The characterization of Mycoplasma synoviae EF-Tu protein and proteins involved in hemadherence and their N-terminal amino acid sequences

An abundant cytoplasmic 43-kDa protein from Mycoplasma synoviae, a major pathogen from poultry, was identified as elongation factor Tu. The N-terminal amino acid sequence (AKLDFDRSKEHVNVGTIGHV) has 90% identity with the sequence of the Mycoplasma hominis elongation factor Tu protein. Monoclonal antibodies reacting with the M. synoviae elongation factor Tu protein also reacted with 43-kDa proteins from the avian Mycoplasma species Mycoplasma gallinarum, Mycoplasma gallinaceum, Mycoplasma pullorum, Mycoplasma cloacale, Mycoplasma iners and Mycoplasma meleagridis, but not with the proteins from Mycoplasma gallisepticum, Mycoplasma imitans or Mycoplasma iowae. In addition, two groups of phase variable integral membrane proteins, pMSA and pMSB, associated with hemadherence and pathogenicity of M. synoviae strains AAY-4 and ULB925 were identified. The cleavage of a larger hemagglutinating protein encoded by a gene homologous to the vlhA gene of M. synoviae generates pMSB1 and pMSA1 proteins defined by mAb 125 and by hemagglutination inhibiting mAb 3E10, respectively. The N-terminal amino acid sequences of pMSA proteins (SENKLI ... and SENETQ ...) probably indicate the cleavage site of the M. synoviae strain ULB 925 hemagglutinin.     

A polymerase chain reaction based method for detecting Mycoplasma/Acholeplasma contaminants in cell culture

A detection system that utilizes a primer mixture in a nested polymerase chain reaction for detecting Mycoplasma contaminants in cell cultures is described. Primers were designed to amplify the spacer regions between the 16S and 23S ribosomal RNA genes of Mycoplasma and Acholeplasma. This detection system was able to detect 20-180 colony forming units per milliliter of sample. Eight commonly encountered Mycoplasma and Acholeplasma contaminants, which include Mycoplasma (M.) arginini, M. fermentans, M. hominis, M. hyorhinis, M. orale, M. pirum, M. salivarium, and Acholeplasma laidlawii, were consistently amplified. Mycoplasma contaminants generated a single DNA band of 236-365 base pairs (bp), whereas A. laidlawii produced a characteristic two-band pattern of 426 and 219 bp amplicons. Species identification could be achieved by size determination and restriction enzyme digestion. Minor cross-reactions were noted with a few closely related gram positive bacteria and DNA from rat cell lines. A Mycoplasma Detection Kit for detecting Mycoplasma contaminants in cell cultures has been developed based on this approach.     

A 41-kDa variable surface protein of Mycoplasma gallisepticum has a counterpart in Mycoplasma imitans and Mycoplasma iowae

Abstract Mycoplasma gallisepticun, M. imitans and M. iowae are three morphologically similar avian Mycoplasma species, and M. gallisepticum and M. imitans have been shown to be antigenically related. Using a monoclonal antibody that binds to the previously described size- and phase-variant integral membrane surface protein PvpA of M. gallisepticum , we have identified in all three avian Mycoplasma species a 41-kDa surface antigen, which in M. gallisepticum and M. imitans was identified as peripheral membrane protein undergoing variation in expression among clonal isolates. Southern blot analysis using the pvpA gene as a probe demonstrated sequence homology with M. imitans and M. iowae genomic DNA and suggested that a pvpA -related gene that may encode the 41-kDa product exists in these two Mycoplasma species. These studies establish (i) that M. iowae is antigenically related to M. gallisepticum and M. imitans , (ii) that the three species share non-ribosomal gene sequences, and (iii) that peripheral membrane proteins contribute to Mycoplasma surface variation.     

