Mycoplasma insons sp. nov., a twisted mycoplasma from green iguanas (Iguana iguana)

Mycoplasma insons sp. nov., first cultured from the choanae and tracheae of healthy green iguanas (Iguana iguana) from El Salvador, was readily distinguished from all previously described mollicutes and assigned to the Mycoplasma fastidiosum phylogenetic cluster by 16S rRNA gene sequence comparisons. Growth inhibition assays distinguished the isolates serologically from the other two members of that cluster. Many M. insons cells exhibit a remarkable twisted rod morphology despite lacking a cell wall. The organism is nonmotile, produces acid from glucose, but does not hydrolyze arginine, esculin, or urea. Mycoplasma insons 16S rRNA gene was also detected by PCR in packed blood cells from culture-negative iguanas. The type strain I17P1(T) has been deposited with the Mollicutes Collection at Purdue University and with the American Type Culture Collection (ATCC BAA-1435) in the USA. A limited number of cultures generated by the authors have also been deposited with the Culture Collection, University of G?teborg, in Sweden (CCUG 53461).     

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.     

Tetrazolium reduction methods for assessment of substrate oxidation and strain differentiation among mycoplasmas, with particular reference to Mycoplasma bovigenitalium and some members of the Mycoplasma mycoides cluster

Aims:  To apply a rapid nitroblue tetrazolium (NBT) reduction assay of substrate metabolism by mycoplasmas that would help to differentiate Mycoplasmas .
Methods and Results:  Growth, substrate preferences and tetrazolium reduction were assessed for 18 strains of Mycoplasma bovigenitalium and Mycoplasma ovine serogroup 11. NBT reduction was detectable in 1 h with 10⁸ CFU ml⁻¹. Use of α-ketobutyrate, lactate and pyruvate to support growth and NBT reduction were correlated: pyruvate was preferred and lactate was used by only four of the 18 strains. Selected members of the Mycoplasma mycoides cluster were also assessed and monotetrazoles tested as alternatives to NBT. The NBT method was applied to a further 19 species.
Conclusions:  This simple and reproducible method requires only small amounts of cells, enabling routine assessment of substrate use within 1 h, and the rapid assignment of numerous mycoplasmas to one of six physiological groups. The four physiological groups of M. bovigenitalium and Mycoplasma serogroup 11 strains were indistinguishable from each other, which supports the view that these belong to the same species.
Significance and Impact of the Study:  Strain-specific substrate-utilization patterns by mycoplasmas can be obtained rapidly and reliably. The method has potential as a large-scale semi-automated procedure to monitor numerous strains and substrates simultaneously.     

First identification and functional characterization of an immunogenic protein in unculturable haemotrophic Mycoplasmas (Mycoplasma suis HspA1)

The antigenic structures of the haemotrophic Mycoplasma suis, an epicellular parasite of porcine erythrocytes, are largely unknown due to its unculturability. In this study, serological proteome and mass spectrometry analyses allowed the characterization of M. suis proteins targeted by the porcine antibody response: two proteins with characteristics of heat shock proteins, two proteins with characteristics of glycolytic enzymes, a RNA helicase- and an actin-like protein. The DnaK-like protein of M. suis (HspA1) was further analysed genetically and functionally. Its encoding gene (M. suis a1 gene) is 1.830 bp in size and corresponds to a 67 kDa protein. Immunoelectron microscopy verified the surface accessibility of HspA1 in M. suis. Recombinant HspA1 expressed in Escherichia coli demonstrated ATPase activity and antigenicity in experimentally infected pigs. In conclusion, this first identification and recombinant expression of an antigenic protein of M. suis provides the basis for the development of vaccines and new in vitro diagnostic assays.     

The phylogeny of Mycoplasma bovis as determined by sequence analysis of the 16S rRNA gene

Abstract The nucleotide sequence of the 16S rRNA gene of Mycoplasma bovis has been determined. Comparisons with other 16S rRNA sequences of mycoplasmas showed that Mycoplasma agalactiae is phylogenetically the closet relative. In total, only eight nucleotides differed between the M. bovis and M. agalactiae 16S rRNA sequences. The phylogenetic position of M. bovis with respect to other mycoplasmas was determined by sequence comparisons and from features in the secondary structure of 16S rRNA.     

