Sequence polymorphisms within the pMGA genes and pMGA antigenic variants in Mycoplasma gallisepticum

Antigenic variants of Mycoplasma gallisepticum major surface lipoprotein, pMGA, are encoded by a large gene family. In this study sequence analyses of the PCR-amplified pMGA genes showed two types of sequences similar to the pMGA1.2 gene in M. gallisepticum strains. They differed in the sequence encoding a proline-rich region (PRR) at the N-terminus of the pMGA protein. The type A genes had sequences similar to the published pMGA1.2 gene sequence of strain S6, whereas the type B genes lacked the second repetitive segment encoding PTPN sequence within PRR and were similar to the published sequence of PG31 strain. Low in vitro passages of M. gallisepticum strains isolated recently in Slovenia from four avian species showed very different expression patterns of pMGA1.2 and pMGA1.9 genes. Among isogenic populations of S6(B) and IHB1 strains a high frequency of pMGA antigenic variants lacking an epitope for monoclonal antibody (mAb) 71 was found. Strain IHB1 clones, which synthesized pMGA recognized by mAb 71, transcribed pMGA genes whose partial sequence encoded the amino acid sequence (262)TNGDEPRSVS of the mAb 71 epitope. Other IHB1 clones synthesized pMGA variants with different isoelectric points, lacking the epitope for mAb 71, but expressing downstream epitopes for other mAbs. Our study suggests that a molecular basis for pMGA antigenic variation lies in the corresponding changes at the DNA level.     

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.     

Phylogeny of Helicobacter felis sp. nov., Helicobacter mustelae, and related bacteria.

Strain CS1T (T = type strain) is a gram-negative, microaerophilic, urease-positive, spiral-shaped bacterium that was isolated from the gastric mucosa of a cat. Additional strains which possessed biochemical and ultrastructural characteristics similar to those of strain CS1T were isolated from the gastric mucosa of cats and dogs. The guanine-plus-cytosine content of the DNA of strain CS1T was 42.5 mol%. The 16S rRNA sequences of strain CS1T, strain DS3 (a spiral-shaped isolate from a dog), and Helicobacter mustelae were determined by direct RNA sequencing, using a modified Sanger method. These sequences were compared with the 16S rRNA sequences of Helicobacter pylori, "Flexispira rappini," Wolinella succinogenes, and 11 species of campylobacters. A dendrogram was constructed based upon sequence similarities. Strains CS1T and DS3 were very closely related (level of similarity, 99.3%). Two major phylogenetic groups were formed; one group consisted of strains CS1T and DS3, H. mustelae, H. pylori, "F. rappini," and W. succinogenes, and the other group contained the true campylobacters. The average level of similarity between members of these two groups was 84.9%. Within the first group, strains CS1T and DS3, H. pylori, and H. mustelae formed a cluster of organisms with an interspecies similarity level of 94.5%. The phylogenetic positions of W. succinogenes and "F. rappini" were just outside this cluster. On the basis of the results of this study, we believe that strains CS1T (= ATCC 49179T) and DS3 represent a new species of the genus Helicobacter, for which we propose the name Helicobacter felis.     

Immune response of chimpanzees infected with human mycoplasmas

Seven chimpanzees were inoculated intra-articularly with one of three Mycoplasma species. Three chimpanzees were inoculated with clinical synovial isolate 1620, and one was inoculated with type strain PG-21 of M. hominis. The fifth animal was inoculated with clinical synovial isolate 2010B, and the sixth was inoculated with the type strain CO of Ureaplasma urealyticum. The seventh animal was inoculated with clinical isolate PI-1428 of M. pneumoniae. The synovial isolates induced intense antibody responses, antibodies recognized more antigens, and the reactive antigens were distinct from the corresponding type strains. Chimpanzees infected with synovial isolate 1620, but not type strain PG-21, recognized antigens of molecular mass 223, 208, 168, 165, 155, 150, 142, 134, 115, 105, 52, 50, 45, 38, and 36 kDa. The chimpanzee infected with synovial isolate 2010B, but not type strain CO, recognized antigens at about 277, 237, 62, 57, and 43 kDa. The major antibody reactive antigens of M. pneumoniae isolate PI-1428 migrated at about 170, 90, 56, 40, 32, and 30 kDa. The reported biologic activities of antigenic proteins derived from these Mycoplasma species are reviewed.     

