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1.
The canine gastric mucosa is known to be a habitat for various Helicobacter species. So far, five Helicobacter species have been described from the canine gastric mucosa, but histological studies have demonstrated a greater variety. In order to gain more information on diversity of canine gastric mucosa colonising helicobacters, biopsy samples of four pet dogs were examined by DNA-based techniques. PCR with a primer pair binding specifically to the 16S rDNA of the species of the genus Helicobacter and generating a fragment of approximately 400 bp indicated the presence of Helicobacter strains in the stomachs of the four dogs. PCR products were cloned into Escherichia coli DH10B and PCR-re-amplified 16S rDNA fragments were subjected to amplified ribosomal DNA restriction analysis (ARDRA) employing restriction enzyme HhaI. Restriction profiles indicated the presence of at least two different Helicobacter species in two dogs. Partial sequences of 16S rDNA of six clones were compared with sequences available in the EMBL data bank. Two sequences obtained from different dogs were identical with the corresponding sequences of Helicobacter pylori strains. Three sequences showed highest but moderate similarity values to H. pylori (96.6-98.0%) and one sequence to Helicobacter salomonis (97.3%). In contrast to previous reports our data implicate that the gastric mucosa of dogs may be colonised by strains of H. pylori or a very closely related species but they also confirm indications for the presence of so far uncultivated species of Helicobacter. 相似文献
2.
副鸡嗜血杆菌16 S rDNA PCR检测方法的建立 总被引：1，自引：0，他引：1
根据副鸡嗜血杆菌的16 S rDNA基因序列设计一对特异性引物XZIC1和XZIC2,对6株副鸡嗜血杆菌进行PCR扩增。结果显示,该对引物对6株副鸡嗜血杆菌均扩增出与预期大小相一致的282bp片段,而对鸡毒支原体、禽巴氏杆菌、鸡传染性支气管炎病毒、鸡新城疫病毒、大肠埃希菌、鸡白痢沙门菌、禽流感病毒(H9)、鸡喉气管炎病毒及葡萄球菌等9种病原体的扩增结果均为阴性。该PCR敏感性结果表明,本方法可以检测到10pg的副鸡嗜血杆菌DNA模板。采用引物XZIC1和XZIC2,对分别用副鸡嗜血杆菌ctcc253、ctcc255、ctcc257、ctcc269株感染SPF鸡的临床病料DNA进行PCR扩增,均可扩增出单一的282bp的片段。 相似文献
3.
Sasaki E Osawa R Nishitani Y Whiley RA 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2004,66(11):1467-1470
A total of 23 Streptococcus gallolyticus strains, consisting of 12 strains from feces of healthy animals and 11 from clinical cases of human or cow mastitis milk, were examined genealogically. Four strains of S. bovis "biotype II/1" and 3 strains of S. equinus, the closely related organisms to S. gallolyticus, were also analyzed for outgroup comparison. Neither the amplified ribosomal DNA restriction analysis (ARDRA) nor the randomly amplified polymorphic DNA (RAPD) analysis that had been designed to recognize S. gallolyticus strains virulent in pigeons could differentiate clinical strains from the others of S. gallolyticus. No correspondence between the DNA profile in either analysis and the host animal species was detected. 相似文献
4.
