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1.  A survey of Mycoplasma agalactiae in dairy sheep farms in Spain 
Background
Contagious Agalactia (CA) is one of the major animal health problems in small ruminants because of its economic significance. Currently, four Mycoplasma spp. have been associated with this syndrome: M. agalactiae, M. mycoides subsp. capri, M. capricolum subsp. capricolum and M. putrefaciens. Their presence has been evaluated in several studies conducted in CA-endemic countries. However, previous Spanish studies have been focused on caprine CA, and there is a knowledge gap regarding which Mycoplasma species are present in sheep flocks from Spain, which has the second highest number of sheep amongst the 27 European Union member states. Consequently, we investigated the presence and geographic distribution of the four CA-causing mycoplasmas in Spanish dairy sheep farms. This is the first time such an investigation has been performed.
Results
Three hundred thirty nine out of 922 sheep flocks were positive for M. agalactiae by real time PCR (36.8%) and 85 by microbiological identification (9.2%). Interestingly, all 597 milk samples assessed for the presence of M. mycoides subsp. capri, M. capricolum subsp. capricolum and M. putrefaciens tested negative. To evaluate the intermittent excretion of the pathogen in milk, we sampled 391 additional farms from 2 to 5 times, resulting that in 26.3% of the cases a previously positive farm tested negative in a later sampling.
Conclusions
M. agalactiae was the only Mycoplasma species detected in the study area showing a high frequency of presence and wide distribution. Therefore, the establishment of a permanent surveillance network is advantageous, as well as the implementation of control and prevention measures to hinder the dissemination of M. agalactiae and to prevent the entrance of other Mycoplasma species.
doi:10.1186/1746-6148-8-171
PMCID: PMC3514350  PMID: 23006445
Mycoplasma agalactiae; Contagious agalactia; Real time PCR; Sheep; Dairy; Spain
2.  Mycoplasma agalactiae Secretion of β-(1→6)-Glucan, a Rare Polysaccharide in Prokaryotes, Is Governed by High-Frequency Phase Variation 
Applied and Environmental Microbiology  2016;82(11):3370-3383.
ABSTRACT
Mycoplasmas are minimal, wall-less bacteria but have retained the ability to secrete complex carbohydrate polymers that constitute a glycocalyx. In members of the Mycoplasma mycoides cluster, which are important ruminant pathogens, the glycocalyx includes both cell-attached and cell-free polysaccharides. This report explores the potential secretion of polysaccharides by M. agalactiae, another ruminant pathogen that belongs to a distant phylogenetic group. Comparative genomic analyses showed that M. agalactiae possesses all the genes required for polysaccharide secretion. Notably, a putative synthase gene (gsmA) was identified, by in silico reconstruction of the biosynthetic pathway, that could be involved in both polymerization and export of the carbohydrate polymers. M. agalactiae polysaccharides were then purified in vitro and found to be mainly cell attached, with a linear β-(1→6)-glucopyranose structure [β-(1→6)-glucan]. Secretion of β-(1→6)-glucan was further shown to rely on the presence of a functional gsmA gene, whose expression is subjected to high-frequency phase variation. This event is governed by the spontaneous intraclonal variation in length of a poly(G) tract located in the gsmA coding sequence and was shown to occur in most of the M. agalactiae clinical isolates tested in this study. M. agalactiae susceptibility to serum-killing activity appeared to be dictated by ON/OFF switching of β-(1→6)-glucan secretion, suggesting a role of this phenomenon in survival of the pathogen when it invades the host bloodstream. Finally, β-(1→6)-glucan secretion was not restricted to M. agalactiae but was detected also in M. mycoides subsp. capri PG3T, another pathogen of small ruminants.
IMPORTANCE Many if not all bacteria are able to secrete polysaccharides, either attached to the cell surface or exported unbound into the extracellular environment. Both types of polysaccharides can play a role in bacterium-host interactions. Mycoplasmas are no exception despite their poor overall metabolic capacity. We showed here that M. agalactiae secretes a capsular β-(1→6)-glucopyranose thanks to a specific glycosyltransferase with synthase activity. This secretion is governed by high-frequency ON/OFF phase variation that might be crucial in mycoplasma host dissemination, as cell-attached β-(1→6)-glucopyranose increases serum-killing susceptibility. Our results provide functional genetic data about mycoplasmal glycosyltransferases with dual functions, i.e., assembly and export of the sugar polymers across the cell membrane. Furthermore, we demonstrated that nonprotein epitopes can be subjected to surface antigenic variation in mycoplasmas. Finally, the present report contributes to unravel the role of secreted polysaccharides in the virulence and pathogenicity of these peculiar bacteria.
doi:10.1128/AEM.00274-16
PMCID: PMC4959233  PMID: 27037120
3.  Variable Lipoprotein Genes of Mycoplasma agalactiae Are Activated In Vivo by Promoter Addition via Site-Specific DNA Inversions  
Infection and Immunity  2003;71(7):3821-3830.
