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1.
We have constructed a physical map of the Mycoplasma agalactiae strain PG2 chromosome analyzing it by pulsed field gel electrophoresis in a contour-clamped homogeneous electric-field system. We mapped 33 cleavage sites generated with SmaI, XhoI, SalI, EclXI and BsiWI restriction endonucleases using double digestions, one- and two-dimensional pulsed electrophoresis, cross-hybridization and linking clones. We have also mapped the loci of some genes by Southern hybridization. 相似文献
2.
S. Tola G. Idini A. M. Rocchigiani D. Manunta P. P. Angioi S. Rocca M. Cocco G. Leori 《Zoonoses and public health》1999,46(3):199-206
Mycoplasma agalactiae and Mycoplasma bovis are closely related species in phylogenetic terms. Pulsed Field Gel Electrophoresis (PFGE) of SmaI, EclXI, Bsi WI, MluI, BssHII, SalI, XhoI, NruI and ApaI digested DNAs were used to analyse and to compare restriction fragment length polymorphism between M. agalactiae and M. bovis and to estimate their genome sizes. SmaI, EclXI and Bsi WI enzymes cleaved DNAs of both microrganisms. MluI, BssHII, SalI, XhoI and NruI digested only M. agalactiae DNA whereas ApaI cut only M. bovis DNA. The total DNA length was established to be 945 ± 8.4 Kb for M. agalactiae and 961 ± 18.9 Kb for M. bovis. 相似文献
3.
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
4.
Genetic and antigenic characterization of the surface lipoprotein P48 of Mycoplasma bovis 总被引：2，自引：0，他引：2
Robino P Alberti A Pittau M Chessa B Miciletta M Nebbia P Le Grand D Rosati S 《Veterinary microbiology》2005,109(3-4):201-209
The presence of a membrane lipoprotein homologous to the P48 of Mycoplasma agalactiae was investigated in different Mycoplasma bovis isolates selected by geographical locations and biological properties. Its potential as a diagnostic tool was also discussed. The presence of a specific signal observed in all M. bovis field isolates probed with a rabbit antiserum raised against the M. agalactiae recombinant P48 demonstrated that this protein is structurally and antigenically conserved within the M. bovis cluster. No signal was detected when testing six different mycoplasma species found in cattle. The p48 gene was identified by PCR approach and partially sequenced. Full length gene sequence was obtained by direct bacterial chromosome sequencing. Five UGAs were selectively mutated into UGG and the full length mutated gene, lacking the signal peptide, was cloned and expressed in Escherichia coli. The purified recombinant antigen (r-P48) was evaluated as a potential marker of infection using a panel of 86 well-characterized sera from experimentally and naturally infected cattle. Specific IgM antibodies were detected within 6-9 days after experimental infection followed by an IgG response lasting from the third/fourth week after contact. Although antibody titers were well below those observed in sheep or goats infected with M. agalactiae, results suggest that M. bovis r-P48 can be used as a specific marker of infection. 相似文献
5.
Arcangioli MA Aslan H Tardy F Poumarat F Le Grand D 《Veterinary journal (London, England : 1997)》2012,192(1):96-100
Mycoplasma bovis is a major cause of respiratory outbreaks in cattle feedlots. In this study pulsed-field gel electrophoresis (PFGE) was used to trace field strains and provide information on M. bovis patterns of spread in calf feedlots. The suitability of KpnI, MluI and SmaI restriction enzymes was assessed on different sets of strains. The discriminative power of the first two enzymes was first assessed using 28 epidemiologically unrelated strains; stability was 100% on multiple isolates from in vivo experimental infection. Thirty-nine field isolates from six feedlots were then evaluated. In contrast to the unique fingerprints displayed by the unrelated strains, the isolates from the feedlots showed identical patterns at the time of the outbreak of respiratory disease and 4 weeks later. The PFGE typing results suggest that M. bovis strains follow a clonal epidemic spread pattern at the herd level and that the same strain persists in calves of the herd after the clinical signs have disappeared. 相似文献
6.