Mycoplasma melaleucae sp. nov., a sterol-requiring mollicute from flowers of several tropical plants

Three sterol-requiring mollicutes from floral surfaces of two tropical plant species (Melaleuca quinquenervia and Melaleuca decora) and a single isolate from a flower of the silk oak (Grevillea robusta) were serologically indistinguishable. Strain M1T (T = type strain), isolated from Melaleuca quinquenervia, was chosen for characterization. Light and electron microscopic observations of strain M1T revealed nonhelical, nonmotile, pleomorphic coccoid cells surrounded by a single cytoplasmic membrane. No evidence of a cell wall was observed. The organism grew well in SP-4 medium, but no sustained growth occurred in conventional mycoplasma media containing horse serum. The optimum temperature for growth was 23 degrees C, but multiplication occurred over a temperature range of 10 to 30 degrees C. Growth was not observed at temperatures above 30 degrees C. Strain M1T and related strains (strains M5, M10, and SO1) catabolized glucose but hydrolyzed neither arginine nor urea. The size of the strain M1T genome was about 561 megadaltons, while the guanine-plus-cytosine content of the DNA was about 27.0 mol%. The organism was serologically unrelated to the type strains of the 80 previously recognized Mycoplasma species or to 18 other unclassified sterol-requiring strains cultivated from animal, plant, or insect sources. Recent sequencing studies of 16S rRNA demonstrated that strain M1T is a member of a clade that contains the type species of the genus Mycoplasma. Strain M1 (= ATCC 49191) is the type strain of Mycoplasma melaleucae sp. nov.     

An evaluation of PCR methods to detect strains of Mycoplasma fermentans.

A panel of 30 putative Mycoplasma fermentans strains, isolated from various sources including human, ovine and cell lines, were tested by a previously described polymerase chain reaction (PCR) to confirm their identity by amplification of a conserved 206 bp region of the insertion sequence IS1550. In addition, the application of another PCR based on the major part of the IS1550 element showed one or two products of different length (1144 and 1341 bp) enabling M. fermentans strains to be divided into two types designated as Type A and Type B. A PCR, which amplifies the macrophage activating lipopeptide gene (malp), supported the identification of all the strains as M. fermentans. Thirteen other species of Mycoplasma from human sources gave negative results in these tests, with the exception of Mycoplasma orale, which was detected by both IS1550-PCRs based on the major part and the conserved 206 bp region of the IS1550 element. This study suggests that all M. fermentans isolates possess both the IS1550 element and the malp gene. In contrast to the IS1550, the malp gene is shown to be species-specific and the use of a malp PCR described here could prove to be a useful adjunct to IS1550 detection as confirmation of the presence of M. fermentans in clinical material.     

Potential pathogens carried by Spanish ibex (Capra pyrenaica hispanica) in southern Spain