Mycoplasma agalactiae, the etiological agent of contagious agalactia of small ruminants, has a family of related genes (avg genes) which encode surface lipoprotein antigens that undergo phase variation. A series of 13 M. agalactiae clonal isolates, obtained from one chronically infected animal over a period of 7 months, were found to undergo major rearrangement events within the avg genomic locus. We show that these rearrangements regulate the phase-variable expression of individual avg genes. Northern blot analysis and reverse transcription-PCR showed that only one avg gene is transcribed, while the other avg genes are transcriptionally silent. Sequence analysis and primer extension experiments with two M. agalactiae clonal isolates showed that a specific 182-bp avg 5′ upstream region (avg-B2) that is present as a single chromosomal copy serves as an active promoter and exhibits a high level of homology with the vsp promoter of the bovine pathogen Mycoplasma bovis. PCR analysis showed that each avg gene is associated with the avg-B2 promoter in a subpopulation of cells that is present in each subclone. Multiple sequence-specific sites for DNA recombination (vis-like), which are presumably recognized by site-specific recombinase, were identified within the conserved avg 5′ upstream regions of all avg genes and were found to be identical to the recombination sites of the M. bovis vsp locus. In addition, a gene encoding a member of the integrase family of tyrosine site-specific recombinases was identified adjacent to the variable avg locus. The molecular genetic basis for avg phase-variable expression appears to be mediated by site-specific DNA inversions occurring in vivo that allow activation of a silent avg gene by promoter addition. A model for the control of avg genes is proposed.
doi:10.1128/IAI.71.7.3821-3830.2003
PMCID: PMC162021  PMID: 12819065
4.  Being Pathogenic, Plastic, and Sexual while Living with a Nearly Minimal Bacterial Genome 
PLoS Genetics  2007;3(5):e75.
Mycoplasmas are commonly described as the simplest self-replicating organisms, whose evolution was mainly characterized by genome downsizing with a proposed evolutionary scenario similar to that of obligate intracellular bacteria such as insect endosymbionts. Thus far, analysis of mycoplasma genomes indicates a low level of horizontal gene transfer (HGT) implying that DNA acquisition is strongly limited in these minimal bacteria. In this study, the genome of the ruminant pathogen Mycoplasma agalactiae was sequenced. Comparative genomic data and phylogenetic tree reconstruction revealed that ∼18% of its small genome (877,438 bp) has undergone HGT with the phylogenetically distinct mycoides cluster, which is composed of significant ruminant pathogens. HGT involves genes often found as clusters, several of which encode lipoproteins that usually play an important role in mycoplasma–host interaction. A decayed form of a conjugative element also described in a member of the mycoides cluster was found in the M. agalactiae genome, suggesting that HGT may have occurred by mobilizing a related genetic element. The possibility of HGT events among other mycoplasmas was evaluated with the available sequenced genomes. Our data indicate marginal levels of HGT among Mycoplasma species except for those described above and, to a lesser extent, for those observed in between the two bird pathogens, M. gallisepticum and M. synoviae. This first description of large-scale HGT among mycoplasmas sharing the same ecological niche challenges the generally accepted evolutionary scenario in which gene loss is the main driving force of mycoplasma evolution. The latter clearly differs from that of other bacteria with small genomes, particularly obligate intracellular bacteria that are isolated within host cells. Consequently, mycoplasmas are not only able to subvert complex hosts but presumably have retained sexual competence, a trait that may prevent them from genome stasis and contribute to adaptation to new hosts.
Author Summary
Mycoplasmas are cell wall–lacking prokaryotes that evolved from ancestors common to Gram-positive bacteria by way of massive losses of genetic material. With their minimal genome, mycoplasmas are considered to be the simplest free-living organisms, yet several species are successful pathogens of man and animal. In this study, we challenged the commonly accepted view in which mycoplasma evolution is driven only by genome down-sizing. Indeed, we showed that a significant amount of genes underwent horizontal transfer among different mycoplasma species that share the same ruminant hosts. In these species, the occurrence of a genetic element that can promote DNA transfer via cell-to-cell contact suggests that some mycoplasmas may have retained or acquired sexual competence. Transferred genes were found to encode proteins that are likely to be associated with mycoplasma–host interactions. Sharing genetic resources via horizontal gene transfer may provide mycoplasmas with a means for adapting to new niches or to new hosts and for avoiding irreversible genome erosion.
doi:10.1371/journal.pgen.0030075
PMCID: PMC1868952  PMID: 17511520
5.  Identification and Subtyping of Clinically Relevant Human and Ruminant Mycoplasmas by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry 
Journal of Clinical Microbiology  2013;51(10):3314-3323.
Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) recently emerged as a technology for the identification of bacteria. In this study, we aimed to evaluate its applicability to human and ruminant mycoplasmal identification, which can be demanding and time-consuming when using phenotypic or molecular methods. In addition, MALDI-TOF MS was tested as a subtyping tool for certain species. A total of 29 main spectra (MSP) from 10 human and 13 ruminant mycoplasma (sub)species were included in a mycoplasma MSP database to complete the Bruker MALDI Biotyper database. After broth culture and protein extraction, MALDI-TOF MS was applied for the identification of 119 human and 143 ruminant clinical isolates that were previously identified by antigenic or molecular methods and for subcultures of 73 ruminant clinical specimens that potentially contained several mycoplasma species. MALDI-TOF MS resulted in accurate (sub)species-level identification with a score of ≥1.700 for 96% (251/262) of the isolates. The phylogenetically closest (sub)species were unequivocally distinguished. Although mixtures of the strains were reliably detected up to a certain cellular ratio, only the predominant species was identified from the cultures of polymicrobial clinical specimens. For typing purposes, MALDI-TOF MS proved to cluster Mycoplasma bovis and Mycoplasma agalactiae isolates by their year of isolation and genome profiles, respectively, and Mycoplasma pneumoniae isolates by their adhesin P1 type. In conclusion, MALDI-TOF MS is a rapid, reliable, and cost-effective method for the routine identification of high-density growing mycoplasmal species and shows promising prospects for its capacity for strain typing.
doi:10.1128/JCM.01573-13
PMCID: PMC3811644  PMID: 23903545
6.  Emergence of Atypical Mycoplasma agalactiae Strains Harboring a New Prophage and Associated with an Alpine Wild Ungulate Mortality Episode 
Applied and Environmental Microbiology  2012;78(13):4659-4668.