Presence of the arginine dihydrolase pathway in Mycoplasma

Barile, Michael F. (Division of Biologics Standards, National Institutes of Health, Bethesda, Md.), Robert T. Schimke, and Donald B. Riggs. Presence of the arginine dihydrolase pathway in Mycoplasma. J. Bacteriol. 91:189-192. 1966.-The presence of the arginine dihydrolase pathway was examined in 61 Mycoplasma strains representing at least 18 Mycoplasma species isolated from nine different sources: human, bovine, avian, murine, swine, goat, canine, sewage, and tissue cell culture origin. Some species were represented by only one or two strains. Different strains of the same species gave the same results. Ten species (56%) were positive. Many nonpathogenic Mycoplasma species (M. hominis, type 1 and 2, M. fermentans, M. salivarium, and M. gallinarum) were positive, whereas most pathogenic species (M. pneumoniae, M. gallisepticum, M. neurolyticum, and M. hyorhinis) were negative. The presence of arginine dihydrolase activity among Mycoplasma species may prove to be useful for purposes of identification and classification.     

Distribution of ecto 5'-nucleotidase on Mycoplasma species associated with arthritis

The enzyme ecto 5'-nucleotidase (5'N) was found to be active on 8/14 strains of Mycoplasma fermentans, K(m) (+/-S.D.) 3.8+/-2.8 microM 5'-AMP, and on the type strain of Mycoplasma pulmonis, K(m) 0.63 microM 5'-AMP. The six M. fermentans strains lacking 5'N activity were related by restriction fragment length polymorphism typing. At pH 8.5, the type strains of Mycoplasma arthritidis, Mycoplasma buccale and Ureaplasma urealyticum showed a relatively non-specific phosphatase activity against 5'-AMP but no activity was shown by the type strains of Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma orale, Mycoplasma penetrans, Mycoplasma pneumoniae and Mycoplasma salivarium at this pH. M. fermentans has been reported from rheumatoid joints, which show a raised 5'N activity on their synovial cells and in their fluid which may be associated directly or indirectly with the mycoplasma.     

Introduction of a validation concept for a PCR-based Mycoplasma detection assay

BACKGROUND: Mycoplasma contamination is amongst the most frequently occurring problems associated with cell cultures. In order to meet the legal requirements (European Pharmacopoeia and FDA) for Mycoplasma testing of cell lines and therapeutics, we have developed a PCR-based method to detect mycoplasms and introduce a validation concept. METHODS: The PCR assay specifically amplifies a 280-bp DNA fragment of the gene coding for the 16S rDNA. Simultaneous amplification of an artificial oligonucleotide containing primer-binding sites allowed control of the efficacy of the PCR. The validation of the PCR assay was performed with two Mycoplasma reference strains, M. orale and M. pneumoniae. The validation concept included (i) cultivation of M. orale and M. pneumoniae in medium with an indicator for bacterial metabolism, (ii) determination of the color-changing units (CCU) in repeated dilution experiments and (iii) correlation of the PCR results with CCU values. RESULTS: The detection range was found to include all Mycoplasma species most commonly found in cell cultures. The analytical sensitivity of the PCR was the CCU equivalent of 100 for M. orale and M. pneumoniae. Probit analysis revealed a detection probability of 9% for a mean concentration of 1222 (935-1844) CCU/mL for M. pneumoniae and 2547 (1584-10,352) CCU/mL for M. orale. DISCUSSION: The validation of the Mycoplasma detection assay supported PCR as an attractive diagnostic tool that will help manage the important issue of Mycoplasma contamination of cell cultures.     

Comparison of polymerase chain reaction assay and Mycoplasma IST 2 test with culture for detection of infections caused by Ureaplasma urealyticum and Mycoplasma hominis

The polymerase chain reaction (PCR) technique and commercial Mycoplasma IST 2 test were compared with culture for the detection of U. urealyticum and M. hominis in 173 clinical samples obtained from patients without clinical symptoms from genito-urinary tract. The presence of U. urealyticum was diagnosed by culture in 24 samples, by PCR in 33 samples and by Mycoplasma IST 2 test in 39 samples. The presence of M. hominis was diagnosed in 26 samples only by Mycoplasma IST 2 test--culture and PCR were negative. The study showed the excellent sensitivity (100%) and good specificity (appropriately 94.0% and 90.0%) for U. urealyticum in PCR and Mycoplasma IST 2 test. The discrepancy of results obtained in Mycoplasma IST 2 test and culture as well as in PCR may suggest the over sensitivity of the commercial test for detection of M. hominis.     