Thomas A Linden A Mainil J Dizier I Baseman JB Kannan TR Fleury B Frey J Vilei EM 《Veterinary microbiology》2004,104(3-4):213-217
An analogue of the adhesin gene p40 of Mycoplasma agalactiae was found in Mycoplasma bovis. Nucleotide sequence analysis of the p40* gene in M. bovis revealed the presence of a large deletion involving a frameshift that causes premature truncation of the translated protein, indicating that p40* exists as a pseudogene in M. bovis. 相似文献
7.
Bashiruddin JB Frey J Königsson MH Johansson KE Hotzel H Diller R de Santis P Botelho A Ayling RD Nicholas RA Thiaucourt F Sachse K 《Veterinary journal (London, England : 1997)》2005,169(2):268-275
Diagnostic differentiation between the ruminant pathogens Mycoplasma agalactiae and Mycoplasma bovis is known to be problematic when only conventional serological and biochemical tests are used. The main reason for this is that both agents share a considerable number of related proteins and common epitopes. DNA-based detection methods offer advantages in terms of specificity and sensitivity. However, there is an urgent need to compare currently used PCR assays because they target different genomic regions and, therefore, may perform differently. In the present work, five laboratories, which use PCR routinely, evaluated the specificity of four different PCR systems for M. agalactiae and three systems for M. bovis on a total of 41 strains of the two Mycoplasma species including six previously unidentified strains. As the vast majority of PCR examinations (97.1% of all tests) correctly identified the strains the specificity of all seven detection systems appears to be high. In four cases, incorrect identification by conventional diagnostic methods was rectified by PCR. Isolates from non-typical hosts, i.e. three M. bovis strains from small ruminants and two M. agalactiae strains from cattle, were characterised by sequencing the 16S and part of the 23S ribosomal RNA genes. 相似文献
8.
A Mycoplasma bovis species-specific PCR assay has been developed with improvement of a previously described method (Ghadersohi et al., 1997). This test and its semi-nested version (Hayman and Hirst, 2003) did not function at all in our hands. A new reverse primer (Mbr2) was designed using previously published sequence data. For testing specificity, DNA was extracted from the most frequently occurring mycoplasma species and bacteria of bovine origin. The new PCR detected only Mycoplasma bovis. Moreover, no cross-reaction was observed with the genetically closest relative species, M. agalactiae. The target organism could be detected in a dose as low as 150 CFU ml(-1) in broth cultures using ethidium-bromide-stained agarose gels. 相似文献
9.
A single radial haemolysis in gel technique is described for assaying antibody to Mycoplasma bovis (M agalactiae subsp bovis) in bovine sera. The test, which should be particularly useful for screening large numbers of serum samples, is sensitive, simple to perform and highly reproducible. 相似文献
10.
Mycoplasma agalactiae subsp. bovis in pneumonia and arthritis of the bovine. 总被引：1，自引：1，他引：0 
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下载免费PDF全文 E V Langford 《Canadian journal of veterinary research》1977,41(1):89-94
The pneumonic lungs of 42 cattle from 26 feedlots were examined for the presence of mycoplasma, pathogenic bacteria and viruses. Four animals representative of two lots failed to yield mycoplasma. One of these yielded the virus of infectious bovine rhinotracheitis and Pasteurella hemolytica, the other yielded only P. P. multocida. Nine animals in eight lots yielded Mycoplasma sp.: five of these were M. bovirhinis, two were M. arginini and two were untypable. All of these animals yielded one or more of P. hemolytica, P. multiocida, infectious bovine rhinotracheitis virus or bovine virus diarrhea virus. Twenty-five of 29 animals in 16 lots yieled M. agalactiae subsp. bovis from lung tissues. The same organism was recovered from the arthritic joints of 12 of these animals. Eight of the 25 animals yielded no other pathogen and all of these had not received any treatment. Nine of the 25 M. agalactiae subsp. bovis positive animals also yielded one or more of P. hemolytica, P. multocida, Corynebacterium pyogenes or infectious bovine rhinotracheitis virus. Bacteriological and virological studies were not completed for the remaining eight of the 25 positive animals. In five lots of cattle which had not received medication for pneumonia and for arthritis only M. agalactiae subsp. bovis was recovered. Twenty-five grossly normal lungs obtained from normal cattle at the time of slaughter were cultured and all were negative. The possible role of M. agalactiae subsp. bovis in pneumonia and arthritis was discussed. 相似文献
11.
