEEC, most frequently found in sheep (19.2%) and pigs (17.6), followed by cattle (10.4%), dogs (7.2%), cats (6.5%) and poultry (2.3%). The 97 AEEC strains from animals were grouped into 44 serotypes. Only four E. coli serotypes (O2:H8, O26:[H11], O109:[H25] and O145:[H28] were found in more than one animal host species. AEEC O26:[H11] strains were most frequently isolated (13.4%) being present in cattle, poultry, pigs and sheep. A search for virulence markers associated with enterohemorrhagic E. coli (EHEC) revealed Shiga-toxin genes in three (3.1%) AEEC strains from sheep. Bundle forming pili genes as a trait of typical enteropathogenic E. coli (EPEC) were detected in four (4.1%) strains from dogs and cats. The remaining 90 AEEC strains were classified as atypical EPEC. Typing of intimin genes revealed intimin beta being present in 51.5% of the strains, followed by intimins theta (23.7%), epsilon (6.2%), kappa (5.2%), zeta (5.2%), alpha, eta and iota (each 1.0%). Our data indicate that domestic animals and pets constitute an important natural reservoir of AEEC strains, and some of these (O26:[H11], O103:H2, O128:H2, O145:[H28] and O177:[H11]) are known to occur as pathogens in humans.     

Comparative investigation on different turkey rhinotracheitis (TRT) virus isolates from different countries.

The present investigation was carried out to compare the antigenic relationship between TRT virus isolates from different countries. The obtained results showed that all virus isolates shared similar physiochemical properties. In virus neutralisation tests marked two way cross reactions between BUT 1 = 8544 (England), STG 761/88 and STG 854/88 (Germany) could be detected. On the other hand VCO 3 isolate (France) showed only partial reaction. Also the SDS-poly acrylamide gel electrophoresis (SDS-PAGE) profiles of three TRT viruses (one from England and two from Germany) were very similar, while the VCO 3 strain from France showed some variation.     

Characterization of a new gene (SLC10) with a spliced leader from Taenia solium

An unknown gene, SLC10, was cloned by spliced leader-based polymerase chain reaction from Taenia solium. The full length of SLC10 was found to be 635 bp, encoding an 18.223 kDa protein. ELISA results showed that none of 70 normal and 75 cysticercosis sera samples reacted with purified recombinant SLC10 protein. Using an immunohistochemical method, it was revealed that the native SLC10 protein distributed extensively in inner cyst walls but not in the scolex in Cysticercus cellulosae. Together with predicted results, it is suggested that the SLC10 protein is a non-secretory structural protein, which is not involved in induction of the host's immune reactions against infection at least at the larval stage.     

Characterization of in vivo-acquired resistance to macrolides of Mycoplasma gallisepticum strains isolated from poultry

ABSTRACT: The macrolide class of antibiotics, including tylosin and tilmicosin, is widely used in the veterinary field for prophylaxis and treatment of mycoplasmosis. In vitro susceptibility testing of 50 strains of M. gallisepticum isolated in Israel during the period 1997-2010 revealed that acquired resistance to tylosin as well as to tilmicosin was present in 50% of them. Moreover, 72% (13/18) of the strains isolated from clinical samples since 2006 showed acquired resistance to enrofloxacin, tylosin and tilmicosin. Molecular typing of the field isolates, performed by gene-target sequencing (GTS), detected 13 molecular types (I-XIII). Type II was the predominant type prior to 2006 whereas type X, first detected in 2008, is currently prevalent. All ten type X strains were resistant to both fluoroquinolones and macrolides, suggesting selective pressure leading to clonal dissemination of resistance. However, this was not a unique event since resistant strains with other GTS molecular types were also found. Concurrently, the molecular basis for macrolide resistance in M. gallisepticum was identified. Our results revealed a clear-cut correlation between single point mutations A2058G or A2059G in domain V of the gene encoding 23S rRNA (rrnA, MGA_01) and acquired macrolide resistance in M. gallisepticum. Indeed, all isolates with MIC ≥ 0.63 μg/mL to tylosin and with MIC ≥ 1.25 μg/mL to tilmicosin possess one of these mutations, suggesting an essential role in decreased susceptibility of M. gallisepticum to 16-membered macrolides.     

Polymorphisms in the canine glucocorticoid receptor alpha gene (NR3C1α)

Corticosteroids are one of the most extensively used class of therapeutic agents in dogs. In human patients, response to corticosteroid therapy has been correlated with the presence of certain polymorphisms of the glucocorticoid receptor gene (NR3C1). Depending on the polymorphism present, patients may show either increased sensitivity to glucocorticoid‐induced adverse effects or resistance to their therapeutic effects. Because response to corticosteroid therapy in dogs can also be variable and unpredictable, we hypothesized that genetic variability exists in the canine NR3C1 gene. The aim of this study was to sequence the coding regions of the canine NR3C1 gene in a representative sample of dogs. Samples from 97 dogs from four previously identified genetic groupings of domestic breeds (Asian/Ancient, Herding, Hunting, and Mastiff) were sequenced and evaluated. Four exons contained polymorphisms and four exons showed no variation from the reference sequence. A total of six single nucleotide polymorphisms (SNPs) were identified including four synonymous SNPs and two nonsynonymous SNPs (c.811A>T and c.2111T>C). No dogs were homozygous for either variant allele, while 23 dogs were heterozygous for the c.811A>T allele and 2 were heterozygous for c.2111T>C allele. The amino acid changes caused by c.811A>T (serine to cysteine) and c.2111T>C (isoleucine to threonine) were both predicted by in silico analysis to be ‘probably damaging’ to structure and function of the resulting protein. We conclude that NR3C1 polymorphisms occur in dogs and may cause individual variation in response to corticosteroid therapy.