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Abstract Background Contagious agalactia (CA) of sheep and goats caused by Mycoplasma agalactiae is a widely occurring economically important disease that is difficult to control. The ELISA is commonly used for the serological detection of CA but it has some limitations and the performance of the available tests have not been properly evaluated. Two commercial ELISA kits are widely used, one involving a fusion protein as target antigen and the other a total antigen. The objectives were to compare these tests by evaluating: i. Their diagnostic sensitivity and specificity, the relevance of the recommended cut-off points, the correlation between the two tests, and, the correlation between serology data and the milk shedding of M. agalatiae; ii. The influence of extrinsic factors such as the targeted animal species, geographical origin of the samples, intra-specific variability of M. agalactiae and concurrent mycoplasma infections. A sample of 5900 animals from 211 farms with continuous CA monitoring for 20 years and no prior vaccination history was used. The infection status was known from prior bacteriological, epidemiological and serological monitoring with a complementary immunoblotting test. Results The average diagnostic sensitivity was 56% [51.8–59.8] for the fusion protein ELISA and 84% [81.3–87.2] for the total antigen ELISA, with noteworthy flock-related variations. The average diagnostic specificity for the fusion protein ELISA was 100% [99.9–100], and for the total antigen ELISA differed significantly between goats and sheep: 99.3% [97.4–99.9] and 95.7% [93.8–97.2] respectively. Experimental inoculations with different M. agalactiae strains revealed that the ELISA kits poorly detected the antibody response to certain strains. Furthermore, test performances varied according to the host species or geographical origin of the samples. Finally, the correlation between milk shedding of M. agalactiae and the presence of detectable antibodies in the blood was poor. Conclusions These serological tests are not interchangeable. The choice of a test will depend on the objectives (early detection of infection or disease control program), on the prevalence of infection and the control protocol used. Given the variety of factors that may influence performance, a preliminary assessment of the test in a given situation is recommended prior to widespread use.

The bacterial secretome comprises polypeptides expressed at the cell surface or released into the extracellular environment as well as the corresponding secretion machineries. Despite their reduced coding capacities, Mycoplasma spp. are able to produce and release several components into their environment, including polypeptides, exopolysaccharides and extracellular vesicles. Technical difficulties in purifying these elements from the complex broth media used to grow mycoplasmas have recently been overcome by optimizing growth conditions and switching to chemically defined culture media. However, the secretion pathways responsible for the release of these structurally varied elements are still poorly described in mycoplasmas. We propose the use of the term ‘releasome,’ instead of secretome, to refer to molecules released by mycoplasmas into their environment. The aim of this review is to more precisely delineate the elements that should be considered part of the mycoplasmal releasome and their role in the interplay of mycoplasmas with host cells and tissues.

Contagious agalactia is associated with mastitis, keratoconjunctivitis, arthritis, pneumonia, and septicemia in small ruminants in countries with large dairy industries worldwide. The causative agents belong to four (sub)species of the Mycoplasma genus that have remained essentially susceptible to antimicrobials, including to the widely-used tetracycline family. However, some clinical isolates have been detected that show increased minimum inhibitory concentrations of tetracyclines, although they do not harbor the mutation in the 16SrRNA gene usually associated with resistance. The present work aimed to assess whether efflux pumps, infrequently described in mycoplasmas, could participate in the observed moderate loss of susceptibility. General efflux mechanisms were measured (i) using the fluorescence property of ethidium bromide when accumulated intracellularly and intercalated in the mycoplasma genomes, its active extrusion resulting in a temperature-dependent decrease in fluorescence and (ii) monitoring the growth inhibition of mycoplasmas by subinhibitory concentrations of tetracycline with or without reserpine, a known inhibitor of efflux in other bacteria. Both methods revealed non-specific efflux phenomena in most of the isolates tested, although their efficacy was difficult to quantify. This property could contribute to the acquisition of mutations conferring resistance by maintaining intracellular concentrations of tetracyclines at subinhibitory levels.

Release of extracellular vesicles (EV) by Gram-negative and positive bacteria is being frequently reported. EV are nano-sized, membrane-derived, non-self-replicating, spherical structures shed into the extracellular environment that could play a role in bacteria-host interactions. Evidence of EV production in bacteria belonging to the class Mollicutes, which are wall-less, is mainly restricted to the genus Acholeplasma and is scanty for the Mycoplasma genus that comprises major human and animal pathogens. Here EV release by six Mycoplasma (sub)species of clinical importance was investigated. EV were obtained under nutritional stress conditions, purified by ultracentrifugation and observed by electron microscopy. The membrane proteins of EV from three different species were further identified by mass spectrometry as a preliminary approach to determining their potential role in host-pathogen interactions. EV were shown to be released by all six (sub)species although their quantities and sizes (30-220 nm) were very variable. EV purification was complicated by the minute size of viable mycoplasmal cells. The proteins of EV-membranes from three (sub)species included major components of host-pathogen interactions, suggesting that EV could contribute to make the host-pathogen interplay more complex. The process behind EV release has yet to be deciphered, although several observations demonstrated their active release from the plasma membrane of living cells. This work shed new light on old concepts of "elementary bodies" and "not-cell bound antigens".