Rinaldi L Perugini AG Capuano F Fenizia D Musella V Veneziano V Cringoli G 《Veterinary parasitology》2005,131(3-4):247-253
Isolates of the rumen fluke Calicophoron daubneyi (Digenea: Paramphistomidae) from various hosts and three locations in southern Italy were characterized genetically. The second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) plus flanking 5.8S and 28S sequence (ITS-2+) was amplified from individual rumen flukes by PCR. PCR-linked restriction fragment length polymorphism (PCR-RFLP) analysis was performed using four different restriction endonucleases, and PCR products were sequenced. The PCR analyses from all the C. daubneyi specimens produced identical fragments, and the PCR-RFLP analyses did not show, with respect to any of the four restriction endonucleases, any differences between the C. daubneyi specimens. The sequence analyses of the ITS-2+ from each of the C. daubneyi specimens showed them all to be 428 bp, and composed of the entire ITS-2 sequence (282 bp) plus the two partial flanking conserved sequences, 5.8S (99 bp) and 28S (47 bp). No intra-specific variation was observed in the nucleotide composition of the ITS-2+ (homology=100%). There was, however, an observable inter-specific variation between the ITS-2+ of C. daubneyi and the ITS-2+ of both Calicophoron calicophorum (homology=97.2 %) and Calicophoronmicrobothrioides (homology=97.4 %), both previously deposited in the GenBank. The finding of the present study shows that, as has already demonstrated for other parasitic helminths, ITS-2 can serve as an effective genetic marker for the molecular identification of paramphistomes, and as a useful tool for developing molecular epidemiological techniques for the study of C. daubneyi transmission patterns and prevalence in definitive and intermediate hosts. 相似文献
5.
Sasaki Y Yamamoto K Kojima A Norimatsu M Tamura Y 《Research in veterinary science》2000,69(3):289-294
In cattle, sheep, and other ruminants, clostridial myonecrosis (gas gangrene) is mostly caused by Clostridium chauvoei, C septicum, C novyi and C sordellii. A polymerase chain reaction (PCR) system using common primers designed from multiple alignment of the 16S rRNA and 23S rRNA genes of Clostridium species was developed to identify pathogenic clostridia. The PCR was performed with total DNA from 26 strains which included seven different Clostridia species. These bacteria were differentiated at species level by the different PCR product patterns. To characterise the 16S-23S rDNA spacer regions of these clostridia further, most PCR products of these bacteria were sequenced. The smallest PCR products of each bacterium represented the fundamental 16S-23S rDNA spacer region; larger PCR products of each bacterium were caused by insertion sequences, i.e. tRNA gene sequences. The authors' observations indicate that the PCR patterns of the 16S-23S rDNA spacer regions have the potential to be used as an identification marker of pathogenic clostridia in gas gangrene. 相似文献
6.
Eight strains of Taylorella equigenitalis were identified by a polymerase chain reaction using a primer pair specific to the 16S rDNA of T equigenitalis. These eight strains were chosen because they had previously been shown to represent eight distinct genotypes by pulsed-field gel electrophoresis analysis after separate digestion of the genomic DNA with ApaI or NotI. The eight strains could be classified into six or seven types by random amplified polymorphic DNA analysis using different kinds of primers. Amplified rDNA restriction analysis after separate digestion with five restriction enzymes, including AluI and MboI, of the 1,500 bp fragments of rDNA amplified by polymerase chain reaction did not discriminate the genomic variations among the eight strains of T equigenitalis. Thus, pulsed-field gel electrophoresis was shown to discriminate these eight organisms better than random amplified polymorphic DNA analysis, while amplified rDNA restriction analysis was found to be unsuitable for subtyping T equigenitalis. 相似文献
7.
Kokotovic B Friis NF Ahrens P 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2002,49(5):245-252
Mycoplasma hyospnoviae strains from Denmark, Germany, Japan, Sweden, the Netherlands and the UK were examined for variations in the genomic DNA and within the 16S ribosomal RNA (rRNA) gene. Variations in the chromosomal DNA among 57 isolates recovered from the respiratory tract and joints of pigs, were investigated by analysis of amplified fragment length polymorphisms of the Bg/II and MfeI restriction sites and by pulsed-field gel electrophoresis of a BssHII digest of chromosomal DNA. Both methods allowed unambiguous differentiation of the analysed strains and showed similar discriminatory potential for the differentiation of M. hyosynoviae isolates. Concordant results obtained with the two whole-genome fingerprinting techniques evidence the considerable intraspecies genetic heterogeneity of M. hyosynoviae. Sixteen field strains of M. hyosynoviae and the type strain S16(T) were further examined for variation within the 16S rRNA gene. Ten field strains possessed the 16S rDNA sequences identical to the type strain, while the remaining six strains had sequences that differed by one to two nucleotides from that obtained from the type strain. 相似文献
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Wakeley PR Errington J Hannon S Roest HI Carson T Hunt B Sawyer J Heath P 《Veterinary microbiology》2006,118(3-4):247-254
A discriminatory real time PCR for the detection of Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM), and the related species T. asinigenitalis was developed for the direct examination of genital swabs. The 112bp amplicons produced from the two species were discriminated from each other using TaqMan probes labelled with different fluorophores. The TaqMan PCR was shown to be specific for the 16S ribosomal DNA of the two species of taylorella and did not cross-hybridise with the 16S ribosomal DNA of other bacteria tested. Direct amplification from genital swabs was shown to be equally sensitive to that of culture methods. Prevalence in a sample set from The Netherlands was shown to be equivalent to that demonstrated by culture. A companion real time PCR that amplified a fragment of the 16S rDNA gene of equine commensal bacteria was developed to ensure bacterial DNA was extracted from swab material supplied for testing. The use of a rapid and reliable real time PCR for the organism causing CEM should aid the control of this disease. 相似文献
10.
