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1.
Mycoplasma is the common name for the smallest free-living microorganisms, the Mollicutes. Mycoplasma hyopneumoniae is of great importance in veterinary medicine, causing enzootic pneumonia in pigs. M hyorhinis can cause polyserositis and may cause pneumonia in piglets. Oligonucleotides complementary to variable regions of 16S rRNA from these mycoplasmas were designed and used as probes for detection and identification of these mycoplasmas. The probe complementary to 16S rRNA of M hyorhinis gave a very weak cross-hybridisation with M hyosynoviae in filter hybridisation experiments, but not with any of the other porcine mycoplasmas tested. Three oligonucleotide probes complementary to M hyopneumoniae 16S rRNA were tested. One of the probes (Mhp6/30) was found to be specific to M hyopneumoniae, but the other two gave cross-hybridisation with M flocculare. Using the Mhp6/30 probe in direct filter hybridisation experiments, it proved possible to detect M hyopneumoniae in lung biopsies from experimentally infected pigs.  相似文献   

2.
败血支原体16S rRNA基因的克隆与核酸序列分析   总被引:5,自引:0,他引:5  
从败血支原体D9604株培养物中直接快速提取染色体DNA;构建了MG基因文库,以PCR扩增16S rRNA基因全长片估并对其进行了核苷酸序列分析。结果表明,D9604株与A5969株只有5个核苷酸的差异,同源性为99.67%,16Sr RNA基因具有高度的保守性。  相似文献   

3.
鸡毒支原体(Mycoplasma gallisepticum,MG)是禽类主要病原菌之一,其感染引起鸡慢性呼吸道病(CRD)和火鸡传染性窦炎。目前预防鸡毒支原体感染仍主要依靠抗菌药物,但长期广泛应用抗菌药物造成鸡毒支原体耐药性不断发展。有调查表明,鸡毒支原体临床株对四环素类、大环内酯类及氟喹诺酮类等抗菌药物已产生了耐药性,造成药物疗效下降甚至失效。由于支原体分离培养困难,国内外对动物源支原体耐药性研究十分有限,至今未见有鸡毒支原体大环内酯类抗生素耐药机制的研究报道。本文以鸡毒支原体S6株为模式菌,研究红霉素和替米考星在体外诱导鸡毒支原体的耐药情况,并分析耐药菌株23S rRNA基因V域的耐药突变特征。  相似文献   

4.
The present paper compares the fluorescence-serological detection of M. hyopneumoniae and M. hyorhinis in frozen sections with cultural procedures. In 161 lungs of swine the agent of enzootic pneumonia, M. hyopneumoniae, was found almost exclusively in frozen sections (22 times). Only one strain was cultivable. M. hyorhinis was detected 59 times by culture but only 18 times in the sections. For the detection of M. hyopneumoniae therefore the fluorescence-serological investigation of lung sections is the preferable method.  相似文献   

5.
6.
作者拟探讨禽源支原体对替米考星耐药的分子机制.体外诱导得到鸡毒支原体(R株、PG31株和S6株)、鸡滑液支原体、衣阿华支原体的替米考星耐药株;PCR扩增原始敏感株和诱导耐药株的23S rRNA基因V域,测序分析耐药相关碱基突变情况.结果鸡毒支原体R株、PG31株、S6株、鸡滑液支原体、衣阿华支原体分别通过9代、8代、6代、14代、9代诱导获得替米考星耐药(≥128 μg·mL-1)株;3种禽源支原体替米考星诱导耐药株均对大环内酯类药物表现交叉耐药,23S rRNA基因发生A2503T突变的诱导耐药株对截短侧耳素类、氯霉素类药物的敏感性明显降低.诱导获得的替米考星耐药鸡毒支原体R株发生了A2058G和A2503T突变,PG31株发生了A2058G和A2059G突变,S6株发生了A2058G和A2503T突变;而诱导获得的替米考星耐药鸡滑液支原体发生了G2162A突变,衣阿华支原体发生了A2059C突变.本研究表明鸡毒支原体和衣阿华支原体在体外较易经替米考星诱导产生耐药性,而鸡滑液支原体相对较难.菌株23S rRNA基因V域2 058、2 059、2 503位点的碱基突变与替米考星耐药表型有密切关系.  相似文献   

7.
8.
This study attempted to determine whether one multiplex polymerase chain reaction (PCR) is an effective adjunct method for diagnosing Mycoplasma hyopneumoniae and Mycoplasma hyorhinis infection, and whether M. hyorhinis should be considered as an enzootic pneumonia or porcine respiratory disease complex pathogen in Taiwan. To our knowledge, this study is the first to isolate and identify M. hyorhinis as a porcine pathogen in Taiwan. A novel isolation method and a multiplex PCR test were applied to detect and isolate M. hyorhinis. The correlation of M. hyorhinis with swine pneumonia was also examined using a challenge test. Based on weight, 18 pigs were assigned to three groups and housed throughout the study in a specific-pathogen-free (SPF) facility and provided with aseptic feed and water. Groups 1 (n=6) and 2 (n=6) were challenged with 5mL M. hyorhinis culture via tracheal intubation on day 1. The M. hyorhinis strains ATIT-1, -3, and-7 were used to infect group 1 and the strain ATCC 27717 was used for group 2. Culture medium was replaced by phosphate-buffered saline in group 3 (n=6). All pigs were slaughtered on day 28, and their lungs were removed for examination of lesions. Of the six pigs in group 1 challenged with wild-type strains, two had typical mycoplasma pneumonia lesions. No gross lung lesions were observed in groups 2 and 3. Although further examination is necessary to confirm that wild-type strains can cause pneumonia, it appears that M. hyopneumoniae is no longer the only mycoplasma pathogen implicated in the diagnosis of swine enzootic pneumonia (SEP).  相似文献   