The bacterium Mycoplasma agalactiae is responsible for contagious agalactia (CA) in small domestic ruminants, a syndrome listed by the World Organization for Animal Health and responsible for severe damage to the dairy industry. Recently, we frequently isolated this pathogen from lung lesions of ibexes during a mortality episode in the French Alps. This situation was unusual in terms of host specificity and tissue tropism, raising the question of M. agalactiae emergence in wildlife. To address this issue, the ibex isolates were characterized using a combination of approaches that included antigenic profiles, molecular typing, optical mapping, and whole-genome sequencing. Genome analyses showed the presence of a new, large prophage containing 35 coding sequences (CDS) that was detected in most but not all ibex strains and has a homolog in Mycoplasma conjunctivae, a species causing keratoconjunctivitis in wild ungulates. This and the presence in all strains of large integrated conjugative elements suggested highly dynamic genomes. Nevertheless, M. agalactiae strains circulating in the ibex population were shown to be highly related, most likely originating from a single parental clone that has also spread to another wild ungulate species of the same geographical area, the chamois. These strains clearly differ from strains described in Europe so far, including those found nearby, before CA eradication a few years ago. While M. agalactiae pathogenicity in ibexes remains unclear, our data showed the emergence of atypical strains in Alpine wild ungulates, raising the question of a role for the wild fauna as a potential reservoir of pathogenic mycoplasmas.
doi:10.1128/AEM.00332-12
PMCID: PMC3370481  PMID: 22522685
7.  The Origin of the ‘Mycoplasma mycoides Cluster’ Coincides with Domestication of Ruminants 
PLoS ONE  2012;7(4):e36150.
The ‘Mycoplasma mycoides cluster’ comprises the ruminant pathogens Mycoplasma mycoides subsp. mycoides the causative agent of contagious bovine pleuropneumonia (CBPP), Mycoplasma capricolum subsp. capripneumoniae the agent of contagious caprine pleuropneumonia (CCPP), Mycoplasma capricolum subsp. capricolum, Mycoplasma leachii and Mycoplasma mycoides subsp. capri. CBPP and CCPP are major livestock diseases and impact the agricultural sector especially in developing countries through reduced food-supply and international trade restrictions. In addition, these diseases are a threat to disease-free countries. We used a multilocus sequence typing (MLST) approach to gain insights into the demographic history of and phylogenetic relationships among the members of the ‘M. mycoides cluster’. We collected partial sequences from seven housekeeping genes representing a total of 3,816 base pairs from 118 strains within this cluster, and five strains isolated from wild Caprinae. Strikingly, the origin of the ‘M. mycoides cluster’ dates to about 10,000 years ago, suggesting that the establishment and spread of the cluster coincided with livestock domestication. In addition, we show that hybridization and recombination may be important factors in the evolutionary history of the cluster.
doi:10.1371/journal.pone.0036150
PMCID: PMC3338596  PMID: 22558362
8.  Highly Dynamic Genomic Loci Drive the Synthesis of Two Types of Capsular or Secreted Polysaccharides within the Mycoplasma mycoides Cluster 
Mycoplasmas of the Mycoplasma mycoides cluster are all ruminant pathogens. Mycoplasma mycoides subsp. mycoides is responsible for contagious bovine pleuropneumonia and is known to produce capsular polysaccharide (CPS) and exopolysaccharide (EPS). Previous studies have strongly suggested a role for Mycoplasma mycoides subsp. mycoides polysaccharides in pathogenicity. Mycoplasma mycoides subsp. mycoides-secreted EPS was recently characterized as a β(1→6)-galactofuranose homopolymer (galactan) identical to the capsular product. Here, we extended the characterization of secreted polysaccharides to all other members of the M. mycoides cluster: M. capricolum subsp. capripneumoniae, M. capricolum subsp. capricolum, M. leachii, and M. mycoides subsp. capri (including the LC and Capri serovars). Extracted EPS was characterized by nuclear magnetic resonance, resulting in the identification of a homopolymer of β(1→2)-glucopyranose (glucan) in M. capricolum subsp. capripneumoniae and M. leachii. Monoclonal antibodies specific for this glucan and for the Mycoplasma mycoides subsp. mycoides-secreted galactan were used to detect the two polysaccharides. While M. mycoides subsp. capri strains of serovar LC produced only capsular galactan, no polysaccharide could be detected in strains of serovar Capri. All strains of M. capricolum subsp. capripneumoniae and M. leachii produced glucan CPS and EPS, whereas glucan production and localization varied among M. capricolum subsp. capricolum strains. Genes associated with polysaccharide synthesis and forming a biosynthetic pathway were predicted in all cluster members. These genes were organized in clusters within two loci representing genetic variability hot spots. Phylogenetic analysis showed that some of these genes, notably galE and glf, were acquired via horizontal gene transfer. These findings call for a reassessment of the specificity of the serological tests based on mycoplasma polysaccharides.
doi:10.1128/AEM.02892-14
PMCID: PMC4277593  PMID: 25398856
9.  Mycoplasma Taxonomy Studied by Electrophoresis of Cell Proteins 
Journal of Bacteriology  1968;96(3):687-694.
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.