To differentiate the morphologically similar pinworms of the common laboratory rodents, such as Syphacia obvelata and Syphacia muris, we amplified and sequenced the region spanning the internal transcribed spacer 1 (ITS-1), 5.8S gene, and ITS-2 of the ribosomal DNA followed by designing of species-specific primers for future use in the identification of the worms. It was observed that S. obvelata, S. muris and Aspiculuris tetraptera can be differentiated from each other based on their rDNA sequences. This is the first report of the ITS-1, 5.8S, and ITS-2 of the rDNA of the three aforementioned rodent pinworm species. The use of restriction endonucleases, AluI or RsaI, further allowed the delineation of the three species. Moreover, we also constructed species-specific primers that were designed for unique regions of the ITS-2 of the three species. This approach allowed their specific identification with no amplicons being amplified from heterogenous DNA samples, and sequencing confirmed the identity of the sequences amplified. Thus, the use of these specific primers along with PCR-RFLP can serve as useful tools for the identification of pinworms in rats, mice, and wild rodents. 相似文献
11.
根据NCBI中已报道的嗜麦芽窄食单胞菌16S rDNA基因序列设计引物,对两株来源于猪“高热病”病料中的疑似嗜麦芽窄食单胞菌PSM-5、PSM-6进行PCR扩增。将扩增产物克隆至pMD18-T载体上,并转化到DH5α中。抽取质粒模板,进行PCR和双酶切鉴定并测序,从分子水平上予以鉴定。两株菌的16S rDNA基因扩增片段的核苷酸序列（GenBank登录号为GQ267816和GQ267817）与已经报道的嗜麦芽窄食单胞菌分离株的16S rDNA序列的相似性均在96.78%以上,最高达99.93%。本试验对通过16S rDNA鉴定嗜麦芽窄食单胞菌提供一定参考,同时对猪“高热病”的诊断治疗奠定细菌学方面的基础,对其发病机制的探讨提供依据。 相似文献
12.
Strains of the bacterium Ornithobacterium rhinotracheale (ORT), a causal agent of respiratory diseases in birds, were microbiologically isolated, identified, and molecularly characterized. Blood-enriched culture media and biochemistry tests were used for microbiologic identification. Polymerase chain reaction (PCR) and repetitive extragenic palindromic PCR (rep-PCR) techniques were used for molecular identification and characterization, respectively, of the microorganism. ORT strains were isolated in enriched media from the trachea and air sacs of broilers, breeders, and layers from several geographic zones of Peru. Of the original 75 strains isolated from 75 clinical samples from which ORT was recovered during 1998-2000, 25 were selected for further study based on ORT as the primary pathogenic isolate (no other pathogens were detected). Selected isolates were molecularly identified and characterized by PCR using specific primers designed from the conserved zones of the 16S ribosomal genes. Primers used for the identification of ORT produced a specific fragment of 784 base pair (bp), which did not appear in Haemophilus paragallinarum or Pasteurella multocida, microorganisms with similar morphologic and biochemical characteristics that produce dinical signs identical to those of ORT. All 25 strains of ORT tested with rep-PCR had a genetic profile similar to that of ORT American Type Culture Collection 51463, indicating the presence of only one genotype in the ORT strains studied. 相似文献
13.