The Spanish ibex (Capra pyrenaica hispanica) population of southern Spain was surveyed for potential pathogens associated with the conjunctiva, external ear canal, as well as reproductive and upper respiratory tracts. We sampled 321 ibex (131 adult males, 100 adult females, and 90 yearlings); these included 271 apparently healthy animals and 50 that were naturally infected with Sarcoptes scabiei. A total of 688 bacterial isolates were identified (377 gram-negatives, 225 gram-positives, and 86 Mycoplasma spp.); sex, age, location, infection with S. scabiei, and disposition of the animal (free-ranging versus captive) were evaluated as risk factors for infection. Infections with Mycoplasma agalactiae and Mycoplasma arginini were associated with age, having a higher frequency of isolation in young animals. With Escherichia coli, Mannheimia haemolytica, Pasteurella multocida biotype A, and Staphylococcus aureus, significantly higher isolation rates were associated with adults. The isolation frequency for E. coli was higher in females, whereas Moraxella bovis isolations were mostly associated with males. The presence of mange increased the risk of infection with both Streptococcus equi subsp. zooepidemicus and M. haemolytica. The geographic origin of sampled animals was related to the isolation of Branhamella ovis, M. agalactiae, and all Pasteurella sp. Isolations of M. haemolytica, P. multocida biotype A, E. coli, and B. ovis were more prevalent in samples from free-ranging rather than captive animals. Of the gram-positive bacteria, S. aureus represented the predominant species isolated from nasal, vaginal, and ocular samples. Mycoplasma agalactiae and M. arginini were the predominant Mycoplasma spp., and both were associated most often with the external ear canal. The most frequently isolated gram-negative bacteria included E. coli, M. haemolytica, P. multocida biotype A, and B. ovis. Isolation rates of gram-negative species varied by source. In nasal samples, M. haemolytica and P. multocida biotype A were isolated most frequently, whereas in ocular and vaginal samples, B. ovis and E. coli, respectively, were most frequently isolated.     

Mycoplasma taxonomy studiedy electrophoresis of cell proteins

The electrophoretic patterns of cell proteins in polyacrylamide gels were used for the study of several taxonomic problems in the Mycoplasmatales. The patterns of five Mycoplasma hominis strains showed marked differences that corresponded with their known serological and nucleic acid heterogeneity. The patterns of three M. mycoides var. mycoides strains isolated in different countries were essentially identical. The electrophoretic patterns of several caprine strains resembled those of M. mycoides var. mycoides, supporting their classification as M. mycoides var. capri. Strain B3, a swine isolate, accordingly was tentatively identified as M. mycoides var. capri. The bovine mastitis strain M. agalactiae var. bovis possessed a pattern basically similar to that of the goat mastitis strain M. agalactiae, supporting the inclusion of both strains in one species. Three M. pulmonis strains isolated from rats or tissue cultures showed nearly identical patterns. The pattern of the toxigenic M. neurolyticum (Sabin A) strain resembled but was not identical with that of the nontoxigenic PG28 strain. The avian Mycoplasma species, M. gallisepticum, M. meleagridis, M. synoviae, M. gallinarum, and M. iners showed easily distinguishable and specific patterns, supporting their present classification in different species. Several improvements in the electrophoretic technique are described, and its advantages and limitations as a taxonomic tool are discussed.     

A Serologic Variant of Mycoplasma Hyorhinis Recovered from the Conjunctiva of Swine

In an examination of conjunctival samples from 40 piglets for mycoplasmas, 17 isolates were obtained. Eight could be identified as Mycoplasma hyorhinis, three as Mycoplasma flocculare, and one as Acholenlasma sp. Five strains were not readily identifiable, but together with two previously recovered strains they were found to represent a distinct serogroup. All seven strains were glucose and phosphatase positive. Incubation in a CO2-enriched atmosphere led to enhancement of the growth on solid medium. The serogroup was serologically related to M. hyorhinis, but not to a number of other glucose fermenting species of mycoplasma, and it may therefore be regarded as a new subspecies of M. hyorhinis.     

Mycoplasma phocarhinis sp. nov. and Mycoplasma phocacerebrale sp. nov., two new species from harbor seals (Phoca vitulina L.)

A total of 120 mycoplasma strains were recovered from 97 of 265 diseased seals investigated during the seal epidemic in the North Sea and in the Baltic Sea in 1988. Mycoplasmas were isolated from the respiratory tracts (including lungs), hearts, brains, and eyes of the seals. Thirty strains were filter cloned and investigated for their morphological, biochemical, and serological characteristics compared with the characteristics of previously described species. The results of an indirect immunofluorescence test, a growth inhibition test, and an immunobinding assay showed that these strains belong to two new species, for which the names Mycoplasma phocarhinis and Mycoplasma phocacerebrale are proposed. M. phocarhinis (17 strains) did not ferment glucose or hydrolyze arginine but did reduce tetrazolium chloride and potassium tellurite and produced films and spots. M. phocacerebrale (13 strains) metabolized arginine but not glucose and produced phosphatase but did not reduce tetrazolium chloride and potassium tellurite. Both species lysed sheep erythrocytes but did not absorb sheep or guinea pig erythrocytes. The type strain of M. phocarhinis is strain 852 (= ATCC 49639), and the type strain of M. phocacerebrale is strain 1049 (= ATCC 49640).     