Suspected utility of enzymes with multiple activities in the small genome Mycoplasma species: the replacement of the missing "household" nucleoside diphosphate kinase gene and activity by glycolytic kinases

The small genome Mollicutes whose DNAs are completely sequenced (Mycoplasma genitalium, Mycoplasma pneumoniae, Mycoplasma pulmonis, and Ureaplasma urealyticum [parvum]) lack a gene (ndk) for the presumably essential nucleoside diphosphate kinase (NDPK). We hypothesized that other activities might replace NDPK activity. We found in M. genitalium G37(T), Mycoplasma pneumoniae FH(T), Mycoplasma fermentans PG18(T), and Mycoplasma capricolum subsp. capricolum Kid(T) that their 6-phosphofructokinases (6-PFKs), phosphoglycerate kinases (PGKs), pyruvate kinases (PKs), and acetate kinases (AKs), besides reactant ADP/ATP, could use other ribo- and deoxyribo-purine and pyrimidine NDPs and NTPs. These activities could compensate for the absence of an orthologous ndk gene in the Mycoplasmataceae. They suggest a metabolically varied and consequential role for unrelated and perhaps unsuspected "replacement" or compensatory enzymes that may confound metabolic prediction. We partially purified and biochemically characterized the PKs, 6-PFKs, PGKs, and AKs from M. capricolum subsp. capricolum Kid(T) and M. fermentans PG18(T).     

Experimental pathogenicity of Mollicutes of bovine udder origin in hamster tracheal ring organ culture.

Hamster tracheal-ring organ culture was employed to examine pathogenic effects of 8 isolates of Mollicutes of bovine udder origin. The tested Mollicutes could be categorized into two groups: (i) Mycoplasma F-38, M. mycoides var. capri, M. bovigenitalium mixed with M. bovirhinis, and M. bovigenitalium mixed with M. bovirhinis and Mycoplasma F-38 produced significant ciliostatic effect and infiltration of neutrophils and lymphocytes in lamina propria/subepithelium, hyperplasia and desquamation of epithelial lining cells and loss of cilia; and (ii) A. laidlawii, A. axanthum, an unidentified Acholeplasma and a mixed isolate of M. bovis, M. bovigenitalium, Mycoplasma F-38 and A. laidlawii showed insignificant ciliostatic effects and produced mild histopathological lesions. This correlates with the disease causing potentials of the strains.     

泌尿生殖道支原体感染检测及药敏试验

目的了解本地区泌尿生殖道解脲脲原体（Uu）和人型支原体（Mh）感染状况及药物敏感性,指导临床医生合理应用抗生素。方法应用法国生物梅里埃公司提供的IST试剂盒进行支原体鉴定及9种药物敏感检测,并对结果进行统计分析。结果1210例门诊患者检出支原体阳性683例,总感染率为56.4%,其中Uu单独感染占628例（占51.9%）,Mh单独感染14例（占1.2%）,Uu和Mh混合感染41例（占3.4%）。交沙霉素和原始霉素敏感率最高,对Uu分别为98.5%和97.0%,对Uu和Mh混合感染率都为100%;氧氟沙星敏感率最低,分别为1.5%和0.0%。结论泌尿生殖道系统感染主要由解脲支原体引起,交沙霉素和原始霉素敏感率最高,氧氟沙星敏感率最低。临床应选用培养敏感的抗菌药物,提高治疗效果。     

Isolation of Mycoplasma gallopavonis from free-ranging wild turkeys in coastal North Carolina seropositive and culture-negative for Mycoplasma gallisepticum.