Images
PMCID: PMC252360  PMID: 5732504
10.  The indirect hemagglutination test for the detection of antibodies in cattle naturally infected mycoplasmas. 
Stable mycoplasma antigens for the indirect hemagglutination test (IHA) were prepared employing glutaraldehyde treated sheep erythrocytes sensitized with Mycoplasma agalactiae subsp. bovis and Mycoplasma bovigenitalium antigens. Employing these antigens mycoplasma antibodies were detected in sera from cattle which had mastitic symptoms due to natural infection with either M. agalactiae subsp. bovis or M. bovigenitalium. A total of 200 cows from four herds were examined at varying intervals for the presence of M. agalactiae subsp. bovis and for the detection of antibody using growth inhibition and IHA tests. Mycoplasmas were isolated from 37 animals. Growth inhibiting antibody was detected from 56 of the 200 animals. In the IHA tests, antibody titer greater than or equal to 1:80 were detected in 148 animals, 76 of these having antibody titers greater than or equal to 1:160, while sera of 116 normal control animals had no growth inhibiting antibody and none had IHA antibody titers greater than 1:40. M. bovigenitalium was isolated from the milk of three of 26 animals in a fifth herd during an outbreak of mastitis. Growth inhibiting antibodies were demonstrated in the sera of ten of the 26 animals. However, the IHA test detected antibody titers of greater than or equal to 1:160 in 13 animals and of 1:80 in one of the 26 animals. To determine the specificity of the IHA tests, M. agalactiae subsp. bovis and M. bovigenitalium antigens were reacted with rabbit hyperimmune typing sera produced against 12 species of bovine mycoplasmatales. Homologous antisera showed IHA antibody titers of 1:1280 and 1:2560 against M. agalactiae subsp. bovis and M. bovigenitalium respectively, whereas heterologous antisera showed IHA antibody titers of less than or equal to 1:20. Also eight type-specific bovine antisera were reacted with M agalactiae subsp. bovis and M. bovigenitalium antigens in homologous and heterologous tests. Homoogous reactions showed IHA antibody titers greater than or equal to 1:320, whereas heterologous reactions showed IHA titers of less than or equal to 1:20. This IHA test promises to be useful for the detection of bovine mycoplasma antibodies in sera from cattle infected with M. agalactiae subsp. bovis or M. bovigenitalium. Thes test is sensitive, reproducible and specific and the technique is relatively simple and rapid. The antigens were stable for at least seven months.
PMCID: PMC1277514  PMID: 1000374
11.  Further studies on caprine and ovine mycoplasmas related to Mycoplasma mycoides subsp. mycoides 
The Journal of Hygiene  1980;85(2):247-256.
Nine caprine and ovine mycoplasma strains, said to be indistinguishable serologically from Mycoplasma mycoides subsp. mycoides (the causative organism of contagious bovine pleuropneumonia; CBPP) were examined in mice by (1) a mycoplasmaemia test, and (2) a cross-protection test. Of the nine strains, two from goats belonged to a small colony (SC) type; four caprine and three ovine strains belonged to a large colony (LC) type.
The two SC strains — like a single SC strain examined in an earlier study — were indistinguishable from genuine M. mycoides subsp. mycoides as isolated from CBPP. They produced mycoplasmaemia readily. In a cross-protection test, the two SC strains and a CBPP strain immunized completely against each other.
Of the seven LC strains, six — like six LC strains examined in an earlier study — were easily distinguished from genuine M. mycoides subsp. mycoides; except for one that was not tested, all were shown to lack the ability to produce mycoplasmaemia readily. In cross-protection tests all six strains immunized partially but not completely against a CBPP strain.
The seventh LC strain (Mankefår 2833) was exceptional: it produced mycoplasmaemia readily, resembling the SC strains in this respect. Like other LC strains, in cross-protection tests it protected only partially against a CBPP strain. Strain Mankefår 2833 was isolated in ca. 1965 by Brack from a Barbary sheep (Ammotragus lervia) in a German zoo.
The ability of Mankefår 2833 to produce mycoplasmaemia enabled it to be used as a challenge strain in cross-protection tests. For the purpose of such tests the collection of nine mycoplasma strains referred to above was augmented with six LC strains from an earlier study. Partial but not complete protection against Mankefår 2833 was produced by two caprine SC strains, one CBPP strain, and nine LC strains. Three further LC strains gave protection that may have been as strong as that produced by the homologous strain, but confirmatory experiments are needed. A strain of M. mycoides subsp. capri gave no protection against Mankefår 2833.
PMCID: PMC2133930  PMID: 7005327
12.  Sheep primary cells as in vitro models to investigate Mycoplasma agalactiae host cell interactions 
Pathogens and Disease  2015;73(7):ftv048.
Appropriate infection models are imperative for the understanding of pathogens like mycoplasmas that are known for their strict host and tissue specificity, and lack of suitable cell and small animal models has hindered pathogenicity studies. This is particularly true for the economically important group of ruminant mycoplasmas whose virulence factors need to be elucidated for designing effective intervention strategies. Mycoplasma agalactiae serves as a useful role model especially because it is phylogenetically very close to M. bovis and causes similar symptoms by as yet unknown mechanisms. Here, we successfully prepared and characterized four different primary sheep cell lines, namely the epithelial and stromal cells from the mammary gland and uterus, respectively. Using immunohistochemistry, we identified vimentin and cytokeratin as specific markers to confirm the typical cell phenotypes of these primary cells. Furthermore, M. agalactiae’s consistent adhesion and invasion into these primary cells proves the reliability of these cell models. Mimicking natural infections, mammary epithelial and stromal cells showed higher invasion and adhesion rates compared to the uterine cells as also seen via double immunofluorescence staining. Altogether, we have generated promising in vitro cell models to study host–pathogen interactions of M. agalactiae and related ruminant pathogens in a more authentic manner.
The study is an important step forward in developing in vitro models that will facilitate analyses of Mycoplasma agalactiae and related ruminant mycoplasmas' host–pathogen interactions at the molecular level.