Presence of anaplerotic reactions and transamination, and the absence of the tricarboxylic acid cycle in mollicutes

Cell extracts of the fermentative Mollicutes Acholeplasma laidlawii B-PG9, Acholeplasma morum S2, Mycoplasma capricolum 14, Mycoplasma gallisepticum S6, Mycoplasma pneumoniae FH, Mycoplasma hyopneumoniae J and M. genitalium G-37, and the non-fermentative Mycoplasma hominis PG-21, Mycoplasma hominis 1620 and Mycoplasma bovigenitalium PG-11 were examined for 39 cytoplasmic enzyme activities associated with the tricarboxylic acid (TCA) cycle, transamination, anaplerotic reactions and other enzyme activities at the pyruvate locus. Malate dehydrogenase (EC 4.2.1.2) was the only TCA-cycle-associated enzyme activity detected and it was found only in the eight Mycoplasma species. Aspartate aminotransferase (EC 2.6.1.1) activity was detected in all Mollicutes tested except M. gallisepticum S6. Malate synthetase (EC 4.1.3.2) activity, in the direction of malate formation, was found in the eight Mycoplasma species, but not in any of the Acholeplasma species. Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was detected in the direction of oxaloacetate (OAA) formation in both Acholeplasma species, but not in any of the Mycoplasma species. Pyruvate carboxylase (EC 6.4.1.1), pyruvate kinase (EC 2.7.1.40), pyruvate dehydrogenase (EC 1.2.4.1) and lactate dehydrogenase (EC 1.1.1.27) activities were found in all ten Mollicutes tested. No activities were detected in any of the ten Mollicutes for aspartase (EC 4.3.1.1), malic enzyme (EC 1.1.1.40), PEP carboxytransphosphorylase (EC 4.1.1.38), PEP carboxykinase (EC 4.1.1.32) or pyruvate orthophosphate dikinase (EC 2.7.9.1). In these TCA-cycle-deficient Mollicutes the pyruvate-OAA locus may be a point of linkage for the carbons of glycolysis, lipid synthesis, nucleic acid synthesis and certain amino acids. CO2 fixation appears obligatory in the Acholeplasma species and either CO2 fixation or malate synthesis appears obligatory in the Mycoplasma species.     

Reiterated DNA sequences defining genomic diversity within the species Mycoplasma hyorhinis

Genomic restriction fragments isolated from Mycoplasma hyorhinis and Mycoplasma hyopneumoniae were shown by DNA hybridization and nucleotide sequence analyses to contain sequences common to these two species, as well as another porcine-derived mycoplasma, Mycoplasma flocculare. Intraspecies hybridization experiments using these fragments as probes indicated that the sequence is highly redundant in several strains of M. hyorhinis, but that there is diversity in the sizes of restriction fragments detected among these strains. In contrast, repetition of the sequence was limited in M. hyopneumoniae and M. flocculare, and no homologies to this repeated element were apparent in mycoplasma species isolated from animal hosts other than the swine. The reiterated sequence may reflect intraspecies genomic diversification in M. hyorhinis and its selective presence in otherwise unrelated species raises the possibility that it has been horizontally transmitted between these organisms.     