Serum samples and choanal cleft swabs were collected from livetrapped and hunter killed wild turkeys (Meleagris gallopavo) from Martin and Bertie counties, North Carolina (USA). Sera were tested for antibodies to Mycoplasma gallisepticum, Mycoplasma synoviae and Mycoplasma meleagridis by hemagglutination inhibition (HI). Sera from 33% (five of 15) of livetrapped turkeys were positive for antibodies to M. gallisepticum by HI, and all were negative for antibodies to M. synoviae and M. meleagridis. Choanal cleft swabs from 22 livertrapped and five hunter killed wild turkeys cultured in Frey's broth medium resulted in 23 mycoplasma isolations. Using direct immunofluorescence, 74% (17/23) were M. gallopavonis, and 26% (six of 23) were unidentified; no isolate was identified as M. gallisepticum, M. synoviae or M. meleagridis.     

Role of arginine deiminase in growth of Mycoplasma hominis.

Arginine has been considered as the major energy source of nonglycolytic arginine-utilizing mycoplasmata. When three strains of Mycoplasma arginini, and one strain each of Mycoplasma arthritidis, Mycoplasma fermentans, Mycoplasma gallinarum, Mycoplasma gallisepticum and Mycoplasma hominis were grown in the medium with high arginine concentration (34 mM) compared with low arginine (4 mM), both the protein content of the organisms and the specific activity of arginine deiminase increased. M. fermentans, the one arginine-utilizing species included in the survey which is also glycolytic, showed an increase in protein content but no increase in specific activity of the enzyme. The glycolytic non-arginine-utilizing M. gallisepticum did not show an increase in either parameter. The Km for arginine deiminase from crude cell extracts was 1.66 X 10(-4)M. The enzyme demonstrated a hyperbolic activation curve subject to substrate inhibition and was not affected by the presence of L-histidine. When mycoplasmic protein and arginine deiminase were determined for M. hominis under aerobic and anaerobic conditions, aerobically grown cells exhibited no detectable enzymatic increases until late in log phase. Higher levels of arginine deiminase were observed earlier in the anaerobic growth cycle. The rate of 14CO2 evolution from [guanido-14C]arginine was not altered in arginine-supplemented cells compared with cells grown in low arginine. In addition, CO2 production did not parallel increased arginine deiminase activity. These observations argue that arginine is used only as an alternate energy source in these organisms.     

Molecular identification of Mycoplasma cynos from laboratory beagle dogs with respiratory disease

In this study, we examined a colony of 20 beagle dogs in a laboratory animal facility. Mycoplasma was detected by consensus PCR assay in 1 dog with respiratory and constitutional symptoms. None of the other dogs were affected. The dog was euthanized and necropsied. In postmortem examinations, gray or plum-colored gross lesions were found on the lung, most commonly in the apical and cardiac lobes. Some lesions showed clear demarcation and consolidation. Microscopic examination showed peribronchiolar lymphoid hyperplasia and interstitial thickening, lesions pathognomonic for mycoplasma pneumonia. To identify canine Mycoplasma species, we used species-specific PCR reactions for M. arginini, M. canis, M. cynos, M. edwardii, M. felis, M. gateae, M. maculosum, M. molare, M. opalescens, M. spumans, Mycoplasma sp. HRC 689, and M. collis. As the result, we identified Mycoplasma cynos by amplification of DNA extracted from lung tissue of the laboratory beagle dog with respiratory disease.     

鼠肺支原体单克隆抗体的研究

通过杂交瘤技术建立了３株抗鼠肺支原体单克隆抗体细胞株,它们分别是ＢＡ１１,ＢＤ７和Ｂ６１２．试验表明：３株细胞所分泌的抗体均属ＩｇＧ１亚类,都是鼠肺支原体的特异性抗体,与多种其它支原体无交叉反应。     

Complete Genome Sequences of Mycoplasma leachii Strain PG50T and the Pathogenic Mycoplasma mycoides subsp. mycoides Small Colony Biotype Strain Gladysdale

Mycoplasma mycoides subsp. mycoides small colony biotype (SC) is the high-consequence animal pathogen causing contagious bovine pleuropneumonia. We report the complete genome sequences of the pathogenic strain M. mycoides subsp. mycoides SC Gladysdale and a close phylogenetic relative, Mycoplasma leachii PG50(T), another bovine pathogen of the M. mycoides phylogenetic clade.     