Graphical Abstract Figure.The study is an important step forward in developing in vitro models that will facilitate analyses of Mycoplasma agalactiae and related ruminant mycoplasmas' host–pathogen interactions at the molecular level.
doi:10.1093/femspd/ftv048
PMCID: PMC4535462  PMID: 26187893
invasion; adhesion; primary cell culture; immunohistochemistry; ruminant mycoplasmosis; host cell interactions
13.  Suppression-Subtractive Hybridization as a Strategy To Identify Taxon-Specific Sequences within the Mycoplasma mycoides Cluster: Design and Validation of an M. capricolum subsp. capricolum-Specific PCR Assay▿  
Journal of Clinical Microbiology  2008;46(4):1307-1316.
The phylogenetically related Mycoplasma capricolum subsp. capricolum and M. mycoides subsp. mycoides biotype Large Colony are two small-ruminant pathogens involved in contagious agalactia. Their respective contributions to clinical outbreaks are not well documented, because they are difficult to differentiate with the current diagnostic techniques. In order to identify DNA sequences specific to one taxon or the other, a suppression-subtractive hybridization approach was developed. DNA fragments resulting from the reciprocal subtraction of the type strains were used as probes on a panel of M. capricolum subsp. capricolum and M. mycoides subsp. mycoides biotype Large Colony strains to assess their intrataxon specificity. Due to a high intrataxon polymorphism and important cross-reactions between taxa, a single DNA fragment was shown to be specific for M. capricolum subsp. capricolum and to be present in all M. capricolum subsp. capricolum field isolates tested in this study. A PCR assay targeting the corresponding gene (simpA51) was designed that resulted in a 560-bp amplification only in M. capricolum subsp. capricolum and in M. capricolum subsp. capripneumoniae (the etiological agent of contagious caprine pleuropneumonia). simpA51 was further improved to generate a multiplex PCR (multA51) that allows the differentiation of M. capricolum subsp. capripneumoniae from M. capricolum subsp. capricolum. Both the simpA51 and multA51 assays accurately identify M. capricolum subsp. capricolum among other mycoplasmas, including all members of the M. mycoides cluster. simpA51 and multA51 PCRs are proposed as sensitive and robust tools for the specific identification of M. capricolum subsp. capricolum and M. capricolum subsp. capripneumoniae.
doi:10.1128/JCM.01617-07
PMCID: PMC2292954  PMID: 18234866
14.  Mycoplasma mycoides, from "mycoides Small Colony" to "capri". A microevolutionary perspective 
BMC Genomics  2011;12:114.
Background
The Mycoplasma mycoides cluster consists of five species or subspecies that are ruminant pathogens. One subspecies, Mycoplasma mycoides subspecies mycoides Small Colony (MmmSC), is the causative agent of contagious bovine pleuropneumonia. Its very close relative, Mycoplasma mycoides subsp. capri (Mmc), is a more ubiquitous pathogen in small ruminants causing mastitis, arthritis, keratitis, pneumonia and septicaemia and is also found as saprophyte in the ear canal. To understand the genetics underlying these phenotypic differences, we compared the MmmSC PG1 type strain genome, which was already available, with the genome of an Mmc field strain (95010) that was sequenced in this study. We also compared the 95010 genome with the recently published genome of another Mmc strain (GM12) to evaluate Mmc strain diversity.
Results
The MmmSC PG1 genome is 1,212 kbp and that of Mmc 95010 is ca. 58 kbp shorter. Most of the sequences present in PG1 but not 95010 are highly repeated Insertion Sequences (three types of IS) and large duplicated DNA fragments. The 95010 genome contains five types of IS, present in fewer copies than in PG1, and two copies of an integrative conjugative element. These mobile genetic elements have played a key role in genome plasticity, leading to inversions of large DNA fragments. Comparison of the two genomes suggested a marked decay of the PG1 genome that seems to be correlated with a greater number of IS. The repertoire of gene families encoding surface proteins is smaller in PG1. Several genes involved in polysaccharide metabolism and protein degradation are also absent from, or degraded in, PG1.
Conclusions
The genome of MmmSC PG1 is larger than that of Mmc 95010, its very close relative, but has less coding capacity. This is the result of large genetic rearrangements due to mobile elements that have also led to marked gene decay. This is consistent with a non-adaptative genomic complexity theory, allowing duplications or pseudogenes to be maintained in the absence of adaptive selection that would lead to purifying selection and genome streamlining over longer evolutionary times. These findings also suggest that MmmSC only recently adapted to its bovine host.
doi:10.1186/1471-2164-12-114
PMCID: PMC3053259  PMID: 21324191
15.  Selected ethno-medicinal plants from Kenya with in vitro activity against major African livestock pathogens belonging to the “Mycoplasma mycoides cluster” 
Journal of Ethnopharmacology  2016;192:524-534.
Ethnopharmocological relevance
Members of ‘Mycoplasma mycoides cluster’ are important ruminant pathogens in Africa. Diseases caused by these Mycoplasma negatively affect the agricultural sector especially in developing countries through losses in livestock productivity, mortality and international trade restrictions. There is therefore urgent need to develop antimicrobials from alternative sources such as medicinal plants to curb these diseases. In Kenya, smallholder farmers belonging to the Maasai, Kuria and Luo rely on traditional Kenyan herbals to treat respiratory symptoms in ruminants. In the current study extracts from some of these plants were tested against the growth of members of Mycoplasma mycoides cluster.
Aim
This study aimed at identifying plants that exhibit antimycoplasmal activities using an ethnobotanical approach.
Materials and methods
Kenyan farmers of Maasai, Luo and Kuria ethnic groups were interviewed for plant remedies given to livestock with respiratory syndromes. The plant materials were thereafter collected and crude extracts prepared using a mixture of 50% of methanol (MeOH) in dichloromethane (CH2Cl2), neat methanol (MeOH), ethanol (EtOH) and water to yield four crude extracts per plant part. The extracts were tested in vitro against five strains of Mycoplasma mycoides subsp. capri, five strains of Mycoplasma mycoides subsp. mycoides and one strain of Mycoplasma capricolum subsp capricolum using broth micro-dilution assays with an initial concentration of 1 mg/ml. Minimum inhibitory concentration (MIC) of the most active extracts were determined by serial dilution.