Interaction of albumin and phospholipid:cholesterol liposomes in growth of Mycoplasma spp.

Mycoplasma spp., sterol and fatty acid auxotrophs, are conventionally grown in complex media containing high concentrations of serum. Serum supplies the required lipids, but its presence complicates studies on the metabolism and antigenicity of mycoplasmas as well as the membrane dynamics of these organisms. In the present work, fetal bovine serum was replaced with dilipidated albumin and liposomes containing high concentrations of cholesterol. The liposomes were produced from phosphatidylcholine which contained other lipid species, including phosphatidylethanolamine, phosphatidylglycerol, and cholesterol. Other liposomes containing cholesterol and one phospholipid yielded significantly less growth of Mycoplasma gallisepticum, indicating that several phospholipids are required to achieve growth levels comparable to those obtained with complex medium. The sources and concentrations of cholesterol, albumin, phosphatidylcholine, and other phospholipids and the interactions among them were important affectors of mycoplasmal growth. Optimal lipid and albumin conditions established for M. gallisepticum were then used to propagate five diverse Mycoplasma spp. to growth levels which equalled or surpassed those obtained with medium containing 17% fetal bovine serum.     

Chemotaxis away from thiocyanic and isothiocyanic esters in Pseudomonas aeruginosa

Abstract A novel mycoplasmal species designated as Mycoplasma penetrans has recently been isolated from patients infected with human immunodeficiency virus. The 16S rRNA gene from this mycoplasma was cloned and its nucleotide sequence determined. This sequence was aligned with previously published homologous sequences from several mycoplasmas and with related Gram-positive bacteria and a phylogenetic tree was constructed. The results indicate that M. penetrans belongs to the evolutionary group Pneumoniae.     

One Test Microbial Diagnostic Microarray for Identification of Mycoplasma mycoides subsp. mycoides and Other Mycoplasma Species

The present study describes the use of microarray technology for rapid identification and differentiation of Mycoplasma mycoides subsp. mycoides from other mycoplasmas that may be pathogenic to ruminants, including those of the Mycoplasma mycoides cluster, genetically and antigenically strictly correlated with Mycoplasma mycoides subsp. mycoides. A microarray containing genetic sequences of 55 different bacterial species from Acholeplasma, Mycoplasma, Spiroplasma and Ureaplasma genera was constructed. Sequences to genes of interest were collected in FASTA format from NCBI. The collected sequences were processed with OligoPicker software. Oligonucleotides were then checked for their selectivity with BLAST searches in GenBank. The microarray was tested with ATCC/NCTC strains of Mycoplasma spp. of veterinary importance in ruminants including Mycoplasma belonging to the mycoides cluster as well as Mycoplasma mycoides subsp. mycoides and Mycoplasma mycoides subsp. capri field strains. The results showed that but one ATCC/NCTC reference strains hybridized with their species-specific sequences showed a profile/signature different and distinct from each other. The heat-map of the hybridization results for the nine genes interrogated for Mycoplasma mycoides subsp. mycoides demonstrated that the reference strain Mycoplasma mycoides subsp mycoides PG1 was positive for all of the gene sequences spotted on the microarray. CBPP field, vaccine and reference strains were all typed to be M. mycoides subsp. mycoides, and seven of the nine strains gave positive hybridization results for all of the nine genes. Two Italian strains were negative for some of the genes. Comparison with non-Mycoplasma mycoides subsp. mycoides reference strains showed some positive signals or considerable homology to Mycoplasma mycoides subsp. mycoides genes. As expected, some correlations were observed between the strictly genetically and antigenically correlated Mycoplasma mycoides subsp. mycoides and Mycoplasma mycoides subsp. capri strains. Specifically, we observed that some Italian Mycoplasma mycoides subsp. mycoides strains were positive for two out of the three Mycoplasma mycoides subsp. capri genes, differently from what has been observed for other European or African Mycoplasma mycoides subsp. mycoides strains. This study highlighted the use of microarray technology as a simple and effective method for a single-step identification and differentiation of Mycoplasma mycoides subsp. mycoides from other mycoplasmas that may be pathogenic to ruminants, including those of the Mycoplasma mycoides cluster, genetically and antigenically strictly correlated with Mycoplasma mycoides subsp. mycoides. The opportunity to discriminate several mycoplasmas in a single analysis enhances diagnostic rapidity and may represent a useful tool to screen occasionally mycoplasmas affecting animal farming in territories where diagnostic laboratory support is limited. The heat-map of the hybridization results of the comparative genomic hybridizations DNA-designed chip clearly indicates that the microarray performs well for the identification of the tested Mycoplasma mycoides subsp. mycoides reference and field strains, discriminating them from other mycoplasmas.     