High prevalence of Mycoplasma infections among European chronic fatigue syndrome patients. Examination of four Mycoplasma species in blood of chronic fatigue syndrome patients

Prevalence of Mycoplasma species infections in chronic fatigue syndrome (CFS) has been extensively reported in the scientific literature. However, all previous reports highlighted the presence of Mycoplasmas in American patients. In this prospective study, the presence of Mycoplasma fermentans, M. penetrans, M. pneumoniae and M. hominis in the blood of 261 European CFS patients and 36 healthy volunteers was examined using forensic polymerase chain reaction. One hundred and seventy-nine (68.6%) patients were infected by at least one species of Mycoplasma, compared to two out of 36 (5.6%) in the control sample (P<0.001). Among Mycoplasma-infected patients, M. hominis was the most frequently observed infection (n=96; 36.8% of the overall sample), followed by M. pneumoniae and M. fermentans infections (equal frequencies; n=67; 25.7%). M. penetrans infections were not found. Multiple mycoplasmal infections were detected in 45 patients (17.2%). Compared to American CFS patients (M. pneumoniae>M. hominis>M. penetrans), a slightly different pattern of mycoplasmal infections was found in European CFS patients (M. hominis>M. pneumoniae, M. fermentansz.Gt;M. penetrans).     

Complete genome sequences of two hemotropic mycoplasmas, Mycoplasma haemofelis strain Ohio2 and Mycoplasma suis Strain Illinois

We report the complete and fully assembled genomes of Mycoplasma haemofelis strain Ohio2 and Mycoplasma suis strain Illinois, which are the first available genomes of these uncultivatable hemoplasma species. The single circular chromosomes of 1,152,484 bp and 742,431 bp for M. haemofelis and M. suis, respectively, are typical of mycoplasma species, having reduced size and low G+C content (38.8% for M. haemofelis and 31.1% for M. suis). Their metabolic pathways are reduced, with evidence of adaption to the blood environment.     

The occurrence of Mycoplasma phocicerebrale, Mycoplasma phocidae, and Mycoplasma phocirhinis in grey and common seals (Halichoerus grypus and Phoca vitulina) in the United Kingdom

Following the isolation of Mycoplasma phocicerebrale from the flipper wound of a grey seal (Halichoerus grypus) in Cornwall, UK, surveillance for Mycoplasma species was extended to include other seals rescued or found dead around the UK. Mycoplasma phocicerebrale was frequently detected from the teeth of seals and from infected wounds and respiratory tracts. Mycoplasma phocirhinis, Mycoplasma phocidae, and some unidentified Mycoplasma species were also detected. Mycoplasma phocicerebrale and M. phocidae were the only bacteria consistently identified from the wound infections, but their role in respiratory and other diseases remains unknown, as other bacteria were also isolated from respiratory sites.     

Interaction of Mycoplasma bovis and Mycoplasma bovigenitalium with preimplantation bovine embryos

In vitro exposures of bovine embryos to Mycoplasma bovis and Mycoplasma bovigenitalium were conducted to determine if these organisms adhered to the zona pellucida-intact (ZP-I) bovine embryo, and standard procedures for washing and treating embryos were evaluated to determine their effectiveness for removing or killing mycoplasmas. Mycoplasma bovis and M. bovigenitalium were isolated from 19 of 19 and 24 of 24 ZP-I embryos, respectively, after in vitro exposure and subsequent washing, thus demonstrating adherence of the two species of Mycoplasma to the ZP. Additionally, M. bovis was isolated from 20 of 20 and 23 of 23 embryos, while M. bovigenitalium was isolated from 25 of 25 and 22 of 22 embryos after antibiotic and trypsin treatment, respectively. It was concluded that neither of the standard procedures currently used for cleansing embryos should be relied upon for insuring freedom from mycoplasmas.