Development of a real-time PCR for detection of Mycoplasma bovis in bovine milk and lung samples. 总被引：1，自引：0，他引：1
Hugh Y Cai Patricia Bell-Rogers Lois Parker John F Prescott 《Journal of veterinary diagnostic investigation》2005,17(6):537-545
A real-time polymerase chain reaction (PCR) assay using hybridization probes on a LightCycler platform was developed for detection of Mycoplasma bovis from individual bovine mastitis milk and pneumonic lung tissues. The detection limit was 550 colony forming units (cfu)/ml of milk and 650 cfu/25 mg of lung tissue. A panel of bovine Mycoplasma and of other bovine-origin bacteria were tested; only M. bovis strains were positive, with a melting peak of 66.6 degrees C. Mycoplasma agalactiae PG2 was also positive and could be distinguished because it had a melting peak of 63.1 degrees C. In validation testing of clinical samples, the relative sensitivity and specificity were 100% and 99.3% for individual milks and 96.6% and 100% for the lung tissue. Using M. bovis real-time PCR, the M. bovis culture-positive milk samples were estimated to contain between 5 x 10(4) and 7.7 x 10(8) cfu/ml and the M. bovis culture-positive lungs between 1 x 10(3) and 1 x 10(9) cfu/25 mg. Isolation, confirmed with the real-time PCR and colony fluorescent antibody test, showed that at the herd level, the proportion of samples positive for M. bovis isolation in mastitis milk samples submitted to the Mastitis Laboratory, Animal Health Laboratory, University of Guelph, Ontario, Canada, was 2.4% (5/201). We conclude that this probe-based real-time PCR assay is a sensitive, specific, and rapid method to identify M. bovis infection in bovine milk and pneumonic lungs. 相似文献
12.
试验旨在研究牛支原体（Mycoplasma bovis,M.bovis）武威株二氢硫辛酰胺转乙酰酶（PDHc-E2）基因序列特征及其在牛支原体细胞中的位置。参照GenBank中牛支原体HB0801株pdhc基因（登录号：CP002058.1）设计引物,应用PCR扩增获得牛支原体武威株pdhc基因,在测序及序列分析的基础上,应用Overlap PCR完成点突变后将其克隆至pET-28a（+）中,构建原核表达载体pET-pdhc。pET-pdhc转化大肠杆菌Rosetta（DE3）感受态细胞后经IPTG诱导获得融合蛋白,将纯化蛋白免疫新西兰兔制备多抗血清,应用iELISA和Western blotting对牛支原体武威株PDHc-E2在细胞内的分布进行初步研究。结果显示,牛支原体武威株pdhc基因CDS全长735 bp,编码244个氨基酸,与国内牛支原体分离株HB0801、Hubei-1、CQ-W70、NM2012等基因序列完全一致,与国际标准株PG45同源性为99.2%,与无乳支原体（M.agalactiae）同源性为90.9%~91.2%,与加利福尼亚支原体（M.californicum）ST6株的同源性仅为78.4%,基因序列非常保守;通过Overlap PCR将该基因中4个编码色氨酸的TGA密码子突变为TGG,且完成点突变后的基因在大肠杆菌中成功表达,重组蛋白大小约为29 ku,主要以可溶性形式存在,iELISA结果显示,重组蛋白PDHc-E2具有较高的免疫原性,可刺激新西兰兔产生高水平的抗体,血清效价高达1：100 000;亚细胞定位结果表明,制备的多抗血清与重组蛋白PDHc-E2、牛支原体全菌蛋白、牛支原体膜蛋白、牛支原体胞浆蛋白均能发生特异性结合,说明该蛋白在牛支原体细胞膜和细胞质中均有分布,为膜相关蛋白,但在细胞质中的分布多于细胞膜。本研究结果为进一步研究牛支原体的生物学功能提供了理论依据。 相似文献
13.