Conserved terminal organelle morphology and function in Mycoplasma penetrans and Mycoplasma iowae

Within the genus Mycoplasma are species whose cells have terminal organelles, polarized structures associated with cytadherence and gliding motility. Mycoplasma penetrans, found mostly in HIV-infected patients, and Mycoplasma iowae, an economically significant poultry pathogen, are members of the Mycoplasma muris phylogenetic cluster. Both species have terminal organelles that interact with host cells, yet the structures in these species, or any in the M. muris cluster, remain uncharacterized. Time-lapse microcinematography of two strains of M. penetrans, GTU-54-6A1 and HF-2, and two serovars of M. iowae, K and N, show that the terminal organelles of both species play a role in gliding motility, with differences in speed within and between the two species. The strains and serovars also differed in their hemadsorption abilities that positively correlated with differences in motility speeds. No morphological differences were observed between M. penetrans and M. iowae by scanning electron microscopy (SEM). SEM and light microscopy of M. penetrans and M. iowae showed the presence of membranous filaments connecting pairs of dividing cells. Breaking of this filament during cell division was observed for M. penetrans by microcinematography, and this suggests a role for motility during division. The Triton X-100-insoluble fractions of M. penetrans and M. iowae consisted of similar structures that were unique compared to those identified in other mycoplasma species. Like other polarized mycoplasmas, M. penetrans and M. iowae have terminal organelles with cytadherence and gliding functions. The difference in function and morphology of the terminal organelles suggests that mycoplasmas have evolved terminal organelles independently of one another.     

DNA repair in reduced genome: the Mycoplasma model

The occurrence of bacteria with a reduced genome, such as that found in Mycoplasmas, raises the question as to which genes should be enough to guarantee the genomic stability indispensable for the maintenance of life. The aim of this work was to compare nine Mycoplasma genomes in regard to DNA repair genes. An in silico analysis was done using six Mycoplasma species, whose genomes are accessible at GenBank, and M. synoviae, and two strains of M. hyopneumoniae, whose genomes were recently sequenced by The Brazilian National Genome Project Consortium and Southern Genome Investigation Program (Brazil) respectively. Considering this reduced genome model, our comparative analysis suggests that the DNA integrity necessary for life can be primarily maintained by nucleotide excision repair (NER), which is the only complete repair pathway. Furthermore, some enzymes involved with base excision repair (BER) and recombination are also present and can complement the NER activity. The absence of RecR and RecO-like ORFs was observed only in M. genitalium and M. pneumoniae, which can be involved with the conservation of gene order observed between these two species. We also obtained phylogenetic evidence for the recent acquisition of the ogt gene in M. pulmonis and M. penetrans by a lateral transference event. In general, the presence or nonexistence of repair genes is shared by all species analyzed, suggesting that the loss of the majority of repair genes was an ancestral event, which occurred before the divergence of the Mycoplasma species.     