Results
Extracts from five plants namely: Solanum aculeastrum, Albizia coriaria, Ekebergia capensis, Piliostigma thonningii and Euclea divinorum exhibited the highest activities against the Mycoplasma strains tested. Mycoplasma mycoides subsp. mycoides were more susceptible to these extracts than Mycoplasma mycoides subsp. capri and Mycoplasma capricolum susp. capricolum. The activities of the crude extracts varied with the solvent used for extraction. The MICs mean values of the active extracts varied from 0.02 to 0.6 mg/ml.
Conclusions
The results suggested that these plants could potentially contain antimicrobial compounds that might be useful for the treatment of respiratory diseases in ruminants. Future work should focus on the isolation and identification of the active compounds from the plant extracts that showed interesting activities and evaluation of their antimicrobial and cytotoxic potential.
Graphical abstract
fx1
Highlights
•Ethno-medicinal Plants from Kenyan Flora for treating ruminants respiratory symptoms.•Solanumaculeastrum and Albiziacoriariawith highest antimycoplasmal activities.•The MICs mean values of the active extracts varied from 0.02–0.6 mg/ml.
doi:10.1016/j.jep.2016.09.034
PMCID: PMC5081062  PMID: 27649681
Mycoplasma mycoides; Ethnobotany; Antimicrobial activity; Livestock; Ethno-medicinal from plants from Kenya
17.  Distribution and diversity of mycoplasma plasmids: lessons from cryptic genetic elements 
BMC Microbiology  2012;12:257.
Background
The evolution of mycoplasmas from a common ancestor with Firmicutes has been characterized not only by genome down-sizing but also by horizontal gene transfer between mycoplasma species sharing a common host. The mechanisms of these gene transfers remain unclear because our knowledge of the mycoplasma mobile genetic elements is limited. In particular, only a few plasmids have been described within the Mycoplasma genus.
Results
We have shown that several species of ruminant mycoplasmas carry plasmids that are members of a large family of elements and replicate via a rolling-circle mechanism. All plasmids were isolated from species that either belonged or were closely related to the Mycoplasma mycoides cluster; none was from the Mycoplasma bovis-Mycoplasma agalactiae group. Twenty one plasmids were completely sequenced, named and compared with each other and with the five mycoplasma plasmids previously reported. All plasmids share similar size and genetic organization, and present a mosaic structure. A peculiar case is that of the plasmid pMyBK1 from M. yeatsii; it is larger in size and is predicted to be mobilizable. Its origin of replication and replication protein were identified. In addition, pMyBK1 derivatives were shown to replicate in various species of the M. mycoides cluster, and therefore hold considerable promise for developing gene vectors. The phylogenetic analysis of these plasmids confirms the uniqueness of pMyBK1 and indicates that the other mycoplasma plasmids cluster together, apart from the related replicons found in phytoplasmas and in species of the clade Firmicutes.
Conclusions
Our results unraveled a totally new picture of mycoplasma plasmids. Although they probably play a limited role in the gene exchanges that participate in mycoplasma evolution, they are abundant in some species. Evidence for the occurrence of frequent genetic recombination strongly suggests they are transmitted between species sharing a common host or niche.
doi:10.1186/1471-2180-12-257
PMCID: PMC3541243  PMID: 23145790
Mycoplasma,Plasmid,Replication,Rep protein,Gene transfer,Evolution,Expression vector,Mycoplasma mycoides,Mycoplasma capricolum,Mycoplasma yeatsii
18.  The Complete Genome Sequence of Mycoplasma bovis Strain Hubei-1 
PLoS ONE  2011;6(6):e20999.
Infection by Mycoplasma bovis (M. bovis) can induce diseases, such as pneumonia and otitis media in young calves and mastitis and arthritis in older animals. Here, we report the finished and annotated genome sequence of M. bovis strain Hubei-1, a strain isolated in 2008 that caused calf pneumonia on a Chinese farm. The genome of M. bovis strain Hubei-1 contains a single circular chromosome of 953,114 bp with a 29.37% GC content. We identified 803 open reading frames (ORFs) that occupy 89.5% of the genome. While 34 ORFs were Hubei-1 specific, 662 ORFs had orthologs in the M. bovis type strain PG45 genome. Genome analysis validated lateral gene transfer between M. bovis and the Mycoplasma mycoides subspecies mycoides, while phylogenetic analysis found that the closest M. bovis neighbor is Mycoplasma agalactiae. Glycerol may be the main carbon and energy source of M. bovis, and most of the biosynthesis pathways were incomplete. We report that 47 lipoproteins, 12 extracellular proteins and 18 transmembrane proteins are phase-variable and may help M. bovis escape the immune response. Besides lipoproteins and phase-variable proteins, genomic analysis found two possible pathogenicity islands, which consist of four genes and 11 genes each, and several other virulence factors including hemolysin, lipoate protein ligase, dihydrolipoamide dehydrogenase, extracellular cysteine protease and 5′-nucleotidase.
doi:10.1371/journal.pone.0020999
PMCID: PMC3120828  PMID: 21731639
19.  Prevalence of contagious mastitis pathogens in bulk tank milk in Prince Edward Island 
The Canadian Veterinary Journal  2006;47(6):567-572.