Spaková T Elecko J Vasil M Legáth J Pristas P Javorský P 《Polish journal of veterinary sciences》2012,15(2):329-335
The Aerococcus viridans isolates from bovine mastitis in Slovakia were isolated and characterized by classical microbiological and biochemical, and molecular techniques including IGS-PCR and rep-PCR, ARDRA and 16S rDNA gene sequencing. The substantial variability of antibiotic resistance patterns was observed. The majority of strains were resistant to beta-lactam antibiotics, the resistance to tetracycline was observed in 3 tested strains, resistance to lincomycin was found in 4 strains and practically all tested strains were sensitive to neomycin and ciprofloxacin. While variable at a phenotypic level, no significant genetic variability among A. viridans isolates was detected by molecular DNA based methods. The data obtained suggest that a few A. viridans strains spread among cow's population in Slovak farms. 相似文献
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四川白三叶根瘤菌遗传多样性及系统发育研究 总被引：1，自引：0，他引：1
为阐明四川部分地区野生白三叶根瘤菌的遗传多样性及系统发育地位,对分离自四川雅安、康定、泸定、西昌、成都和乐山6个地区白三叶根瘤的69株菌进行系统研究。采用16S rDNA限制性片段长度多态性分析(restriction fragment length polymorphism, RFLP)和16S rDNA基因、持家基因(recA、atpD、glnII)、结瘤基因(nodC)、固氮基因(nifH)系统发育分析的方法进行了研究。结果表明,16S rDNA PCR-RFLP中所有供试菌株产生了4种酶切图谱类型,表现出较为丰富的遗传多样性。持家基因与16S rDNA基因系统发育分析结果基本一致,9株代表菌株主要分布在α-变形菌纲(Alpha-Proteobacteria)的根瘤菌属(Rhizobium),并与豌豆根瘤菌三叶草生物型(R. leguminosarum bv. trifolii) ATCC 14480T的亲缘关系较近。PCR可扩增出nodC和nifH基因片段,但从属于土壤杆菌属(Agrobacterium)的菌株LS1105中则扩增不出这两个基因。所有供试菌株被鉴定到了种的水平,证实了68株为白三叶根瘤菌,并通过不同采样地点菌株之间的比较,发现白三叶与根瘤菌的共生关系因地理分布不同而具有多样性,对于丰富白三叶根瘤菌资源及其开发利用具有重要意义。 相似文献
17.
为探究影响水稻（Oryza sativa L.）秸秆青贮质量的腐败菌种类并筛选适合其青贮的优质乳酸菌,研究了青贮前后水稻秸秆中可培养微生物的变化并分离鉴定2株乳酸菌。使用稀释平板涂布法将青贮前后可培养微生物计数并分离纯化,限制性酶切分析ARDRA技术分析分离纯化后的微生物多样性,结合ARDRA和RAPD-PCR技术对青贮中优势乳酸菌进行分离鉴定。结果表明：青贮前后好氧或兼性厌氧细菌菌落总数由7.6×107 cfu·g-1下降到2.43×106 cfu·g-1,真菌菌落数变化较为明显,从4.43×105 cfu·g-1下降到86 cfu·g-1,乳酸菌菌落数从4.16×105 cfu·g-1上升到6.61×106 cfu·g-1。ARDRA聚类分析及16SrDNA测序显示青贮前存在腐败细菌芽孢杆菌（Bacillus）和致病菌阴沟肠杆菌（Enterobacter）,青贮后腐败细菌主要是芽孢杆菌,腐败真菌主要为青霉菌属（Penicillium）。筛选出的2株乳酸菌分别属于乳杆菌属（Lactobacillus casei）和片球菌属（Pediococcus ethanolidurans）,需进一步验证是否可作为青贮饲料添加剂。水稻秸秆青贮后依然有腐败菌的存在,需进一步提高其青贮发酵工艺,防治有氧恶化。 相似文献
18.