Spontaneous bronchopneumonia in laboratory dogs infected with untyped Mycoplasma spp

Three Mycoplasma spp. were isolated from five colony bred laboratory dogs (Canis familiaris) obtained from a single vendor. Four of these animals were Beagles and one was a mongrel. Three displayed clinical signs of respiratory disease including dyspnea, chronic coughing and moist rales, while the other two dogs were observed during thoracic surgery to have macroscopic lesions suggestive of pneumonia. All five dogs were submitted for diagnostic necropsy during which they were cultured for bacteria and mycoplasma. Mycoplasma spp. having three distinct colonial forms were isolated from the lungs of each of the animals. These three isolates were sent to the National Cancer Institute Diagnostic Microbiology Laboratory and to the National Institutes of Health, NIAID, Mycoplasmology Laboratory. Neither laboratory could serotype these isolates against antisera to 73 Mycoplasma spp., including the common canine mycoplasmas, and nine Acholeplasma spp. Histologically, the bronchopneumonia was characterized by bronchiectasis, purulent bronchiolitis, bronchial and bronchiolar epithelial hyperplasia, chronic non-suppurative peribronchiolitis and perivasculitis, bronchiolitis obliterans, and acute to subacute purulent pneumonia. The similarity between the pathologic findings in these animals and those observed in respiratory mycoplasmosis of other species, e.g. the rat, suggests a causal relationship between the isolated mycoplasmas and the pulmonary disease observed in these dogs.     

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.     

Interaction of albumin and phospholipid:cholesterol liposomes in growth of Mycoplasma spp

Mycoplasma spp., sterol and fatty acid auxotrophs, are conventionally grown in complex media containing high concentrations of serum. Serum supplies the required lipids, but its presence complicates studies on the metabolism and antigenicity of mycoplasmas as well as the membrane dynamics of these organisms. In the present work, fetal bovine serum was replaced with dilipidated albumin and liposomes containing high concentrations of cholesterol. The liposomes were produced from phosphatidylcholine which contained other lipid species, including phosphatidylethanolamine, phosphatidylglycerol, and cholesterol. Other liposomes containing cholesterol and one phospholipid yielded significantly less growth of Mycoplasma gallisepticum, indicating that several phospholipids are required to achieve growth levels comparable to those obtained with complex medium. The sources and concentrations of cholesterol, albumin, phosphatidylcholine, and other phospholipids and the interactions among them were important affectors of mycoplasmal growth. Optimal lipid and albumin conditions established for M. gallisepticum were then used to propagate five diverse Mycoplasma spp. to growth levels which equalled or surpassed those obtained with medium containing 17% fetal bovine serum.     

Mycoplasma bovis shares insertion sequences with Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides SC: Evolutionary and developmental aspects

Three new insertion elements, ISMbov1, ISMbov2 and ISMbov3, which are closely related to ISMag1 (Mycoplasma agalactiae), ISMmy1 and IS1634 (both Mycoplasma mycoides subsp. mycoides SC), respectively, have been discovered in Mycoplasma bovis, an important pathogen of cattle. Southern blotting showed that the genome of M. bovis harbours 6-12 copies of ISMbov1, 11-15 copies of ISMbov2 and 4-10 copies of ISMbov3, depending on the strain. A fourth insertion element, the IS30-like element, is present in 4-8 copies. This high number of IS elements in M. bovis, which represent a substantial part of its genome, and their relatedness with IS elements of both M. agalactiae and M. mycoides subsp. mycoides SC suggest the occurrence of two evolutionary events: (i) a divergent evolution into M. agalactiae and M. bovis upon infection of different hosts; (ii) a horizontal transfer of IS elements during co-infection with M. mycoides subsp. mycoides SC and M. bovis of a same bovine host.     

Isolation and characterization of haloacetic acid-degrading Afipia spp. from drinking water

Haloacetic acids are a class of disinfection byproducts formed during the chlorination and chloramination of drinking water that have been linked to several human health risks. In this study, we isolated numerous strains of haloacetic acid-degrading Afipia spp. from tap water, the wall of a water distribution pipe, and a granular activated carbon filter treating prechlorinated water. These Afipia spp. harbored two phylogenetically distinct groups of α-halocarboxylic acid dehalogenase genes that clustered with genes previously detected only by cultivation-independent methods or were novel and did not conclusively cluster with the previously defined phylogenetic subdivisions of these genes. Four of these Afipia spp. simultaneously harbored both the known classes of α-halocarboxylic acid dehalogenase genes ( deh I and deh II), which is potentially of importance because these bacteria were also capable of biodegrading the greatest number of different haloacetic acids. Our results suggest that Afipia spp. have a beneficial role in suppressing the concentrations of haloacetic acids in tap water, which contrasts the historical (albeit erroneous) association of Afipia sp. (specifically Afipia felis ) as the causative agent of cat scratch disease.     