9.
The sequential development of Mycroplasma hyorhinis and Mycoplasma hyosynoviae was observed in cultures of a swine synovial fluid cell strain. An early transitory filamentous phase was observed with M. hyorhinis infection followed by the development of cell-associated, relatively large, round structures and some ring forms. Infection with M. hyorhinis was characterized by a generalized distribution of the organism and a severe cytopathic effect.

Infection with M. hyosynoviae was represented by the development of circumscribed foci of small pleomorphic structures and a milder effect on the cells. At a high multiplicity of infection, this organism became associated with the cytoplasmic membranes of the cells.

  相似文献   

10.
The intergenic spacer region between the 16S and 23S rRNA genes of mycoplasmas has been used for a genetic marker for identification of the species. Here we show the intergenic spacer regions of two hemotropic mycoplasmas, Mycoplasma haemofelis and 'Candidatus Mycoplasma haemobos (synonym: 'C. M. haemobovis')' are also useful for classification of this particular group of mycoplasms. The spacer region of M. haemofelis and `C. M. haemobos' consisted of 209 and 210 base pairs, respectively, and both lacked the spacer tRNA genes. Phylogenetic analysis suggested a monophyletic relationship among hemoplasmas and M. fastidiosum. A hypothetical secondary structure predicted in the spacer regions tentatively assigned the boxA and boxB motifs peculiar to the members of the genus Mycoplasma. M. haemofelis and 'C. M. haemobos' possessed a stem-loop structure in common, despite the presence of a palindromic nucleotide substitution in the stem region.  相似文献   

11.
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   

12.
In the helix 25 region, 32 French Taylorella asinigenitalis isolates carried at least one 23S rRNA gene not containing intervening sequences (IVSs). No IVSs in the region were identified in three isolates and the other remaining 29 isolates carried one or more IVSs (UCD-1(T)IVS1A, UCD-1(T)IVS1B and UK-1IVS1B) described already and two new kinds of IVS (TaIVS1C and TaIVS1D). In the helix 45 region, no T. asinigenitalis isolates not carrying any IVSs were identified. UK-1IVS2B was identified in the region from 26 isolates. Five new kinds of IVSs (TaIVS2D, E, F, G and H) occurred in the region in the 13 isolates. Distinctly different tandem repeat units (RS48 and RS32 and RS-A, -B and -C) were evident in both regions, respectively, from the French (n=32) and American (n=3) T. asinigenitalis isolates. Thus, several different kinds of tandem repeat units and their combinations in IVSs in both regions within the gene were shown in 32 French isolates.  相似文献   

13.
The aim of this study was to investigate whether direct PCR (DP) gave similar results to culture prior to PCR (CPP) for detecting mycoplasmas in different types of pig tissues. A total of 724 samples obtained from lungs, tonsils, or synovial fluids from 270 slaughtered pigs were used. The history of clinical signs, lung score, and the presence of joint lesions were recorded during sample collection. The rates of detection of Mycoplasma hyopneumoniae, Mycoplasma hyosynoviae, and Mycoplasma hyorhinis using both procedures were evaluated. The overall prevalences of M. hyopneumoniae, M. hyosynoviae, and M. hyorhinis were 40.3%, 12.3%, and 64.6%, respectively, and the detection rate depended on the sample type and the procedure used. With lung tissue, DP gave a higher detection rate for M. hyopneumoniae (77.4%) than CPP (38.5%). M. hyorhinis was detected by CPP at 15.6% and 18.1% and by DP at 31.5% and 5.2%, respectively. The positive rate derived from tonsil from CPP was closed to that of DP. Using synovial fluid could not yield any positive M. hyorhinis from CPP whereas 37.2% was positive from DP. In contrast, using sample tissue from lung and tonsil by CPP could show much higher positive number than that of DP. There was a significant relationship between joint lesion and M. hyorhinis detection by DP (P < 0.05) but not for M. hyosynoviae and M. hyorhinis detected by CPP. We speculated that lung was a proper sample for M. hyopneumoniae and M. hyorhinis detection by DP and CPP, respectively. Tonsil was likely the community of persistent M. hyosynoviae and M. hyorhinis with highly detection by CPP. Synovial fluid was apparently unsuitable for mycoplasmal culture. The accuracy of mycoplasmal detection may depend upon the type of sample relevant to the detection procedure used.  相似文献   