Fitzmaurice J Sewell M King CM McDougall S McDonald WL O'Keefe JS 《New Zealand veterinary journal》2008,56(5):233-236
AIM: To develop a real-time PCR for the detection of Mycoplasma agalactiae, using PCR primers targeting the ma-mp81 gene. METHODS: A group of 15 M. agalactiae isolates, 21 other Mycoplasma spp. isolates and 21 other bacterial isolates was used in evaluation of the assay. RESULTS: All M. agalactiae isolates were detected by the assay and none of the non-target isolates was amplified. The analytical detection limit of the assay was 10 fg of purified genomic DNA and 104 cfu/ml milk inoculated with M. agalactiae. When applied to goat-milk samples collected from three herds free of M. agalactiae infection, the assay had a specificity of 100%. CONCLUSIONS: The assay would be useful in a diagnostic laboratory, providing specific, sensitive and rapid detection of M. agalactiae. 相似文献
14.
Giangaspero M Nicholas RA Hlusek M Bonfini B Osawa T Orusa R Tatami S Takagi E Moriya H Okura N Kato K Kimura A Harasawa R Ayling RD 《Tropical animal health and production》2012,44(3):395-398
Sheep flocks from Hokkaido, Iwate and Aomori, three northern prefectures of Japan, were screened for antibodies to Mycoplasma ovipneumoniae and Mycoplasma agalactiae by ELISA. Sixty four animals out of 246 (26%) were seropositive to M. ovipneumoniae, with positive results obtained from all three prefectures. None of the sera tested were serologically positive to M. agalactiae. 相似文献
15.
Hydrogen peroxide (H2O2) production and oxygen uptake during the oxidation of NADH and L-alpha-glycerophosphate (GP) by lysed cells was determined for the type and field strains of Mycoplasma bovis and M. agalactiae. NADH oxidation by all the strains showed variable production of H2O2 ranging from 0 to 1.21 mol/mol O2 taken up. All strains were unable to oxidize GP, showing absence of GP oxidase activity. Some strains were identified that produced relatively high levels of H2O2 (> 1.0 mol/ mol O2 taken up). In vitro passage of M. bovis strain 119B96 showed reduced H2O2 production: 0.52, 0.16, and 0.07 mol/mol O2 taken up after the 50th, 100th and 200th passages, respectively. SDS-PAGE analysis showed the loss of a protein band of 32 kDa after 50 passages. These preliminary studies show that not only does H2O2 production by potentially pathogenic Mycoplasma spp. vary in the field but also that similar alterations can be induced by passage in culture. In the latter case, at least in one M. bovis strain, this alteration has been shown by SDS-PAGE to be associated with a loss of specific protein production. Further study of these phenomena is essential background for the production of more efficient vaccines for mycoplasmas. 相似文献
16.
牛支原体单克隆抗体的制备与鉴定 总被引：1，自引：1，他引：0
以牛支原体(Mycoplasma bovis)湖北分离株HB0801作为抗原免疫8周龄BALB/c小鼠,利用杂交瘤技术筛选出了6株能稳定分泌抗牛支原体的单克隆抗体细胞株,分别生产腹水并对单抗进行了纯化和特性鉴定。经亚型测定,这些单抗都属IgG类。腹水ELISA效价在1×105~1.6×106。ELISA特异性分析结果表明,6株单抗与临床分离的牛支原体菌株以及ATCC标准株PG45都显阳性反应,但与牛的其他常见病原菌如多杀性巴氏杆菌、化脓隐秘杆菌等都显阴性反应。所有制备的单抗都与无乳支原体有交叉反应,其中两株单抗1A5和1C11只与无乳支原体有交叉反应,与其他支原体无交叉反应。经Western blotting验证,6株单抗分别识别牛支原体全菌蛋白中的不同条带,说明分别针对不同的蛋白抗原。这些牛支原体单克隆抗体为后期建立牛支原体检测方法及致病机理研究奠定了良好基础。 相似文献
17.