Genetic diversity of invasive Oreochromis spp. (tilapia) populations in Guangdong province of China using microsatellite markers

Ten microsatellites were used to assess the genetic diversity and genetic differences of six wild Oreochromis spp. populations in the primary rivers of Guangdong Province of China, where natural populations have been established. Significant genetic differentiation was observed in Oreochromis spp. populations using “FST” and exact tests. The six populations can be divided into two groups based on the genetic similarity indices. The phylogenetic tree indicated that the HUI (Huizhou) and the SG (Shaoguan) populations formed one cluster, whereas the H (Huazhou), M (Meihua), MU (Muzhou), and ZQ (Zhaoqing) populations formed another cluster. The average mean levels of the observed heterozygosity in the six populations were 0.3972, 0.4264, 0.6977, 0.6335, 0.6680, and 0.6829, respectively. The high genetic diversity observed in most wild tilapia populations suggests that most wild populations are large in size, which is a finding that is supported by field investigations.     

Versatile use of oriC plasmids for functional genomics of Mycoplasma capricolum subsp. capricolum

Replicative oriC plasmids were recently developed for several mollicutes, including three Mycoplasma species belonging to the mycoides cluster that are responsible for bovine and caprine diseases: Mycoplasma mycoides subsp. mycoides small-colony type, Mycoplasma mycoides subsp. mycoides large-colony type, and Mycoplasma capricolum subsp. capricolum. In this study, oriC plasmids were evaluated in M. capricolum subsp. capricolum as genetic tools for (i) expression of heterologous proteins and (ii) gene inactivation by homologous recombination. The reporter gene lacZ, encoding beta-galactosidase, and the gene encoding spiralin, an abundant surface lipoprotein of the related mollicute Spiroplasma citri, were successfully expressed. Functional Escherichia coli beta-galactosidase was detected in transformed Mycoplasma capricolum subsp. capricolum cells despite noticeable codon usage differences. The expression of spiralin in M. capricolum subsp. capricolum was assessed by colony and Western blotting. Accessibility of this protein at the cell surface and its partition into the Triton X-114 detergent phase suggest a correct maturation of the spiralin precursor. The expression of a heterologous lipoprotein in a mycoplasma raises potentially interesting applications, e.g., the use of these bacteria as live vaccines. Targeted inactivation of gene lppA encoding lipoprotein A was achieved in M. capricolum subsp. capricolum with plasmids harboring a replication origin derived from S. citri. Our results suggest that the selection of the infrequent events of homologous recombination could be enhanced by the use of oriC plasmids derived from related mollicute species. Mycoplasma gene inactivation opens the way to functional genomics in a group of bacteria for which a large wealth of genome data are already available and steadily growing.     