14.
15.
In routine diagnostic it is difficult to detect a Haemophilus parasuis infection causing a polyserositis. In the presented study, a detection method by means of PCR, from collective swabs of the serosal surfaces from animals, with and without fibrinous serositis, was investigated. A significant association was detected between post mortal findings of fibrinous serositis and molecular biological detection of Haemophilus parasuis and Mycoplasma hyorhinis. However, no significant association existed between cultural detection of Haemophilus parasuis in bronchoalveolar lavage fluid and pathological-anatomical ascertainable fibrinous serositis. A significant association between Haemophilus parasuis and Mycoplasma hyorhinis could be determined in the animals that were included in this study.  相似文献   

16.
The objective of this study was to examine the degree of phenotypic and genotypic diversity between 43 French Taylorella asinigenitalis strains isolated from 22 jacks, two stallions and one mare between 1995 and 2008 by culturing genital swabs obtained during routine diagnosis for contagious equine metritis. This retrospective analysis revealed the existence of T. asinigenitalis species since 1995 and the natural colonization of a mare's genital tract in 2001. Despite the presence of 27 different patterns revealed by the combination of API ZYM, antibiogram and 16S rDNA profiles, we show that T. asinigenitalis is a highly homogeneous species. API ZYM diversity only concerns acid phosphatase and naphthol-AS-BI-phosphohydrolase activity. The majority of strains are susceptible to a wide range of antimicrobial agents but most are streptomycin-resistant (95.5%), ampicillin-resistant (88.4%), and four strains are atypical due to a high degree of resistance to at least eight antimicrobial agents. 16S rDNA sequence analysis showed only two clusters and revealed similarity of 99.3-100% between T. asinigenitalis strains. The geographic origin of the 43 isolates correlates to the two 16S rDNA clusters.  相似文献   

17.
为探讨兽医临床治疗牛支原体肺炎常用药物的有效性,采用形态观察、分子生物学等方法对病原菌进行鉴定,共获得4株牛支原体,继而对分离菌株进行耐药性检测。结果表明,4株牛支原体均对大环内酯类抗生素耐药,对氟喹诺酮类及四环素类抗生素相对敏感。实验结果为兽医临床对牛支原体肺炎的治疗具有一定的指导意义。  相似文献   

18.
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   

19.
Questions about pathogenesis and therapy for Helicobacter infections in dogs could be answered with a simple, noninvasive, sensitive, and specific diagnostic test. We hypothesized that a fecal polymerase chain reaction (PCR) assay would detect Helicobacter and could be useful for assessing therapeutic responses. Paired gastric biopsies and fecal samples were obtained from 39 random source dogs (group 1). Gastric biopsies from each of these dogs had histologic evidence of gastric spiral bacteria, and paired gastric tissue and fecal samples produced a 375-base pair (bp) product when amplified by PCR with Helicobacter-specific primers. Specificity of the PCR product was confirmed by detection of expected 60-, 119-, and 196-bp products following Hinfl digestion. Direct sequencing of amplicons from paired PCR products from gastric biopsy and fecal samples from 8 group I dogs showed that gastric products had the highest homologies with known gastric Helicobacter species, whereas fecal products had the highest homologies with intestinal species. Healthy mixed-breed dogs (group II; n = 8) with histologically confirmed spiral bacteria infection were treated with a 21-day course of metronidazole, amoxicillin, and famotidine. Fecal samples were collected from group II dogs twice before and within 3 days of completion of treatment. The PCR results correctly identified 15/16 pretreatment samples as positive: 1 pretreatment sample was negative. PCR results identified 8/8 posttreatment samples as Helicobacter negative. Fecal PCR is a useful test for detecting Helicobacter infection in dogs. This assay may be useful as a screening test for infection and could be used to address questions relevant to pathogenesis and therapy.  相似文献   

20.
Intraspecific variation in the 16S rRNA genes of 17 Mycoplasma agalactiae and eight Mycoplasma bovis isolates was investigated to determine the degree of sequence variation in these two species and to determine whether the polymorphisms in the 16S rRNA genes could be used for the construction of an evolutionary tree and as epidemiological markers. A high degree of variation was found within isolates (between operons) and between isolates of both species. In contrast to M. capripneumoniae no distinct evolutionary pattern could be seen, probably because there are functional systems for gene conversion in M. agalactiae and M. bovis. However, the non-European isolates of M. agalactiae shared three characteristic nucleotides and European isolates from the same or neighbouring countries were very similar. Differences within isolates included both polymorphic positions and sequence length differences between operons. The amount of variation within isolates of the respective species ranged from zero to seven polymorphisms for M. agalactiae and from zero to four polymorphisms for M. bovis. The high degree of variation suggests the potential for misdiagnosis of species in diagnostic PCR assays based on the 16S rRNA gene sequences. All isolates of both species had a thymidine in position 912 (E. coli numbering) that causes streptomycin resistance in several bacterial species and which is characteristic for the members of the hominis group. As expected, when five M. agalactiae and three M. bovis isolates were tested for streptomycin susceptibility, they all demonstrated streptomycin resistance. M. agalactiae and M. bovis were found to have high intraspecific variation in their 16S rRNA gene and the polymorphisms patterns indicate that gene conversion takes place.  相似文献   

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