Abstract
The purpose of this study was to 1) estimate the herd prevalence of contagious mastitis pathogens in bulk milk from Prince Edward Island (PEI) dairy farms, 2) determine the association between bulk milk culture results and mean bulk milk somatic cell count (BMSCC), and 3) investigate the agreement of repeated bulk milk cultures. Three consecutive bulk milk samples were obtained at weekly intervals from all 258 PEI dairy herds and were cultured using routine laboratory methods. Cumulative prevalence of Staphylococcus aureus, Streptococcus agalactiae, and Mycoplasma spp. (M. bovis and M. alkalescens) was 74%, 1.6%, and 1.9%, respectively. Bulk milk somatic cell count of Staph. aureus-positive herds was higher than that of negative herds. Agreement for Staph. aureus isolation between 3 consecutive tests was moderate (kappa = 0.46). Mycoplasma bovis and M. alkalescens in bulk milk are being reported for the 1st time in PEI ever and in Canada since 1972.
PMCID: PMC1461414  PMID: 16808229
20.  Genetic Diversity and Population Structure of Leishmania infantum from Southeastern France: Evaluation Using Multi-Locus Microsatellite Typing 
PLoS Neglected Tropical Diseases  2016;10(1):e0004303.
In the south of France, Leishmania infantum is responsible for numerous cases of canine leishmaniasis (CanL), sporadic cases of human visceral leishmaniasis (VL) and rare cases of cutaneous and muco-cutaneous leishmaniasis (CL and MCL, respectively). Several endemic areas have been clearly identified in the south of France including the Pyrénées-Orientales, Cévennes (CE), Provence (P), Alpes-Maritimes (AM) and Corsica (CO). Within these endemic areas, the two cities of Nice (AM) and Marseille (P), which are located 150 km apart, and their surroundings, concentrate the greatest number of French autochthonous leishmaniasis cases. In this study, 270 L. infantum isolates from an extended time period (1978–2011) from four endemic areas, AM, P, CE and CO, were assessed using Multi-Locus Microsatellite Typing (MLMT). MLMT revealed a total of 121 different genotypes with 91 unique genotypes and 30 repeated genotypes. Substantial genetic diversity was found with a strong genetic differentiation between the Leishmania populations from AM and P. However, exchanges were observed between these two endemic areas in which it seems that strains spread from AM to P. The genetic differentiations in these areas suggest strong epidemiological structuring. A model-based analysis using STRUCTURE revealed two main populations: population A (consisting of samples primarily from the P and AM endemic areas with MON-1 and non-MON-1 strains) and population B consisting of only MON-1 strains essentially from the AM endemic area. For four patients, we observed several isolates from different biological samples which provided insight into disease relapse and re-infection. These findings shed light on the transmission dynamics of parasites in humans. However, further data are required to confirm this hypothesis based on a limited sample set. This study represents the most extensive population analysis of L. infantum strains using MLMT conducted in France.
Author Summary
In the south of France, the parasite Leishmania infantum is responsible for diseases that primarily affect dogs but can also impact humans. Several endemic areas have been clearly identified in the south of France including the Pyrénées-Orientales, Cévennes (CE), Provence (P), Alpes-Maritimes (AM) and Corsica (CO). In this study, 270 L. infantum isolates from four endemic areas, AM, P, CE and CO, were assessed using Multi-Locus Microsatellite Typing (MLMT), a tool applied for population genetic studies. MLMT revealed a strong genetic differentiation between the Leishmania populations from AM and P with exchanges observed between these two endemic areas. For four patients, the occurrence of disease relapses and re-infections was examined. These findings shed light on the transmission dynamics of parasites in humans. This study represents the most extensive population analysis of L. infantum isolates using MLMT conducted in France.
doi:10.1371/journal.pntd.0004303
PMCID: PMC4726517  PMID: 26808522
21.  Comparative Study of Agar Mediums for Propagation of Ruminant Mycoplasma 
A brain heart infusion agar supplemented with 16.7% rabbit serum (BHIR) was found the most suitable for the culturing of ruminant mycoplasma. Gourlay medium and Perreau medium (4, 5) were not suitable for growth of Mycoplasma mycoides var. mycoides or M. agalactiae, but were satisfactory for M. mycoides var. capri.
Four strains of M. mycoides var. mycoides, three strains of M. agalactiae and three strains of M. mycoides var. capri were grown in our laboratory.
PMCID: PMC1319756  PMID: 4266704
22.  Integrative Conjugative Elements Are Widespread in Field Isolates of Mycoplasma Species Pathogenic for Ruminants 
Comparative genomics have revealed massive horizontal gene transfer (HGT) between Mycoplasma species sharing common ruminant hosts. Further results pointed toward an integrative conjugative element (ICE) as an important contributor of HGT in the small-ruminant-pathogen Mycoplasma agalactiae. To estimate the prevalence of ICEs in ruminant mycoplasmas, we surveyed their occurrence in a collection of 166 field strains representing 4 (sub)species that are recognized as major pathogens. Based on available sequenced genomes, we first defined the conserved, minimal ICE backbone as composed of 4 coding sequences (CDSs) that are evenly distributed and predicted to be essential for ICE chromosomal integration-excision and horizontal transfer. Screening of the strain collection revealed that these 4 CDSs are well represented in ruminant Mycoplasma species, suggesting widespread occurrence of ICEs. Yet their prevalence varies within and among species, with no correlation found with the individual strain history. Extrachromosomal ICE forms were also often detected, suggesting that ICEs are able to circularize in all species, a first and essential step in ICE horizontal transfer. Examination of the junction of the circular forms and comparative sequence analysis of conserved CDSs clearly pointed toward two types of ICE, the hominis and spiroplasma types, most likely differing in their mechanism of excision-integration. Overall, our data indicate the occurrence and maintenance of functional ICEs in a large number of field isolates of ruminant mycoplasmas. These may contribute to genome plasticity and gene exchanges and, presumably, to the emergence of diverse genotypes within pathogenic mycoplasmas of veterinary importance.
doi:10.1128/AEM.03723-14
PMCID: PMC4325163  PMID: 25527550
23.  Comparative genomic and proteomic analyses of two Mycoplasma agalactiae strains: clues to the macro- and micro-events that are shaping mycoplasma diversity 
BMC Genomics  2010;11:86.