Seven teat dip and sanitizer products were tested in vitro and in vivo for mycoplasmacidal activity against Mycoplasma agalactiae subsp. bovis (M. bovimastitidis). Most, but not all products tested appeared to kill the mycoplasma at satisfactory dilutions. These mycoplasma survived longer on teat skin during humid, rainy weather than during warm, dry weather. Acholeplasma laidlawii was frequently found on normal teat skin. 相似文献
18.
Jechlinger W Chopra-Dewasthaly R Glew M Citti C Rosengarten R 《Berliner und Münchener tier?rztliche Wochenschrift》2004,117(11-12):472-479
Compared to other bacterial pathogens, the current knowledge of the molecular basis of pathogenicity of mycoplasmas is limited, and their strategies of infection at the molecular and cellular level remain to be elucidated. Several studies in the past years have shown that pathogenic mycoplasmas are equipped with sophisticated genetic systems, which allow these agents to spontaneously change their surface antigenic make-up. It is implicated that these variable surface components provide the wall-less mycoplasmas with a means to avoid the host immune response and promote host colonization. In Mycoplasma (M.) agalactiae, the agent of "contagious agalactia" in sheep and goats, a pathogenicity island-like locus has recently been identified that contains six distinct but related genes which encode the major immunodominant membrane proteins, the so-called Vpmas. It was shown that these surface-associated proteins vary in expression at an unusual high frequency due to site-specific DNA rearrangements. The previous lack of tools to genetically manipulate M. agalactiae has hampered more refined studies to assess the exact function of Vpmas in M. agalactiae infection and disease. The recent successful introduction of foreign DNA into the M. agalactiae genome therefore represents an important breakthrough which sets up the basis for a variety of follow-up studies assessing the role of Vpmas in molecular pathogenesis. 相似文献
19.
Analysis of heat shock protein 60 encoding genes of mycoplasmas and investigations concerning their role in immunity and infection 总被引：4，自引：0，他引：4
Only little is known about the heat shock proteins (Hsp) and Hsp-encoding genes of mycoplasmas. The aim of this study was to identify and sequence the hsp60 gene of Mycoplasma agalactiae, Mycoplasma arthritidis, Mycoplasma bovis, and Mycoplasma hyopneumoniae, and to investigate the immune response to Hsp60.Fragments of the hsp60 genes of M. agalactiae, M. arthritidis, M. bovis and M. hyopneumoniae representing almost the entire coding region were amplified by PCR. Two fragments of a hsp60 gene were cloned in Escherichia coli and the antibody response of pigs infected with M. hyopneumoniae against the recombinant Hsp60 fusion proteins was analysed. Within the mycoplasmas, the hsp60 genes showed sequence identities of nearly 100%, with the exception of the hsp60 gene of Mycoplasma genitalium, which was determined to be only 76.5-77.7% identical. Identities to Clostridium perfringens, Bacillus subtilis and E. coli were determined between approximately 50 and 60%. The predicted amino acid sequences of Hsp60 showed an identity of 90 to nearly 100% among mycoplasmas and 50-60% to the other bacteria indicated above. Two Hsp60 derived glutathione-S-transferase fusion proteins containing mycoplasma peptides of 28 and 35kDa were isolated. M. hyopneumoniae-ELISA positive porcine convalescent sera reacted strongly with the recombinant Hsp60 fusion proteins in Western immunoblotting indicating for the first time that mycoplasmal Hsp60 is immunogenic in natural infection. 相似文献
20.
The two avian pathogens Mycoplasma gallisepticum and Mycoplasma synoviae were found, by Southern blot hybridization of their digested DNAs, to share genomic nucleotide sequences additional to those of the highly conserved ribosomal RNA genes. The assumption that some of the shared sequences encode for antigens or epitopes common to both mycoplasmas was supported by Western immunoblot analysis of cell proteins of one mycoplasma with specific antiserum to the other mycoplasma. Interestingly, the band patterns of reactive antigens were different for some of the M. gallisepticum strains, supporting the concept that the species is genotypically variable. The results of the present study may explain the cross-reactivity of the two mycoplasmas noted previously in a variety of routine serological tests. 相似文献