滑液支原体醛缩酶的亚细胞定位及免疫原性

【背景】滑液支原体(Mycoplasma synoviae,MS)感染能够引起鸡和火鸡的气囊炎、关节渗出性的滑液囊膜及腱鞘滑膜炎等。有研究表明,许多支原体中与代谢相关的酶类不仅分布在细胞质,也分布于细胞膜表面,通过结合宿主细胞的胞外基质蛋白,协助病原菌黏附入侵宿主细胞。已有报道牛支原体(Mycoplasma bovis)和鸡毒支原体(Mycoplasma gallisepticum)的醛缩酶(Fructose-bisphosphate aldolase,FBA)分布在膜蛋白和胞浆蛋白中,而MS的FBA蛋白还未见相关研究。【目的】对MSFBA蛋白进行生物信息学分析、原核表达、免疫原性分析及亚细胞定位检测,为进一步探索MS代谢相关酶类的生物学功能奠定基础。【方法】通过分析软件PSORTb、SignalP 4.1 Server和TMHMM Server在线预测MSFBA的亚细胞定位、信号肽及跨膜区,并用BLASTn和MEGA 5.0进行同源比对及进化树分析;通过Overlap PCR点突变扩增MS的fba全基因序列,连接表达载体pET-28a(+)并进行原核表达及纯化,获得纯化后的重组MSFBA(rMSFBA)蛋白;用MS阳性血清进行Westernblot鉴定rMSFBA的免疫原性;用纯化的rMSFBA蛋白制备兔多克隆抗体,与MS全菌蛋白、膜蛋白及胞浆蛋白进行Western blot分析,同时对MS全菌进行悬浮免疫荧光分析,检测MSFBA的膜定位情况。【结果】生物信息分析预测MSFBA分布在细胞质中,无信号肽,无跨膜区,在MS种内相似性高达99%,与其他种属FBA相似性在61%-78%之间,进化树显示其与牛鼻支原体(Mycoplasma bovirhinis)、仓鼠支原体(Mycoplasm acricetuli)等的FBA蛋白进化关系较近,与精氨酸支原体(Mycoplasma arginini)、人型支原体(Mycoplasma hominis)等的FBA蛋白进化关系较远;表达rMSFBA蛋白并纯化,经测定其相对分子质量大小约为33kD;rMSFBA能与MS阳性鸡血清特异性结合,证实其具有较好的免疫原性;Western blot显示抗rMSFBA的兔血清能与MS全菌蛋白和胞浆蛋白反应,而与膜蛋白不反应,说明MSFBA蛋白分布于胞浆中;悬浮免疫荧光实验证实MS的细胞膜上未见FBA蛋白分布。【结论】首次报道了滑液支原体的FBA蛋白是一个高度保守的免疫原性蛋白,主要分布在细胞质中,该结果为进一步研究MSFBA蛋白的生物学功能提供了分子基础。     

Phylogenetic relationships of three porcine mycoplasmas, Mycoplasma hyopneumoniae, Mycoplasma flocculare, and Mycoplasma hyorhinis, and complete 16S rRNA sequence of M. flocculare.

The nucleotide sequence of the 16S rRNA gene of Mycoplasma flocculare was determined and was compared with the sequence of a related porcine mycoplasma, Mycoplasma hyopneumoniae. While the overall level of DNA-DNA homology was approximately 11%, sequence alignment of the two 16S rRNA genes yielded a homology value of more than 95%, emphasizing the highly conserved nature of the 16S rRNA gene. Multiple sequence alignments with other mollicutes indicated that M. flocculare, M. hyopneumoniae, and Mycoplasma hyorhinis form a subcluster within the fermentans phylogroup, and this subcluster is distinct from the Mycoplasma pneumoniae phylogroup. Thus, the three mycoplasmas isolated from porcine respiratory systems exhibit phylogenetic similarities.     

Relationships between members of the Mycoplasma mycoides cluster as shown by DNA probes and sequence analysis.

A gene probe, CAP-21, which demonstrated interrelationships between the members of the Mycoplasma mycoides cluster was developed. The probe easily differentiated mycoplasmas in this cluster by clear and predictable hybridization patterns in Southern blots and separated the cluster into four groups. Strains of M. mycoides subsp. mycoides which were capable of causing contagious bovine pleuropneumonia composed one group. Strains of M. mycoides subsp. mycoides which did not cause contagious bovine pleuropneumonia together with strains of M. mycoides subsp. capri composed the second group. Mycoplasma capricolum and the F38 mycoplasmas formed a third group, while the bovine group 7 mycoplasmas composed a separate, fourth group. Further support for the above grouping of the cluster was obtained when amplified DNA analogous to the probe from one representative strain of each of the cluster members was sequenced and these data were used to construct a phylogenic tree. Contagious caprine pleuropneumonia is recognized as an important disease, and the etiological agent of this disease is now known to be the F38 mycoplasma. The CAP-21 probe did not differentiate between M. capricolum and the closely related F38 mycoplasma. A second probe, F38-12, which was capable of distinguishing these two mycoplasmas was made.