Background
While the genomic era is accumulating a tremendous amount of data, the question of how genomics can describe a bacterial species remains to be fully addressed. The recent sequencing of the genome of the Mycoplasma agalactiae type strain has challenged our general view on mycoplasmas by suggesting that these simple bacteria are able to exchange significant amount of genetic material via horizontal gene transfer. Yet, events that are shaping mycoplasma genomes and that are underlining diversity within this species have to be fully evaluated. For this purpose, we compared two strains that are representative of the genetic spectrum encountered in this species: the type strain PG2 which genome is already available and a field strain, 5632, which was fully sequenced and annotated in this study.
Results
The two genomes differ by ca. 130 kbp with that of 5632 being the largest (1006 kbp). The make up of this additional genetic material mainly corresponds (i) to mobile genetic elements and (ii) to expanded repertoire of gene families that encode putative surface proteins and display features of highly-variable systems. More specifically, three entire copies of a previously described integrative conjugative element are found in 5632 that accounts for ca. 80 kbp. Other mobile genetic elements, found in 5632 but not in PG2, are the more classical insertion sequences which are related to those found in two other ruminant pathogens, M. bovis and M. mycoides subsp. mycoides SC. In 5632, repertoires of gene families encoding surface proteins are larger due to gene duplication. Comparative proteomic analyses of the two strains indicate that the additional coding capacity of 5632 affects the overall architecture of the surface and suggests the occurrence of new phase variable systems based on single nucleotide polymorphisms.
Conclusion
Overall, comparative analyses of two M. agalactiae strains revealed a very dynamic genome which structure has been shaped by gene flow among ruminant mycoplasmas and expansion-reduction of gene repertoires encoding surface proteins, the expression of which is driven by localized genetic micro-events.
doi:10.1186/1471-2164-11-86
PMCID: PMC2824730  PMID: 20122262
24.  Phylogeny of the Mycoplasma mycoides cluster as determined by sequence analysis of the 16S rRNA genes from the two rRNA operons. 
Journal of Bacteriology  1996;178(14):4131-4142.
The so-called Mycoplasma mycoides cluster consists of six species or subspecies of mycoplasmas (Mollicutes). These species are pathogenic for ruminants and some of them are of great concern in veterinary medicine. The members of the M. mycoides cluster have two rRNA operons (rrnA and rrnB). The nucleotide sequences of the 16S rRNA genes of 10 strains, representing all of the known species and subspecies of the M. mycoides cluster, were determined by direct automated solid-phase DNA sequencing. The sequences of both rRNA operons were determined by a novel strategy involving in vitro amplification by PCR with one operon-specific primer pair and one general primer pair. Interestingly, sequence differences (polymorphisms) between the two operons were observed for all strains. Two strains of M. capricolum subsp. capripneumoniae were sequenced, and 15 polymorphisms were found in the type strain (F38) and 17 polymorphisms were found in the other strain (4/2LC). Eight polymorphisms were found in the 16S rRNA genes of the M. mycoides subsp. mycoides small-colony type, and sequence length variations in a poly(A) region were observed in the 16S rRNA genes of the two operons of this species. Secondary-structure analysis showed that polymorphisms were present in both stem and loop regions. The nucleotide substitutions in the polymorphic sites of the stem regions often resulted in a change from a canonical to a noncanonical base pairing or vice versa. A compensatory mutation was never observed in the other nucleotide of the base pair. Phylogenetic analysis based on the 16S rRNA sequences indicated that Mycoplasma sp. strain PG50 should be included in the M. capricolum species group. Furthermore, the 16S rRNA sequences of M. mycoides subsp. capri and the M. mycoides subsp. mycoides large-colony type were 99.9% identical. We therefore suggest that these species be reclassified in a common species group (for instance, "Mycoplasma capri") distinct from the M. mycoides subsp. mycoides small-colony type, which formed an intermediate branch between the M. capricolum species group and the M. capri species group.
PMCID: PMC178170  PMID: 8763941
25.  Differentiation of Mycoplasma mycoides subsp. mycoides from certain closely related caprine mycoplasmas by mycoplasmaemia and cross-protection tests in mice. 
The Journal of Hygiene  1979;82(3):407-418.
In recent years, mycoplasma taxonomists have found that numerous mycoplasma strains from goats are serologically indistinguishable from Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia (CBPP), by routinely used tests, e.g. the metabolism- and growth-inhibition tests. As a result, such organisms are now openly referred to as M. mycoides subsp. mycoides. Seven of these so-called M. mycoides subsp. mycoides strains from goats were compared with two strains of M. mycoides subsp. mycoides from CBPP, and with one strain of M. mycoides subsp. capri, by means of two in-vivo tests, namely, (1) a test of the ability of each strain, injected intraperitoneally into mice, to produce mycoplasmaemia, and (2) a cross-protection test in mice. Of the seven strains, only one ('O goat') was indistinguishable from genuine M. mycoides subsp. mycoides; it also had small colonies resembling those of genuine M. mycoides subsp. mycoides. The other six were easily distinguished from genuine M. mycoides subsp. mycoides, and they produced large colonies. These six strains and others like them should no longer be given a name that fails to distinguish them from the causative agent of CBPP. Cross-protection tests showed that the seven goat strains referred to above differed from M. mycoides subsp. capri.
PMCID: PMC2130076  PMID: 376695

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