Akineden O Alber J Lämmler C Weiss R Siebert U Foster G Tougaard S Brasseur SM Reijnders PJ 《Veterinary microbiology》2007,121(1-2):158-162
The present study was designed to identify 15 beta-hemolytic streptococci isolated during a period between 1988 and 2005 from nine harbour seals and six grey seals from various origins of the North Sea. All isolates were identified as Streptococcus equi subsp. zooepidemicus. The bacteria were additionally investigated for relatedness by restriction fragment length polymorphism analysis of PCR amplified 16S-23S rDNA intergenic spacer region and gene szp and by macrorestriction analysis of chromosomal DNA of the strains by pulsed field gel electrophoresis. The molecular analysis yielded identical or closely related patterns within the strains of the present study and with the S. equi subsp. zooepidemicus strains isolated from harbour seals of German North Sea which were investigated previously [Akineden, O., Hassan, A.A., Alber, J., El-Sayed, A., Estoepangestie, A.T.S., L?mmler, C., Weiss, R., Siebert, U., 2005. Phenotypic and genotypic properties of S. equi subsp. zooepidemicus isolated from harbor seals (Phoca vitulina) from the German North Sea during the phocine distemper outbreak in 2002. Vet. Microbiol. 110, 147-152]. This indicates that this single or closely related bacterial clone existed during both phocine distemper virus epidemics in 1988 and 2002 and that a direct transmission of the strains has occurred between two seal species and between seal populations of far distant regions possibly with grey seals as a vector. 相似文献
19.
利用PCR-DGGE技术分析桑天牛成虫肠道菌群结构及优势菌群,获取桑天牛肠道微生物的多样性信息。从桑天牛成虫肠道中提取细菌基因组DNA,以细菌16S rDNA基因通用引物27F/1495R和27F/519r+GC进行V3可变区PCR扩增,将长约500 bp的扩增产物经变性梯度凝胶电泳(DGGE)分离后,进行优势条带分析、DNA回收、克隆、测序等,初步得到分别属于肠杆菌属(Enterobacter)、不动杆菌属(Acinetobacter)、埃希氏菌属(Escherichia)、志贺菌属(Citrobacter)、克雷伯氏菌属(Klebsiella)、柠檬酸菌属(Shigella)、泛菌属(Pantoea)和沙雷氏菌属(Serratia)的8个细菌菌株,其中优势细菌为克雷伯氏菌属的细菌菌株,其次是沙雷氏菌属的细菌菌株。将获取的细菌16S rDNA序列在GenBank数据库中进行BLAST比对分析,相似度在97%以上的有6个菌株,其中有5个菌株与传统方法分离菌株的鉴定结果一致,表明基于16S rDNA的PCR-DGGE技术可用于桑天牛肠道菌群多样性研究。 相似文献
20.
Conventional serological methods for the identification of canine mycoplasma isolates depend on the availability of a panel of species-specific diagnostic antisera and are not always reliable in terms of specificity. To enable simultaneous identification of field isolates, PCR-RFLP analysis of the 16S-23S rRNA intergenic spacer region was used to characterize the type strains of the 12 currently described canine mycoplasmas of the Genus Mycoplasma which represent the "classic" non-hemotropic species. The use of 16S-23S rDNA PCR in the first step of this analysis revealed specific size differences of amplicons which allowed to classify these 12 canine Mycoplasma species into three groups. Depending on the length of the amplicon, subsequent RFLP analysis of PCR products using two restriction endonucleases in a single digest (ApoI/DdeI or TaqI/VspI) generated unique banding patterns. For further evaluation of the 16S-23S rDNA PCR-RFLP assay system as identification and differentiation tool, a total of 262 field isolates collected from the canine genital tract were tested. PCR-RFLP results for 251 field isolates correlated with traditional serological test results. The remaining 11 isolates had an RFLP pattern distinct from the type strains included in this study and were identified by 16S rDNA sequencing as closely related to M. sp. HRC689. The PCR-RFLP assay established in this study enabled a rapid, accurate and easily performed identification and differentiation of all 12 currently described non-hemotropic canine Mycoplasma species. 相似文献