Mycoplasma nasistruthionis sp. nov. and Mycoplasma struthionis sp. nov. isolated from ostriches with respiratory disease

Twelve Mycoplasma (M.) strains isolated from the nose, the trachea, and the lung of ostriches (Struthio camelus) displaying respiratory disease were investigated. Analysis of 16S rRNA gene sequences placed five of these strains within the M. synoviae cluster, and seven strains within the M. hominis cluster of genus Mycoplasma, which was further confirmed by analyses of the 16S-23S rRNA intergenic spacer region, and partial rpoB gene and amino acid sequences. Genomic information as well as phenotypic features obtained by matrix-assisted laser desorption ionization time of flight (MALDI-ToF) mass spectrometry analysis and serological reactions indicated that the strains examined are representatives of two hitherto unclassified species of genus Mycoplasma, for which the names Mycoplasma nasistruthionis sp. nov., with type strain 2F1A^T (= ATCC BAA-1893^T = DSM 22456^T), and Mycoplasma struthionis sp. nov., with type strain 237IA^T (= ATCC BAA-1890^T = DSM 22453^T), are proposed.     

Construction of the physical maps of Mycoplasma hyopneumoniae and Mycoplasma flocculare and the location of rRNA genes on these maps.

The genomes of Mycoplasma flocculare and Mycoplasma hyopneumoniae, two mycoplasmas of the porcine respiratory system, were studied. Based upon antigenic cross-reactivity and DNA-DNA hybridization, these species have given indication of a close genetic relationship. By using field-inversion gel electrophoresis and employing the restriction digest fragments obtained from the gels as the probes, physical maps of the genomes of the two species were constructed. Mycoplasma hyopneumoniae is similar to M. flocculare in having a single set of rRNA genes and the 5S-rRNA gene is separated from the 16S and 23S rRNA genes. Based upon the location of the rRNA genes on the physical maps in both species, the distance between the 5S and the 16S and 23S rRNA genes is at least 150 kbp. Thus, there is further evidence for the close relationship between these organisms.     

Description of Treponema azotonutricium sp. nov. and Treponema primitia sp. nov., the first spirochetes isolated from termite guts

Long after their original discovery, termite gut spirochetes were recently isolated in pure culture for the first time. They revealed metabolic capabilities hitherto unknown in the Spirochaetes division of the Bacteria, i.e., H(2) plus CO(2) acetogenesis (J. R. Leadbetter, T. M. Schmidt, J. R. Graber, and J. A. Breznak, Science 283:686-689, 1999) and dinitrogen fixation (T. G. Lilburn, K. S. Kim, N. E. Ostrom, K. R. Byzek, J. R. Leadbetter, and J. A. Breznak, Science 292:2495-2498, 2001). However, application of specific epithets to the strains isolated (Treponema strains ZAS-1, ZAS-2, and ZAS-9) was postponed pending a more complete characterization of their phenotypic properties. Here we describe the major properties of strain ZAS-9, which is readily distinguished from strains ZAS-1 and ZAS-2 by its shorter mean cell wavelength or body pitch (1.1 versus 2.3 micro m), by its nonhomoacetogenic fermentation of carbohydrates to acetate, ethanol, H(2), and CO(2), and by 7 to 8% dissimilarity between its 16S rRNA sequence and those of ZAS-1 and ZAS-2. Strain ZAS-9 is proposed as the type strain of the new species, Treponema azotonutricium. Strains ZAS-1 and ZAS-2, which are H(2)-consuming, CO(2)-reducing homoacetogens, are proposed here to be two strains of the new species Treponema primitia. Apart from the salient differences mentioned above, the genomes of all three strains were similar in size (3,461 to 3,901 kb), in G+C content (50.0 to 51.0 mol%), and in possession of 2 copies of the gene encoding 16S rRNA (rrs). For comparison, the genome of the free-living spirochete Spirochaeta aurantia strain J1 was analyzed by the same methods and found to have a size of 3,719 kb, to contain 65.6 mol% G+C, and also to possess 2 copies of the rrs gene.     

Characterization of Mycoplasma pulmonis Variants Isolated from Rabbits I. Identification and Properties of Isolates

Mycoplasma showing at least two colony types were isolated from the nares and oropharynx of New Zealand white rabbits. Two strains were purified by single-colony passages and characterized. Morphology by phase-contrast and electron microscopy was typical of Mycoplasmataceae. Both grew anaerobically as well as aerobically, caused hemolysis of guinea pig, sheep, and horse red blood cells, and fermented glucose. These characteristics are shared by members of the species M. pulmonis, commonly isolated from the respiratory tracts of laboratory rats and mice. By use of the growth-inhibition test and agar-gel double-diffusion tests, the two strains were found to be serologically related to each other and to M. pulmonis ATCC 14267 but not to other representative Mycoplasma species from man and animals.     

Screening and evaluation of probiotics as a biocontrol agent against pathogenic Vibrios in marine aquaculture

AIMS: The present work aims at finding potential probionts from marine sources as a biocontrol agent against pathogenic Vibrio species in shrimp larval culture. METHODS AND RESULTS: A total of 109 bacterial strains were isolated from seawater, sediment and marine fish-gut samples, and were screened for their antagonistic activity against Vibrio species. Three strains (Q, Q1 and M) isolated from the marine sediment were found antagonistic against Vibrio strains. Based on 16S ribosomal DNA gene sequence analysis, the strain Q was identified as Paenibacillus spp. (EF012164); Q1 as Bacillus cereus (DQ915582); and the M as Paenibacillus polymyxa (DQ915580). Further, the two bacterial species, Paenibacillus spp. and B. cereus were challenged separately at two different concentrations of 10(4) and 10(5) CFU ml(-1) for probiotic activity in the postlarvae of Penaeus monodon against pathogenic Vibrio harveyi and Vibrio spp. CONCLUSIONS: The present study identified the probiotic activity of Paenibacillus spp., B. cereus and Pa. polymyxa against the pathogenic Vibrios in the postlarvae of P. monodon. SIGNIFICANCE AND IMPACT OF THE STUDY: In vivo study reveals that the marine bacterial species can be used as probionts against pathogenic Vibrios in shrimp larval culture practices.     

First description of two moderately halophilic and psychrotolerant Mycoplasma species isolated from cephalopods and proposal of Mycoplasma marinum sp. nov. and Mycoplasma todarodis sp. nov

Two moderately halophilic and psychrotolerant new Mycoplasma species were isolated from common cephalopods. Three strains were isolated in pure culture from two individual European flying squid (Todarodes sagittatus), and two individual octopuses (Octopus vulgaris). The strains showed optimal growth at 25 °C and a salinity of 3% (w/v) NaCl. Molecular analyses revealed that the isolates belonged to two new, but phylogenetically related species, divergent from all previously described Mollicutes, representing the first marine isolates of the class, and also the first Mycoplasma strains for which NaCl requirement has been demonstrated. A genome search against all available marine metagenomes and 16S rRNA gene databases indicated that these two species represent a novel non-free-living marine lineage of Mollicutes, specifically associated with marine animals. Morphology and physiology were compatible with other members of this group, and genomic and phenotypic analyses demonstrated that these organisms represent two novel species of the genus Mycoplasma, for which the names Mycoplasma marinum sp. nov. and Mycoplasma todarodis sp. nov. are proposed; the type strains are PE^T (DSM 105487^T, CIP 111404^T) and 5H^T (DSM 105,488^T, CIP 111405^T), respectively.     

Genetic Differentiation by Nucleic Acid Homology II. Genotypic Variations Within Two Mycoplasma Species

Somerson, Norman L. (National Institutes of Health, Bethesda, Md.), Paul R. Reich, Barbara E. Walls, Robert M. Chanock, and Sherman M. Weissman. Genetic differentiation by nucleic acid homology. II. Genotypic variations within two Mycoplasma species. J. Bacteriol. 92:311-317. 1966.-A deoxyribonucleic-ribonucleic acid (DNA-RNA) homology technique was used to determine genetic relatedness among the nucleic acids of eight mycoplasmas which were serologically classified as Mycoplasma hominis type 1. The DNA preparations from these organisms were each found to be distinct. No subgrouping of the M. hominis type 1 strains could be demonstrated. In contrast, when the nucleic acids from six serologically related mycoplasmas which were isolated from tissue cultures were studied, the DNA from these species could not be distinguished. The DNA buoyant densities of the tissue culture isolates were similar. These isolates were closely related genetically to a porcine mycoplasma, M. hyorhinis.