Contagious bovine pleuropneumonia is a severe respiratory disease of cattle that is caused by a bacterium of the Mycoplasma genus, namely Mycoplasma mycoides subsp. mycoides (Mmm). In the absence of classical virulence determinants, the pathogenicity of Mmm is thought to rely on intrinsic metabolic functions and specific components of the outer cell surface. One of these latter, the capsular polysaccharide galactan has been notably demonstrated to play a role in Mmm persistence and dissemination. The free exopolysaccharides (EPS), also produced by Mmm and shown to circulate in the blood stream of infected cattle, have received little attention so far. Indeed, their characterization has been hindered by the presence of polysaccharide contaminants in the complex mycoplasma culture medium. In this study, we developed a method to produce large quantities of EPS by transfer of mycoplasma cells from their complex broth to a chemically defined medium and subsequent purification. NMR analyses revealed that the purified, free EPS had an identical β(1->6)-galactofuranosyl structure to that of capsular galactan. We then analyzed intraclonal Mmm variants that produce opaque/translucent colonies on agar. First, we demonstrated that colony opacity was related to the production of a capsule, as observed by electron microscopy. We then compared the EPS extracts and showed that the non-capsulated, translucent colony variants produced higher amounts of free EPS than the capsulated, opaque colony variants. This phenotypic variation was associated with an antigenic variation of a specific glucose phosphotransferase permease. Finally, we conducted in silico analyses of candidate polysaccharide biosynthetic pathways in order to decipher the potential link between glucose phosphotransferase permease activity and attachment/release of galactan. The co-existence of variants producing alternative forms of galactan (capsular versus free extracellular galactan) and associated with an antigenic switch constitutes a finely tuned mechanism that may be involved in virulence.

Abstract Background Mycoplasma bovis (M. bovis) is an emerging bovine pathogen, leading to significant economic losses in the livestock industry worldwide. Infection can result in a variety of clinical signs, such as arthritis, pneumonia, mastitis and keratoconjunctivitis, none of which are M. bovis-specific. Laboratory diagnosis is therefore important. Serological tests to detect M. bovis antibodies is considered an effective indicator of infection in a herd and often used as a herd test. Combined with clinical judgement, it can also be used to implement control strategies and/or to estimate the disease prevalence within a country. However, due to lack of harmonisation of approaches to testing, and serological tests used by different laboratories, comparisons of prevalence data between countries is often difficult. A network of researchers from six European countries designed and participated in an inter-laboratory trial, with the aim of evaluating the sensitivity (Se) and specificity (Sp) of two commercially available ELISA tests (ID Screen® ELISA (IDvet) and BIO K302 ELISA (BIO-X Diagnostics)) for diagnosis of M. bovis infection. Each laboratory received a blinded panel of bovine sera and tested independently, according to manufacturer’s instructions. Western blot analyses (WB) performed by one of the participating laboratories was used as a third diagnostic test in the statistical evaluation of Se and Sp values using latent class analysis. Results The Se of WB, the ID Screen® ELISA and the BIO K302 ELISA were determined to be 91.8, 93.5 and 49.1% respectively, and corresponding Sp of the three tests were 99.6, 98.6 and 89.6%, respectively. Conclusions The present study is, to our knowledge, the first to present an inter-laboratory comparison of the BIO K302 ELISA and the ID Screen® ELISA. Based on our results, the ID Screen® ELISA showed high consistency with WB and performed with higher precision and accuracy than the BIO K302 ELISA.

The genus Mycoplasma, a group of free-living, wall-less prokaryotes includes more than 100 species of which dozens are primary pathogens of humans and domesticated animals. Mycoplasma species isolated from wildlife are rarely investigated but could provide a fuller picture of the evolutionary history and diversity of this genus. In 2013 several isolates from wild Caprinae were tentatively assigned to a new species, Mycoplasma (M.) feriruminatoris sp. nov., characterized by an unusually rapid growth in vitro and close genetic proximity to ruminant pathogenic species. We suspected that atypical isolates recently collected from Alpine ibex in France belonged to this new species. The present study was undertaken to verify this hypothesis and to further characterize the French ibex isolates. Phylogenetic analyses were performed to identify the isolates and position them in trees containing several other mycoplasma species pathogenic to domesticated ruminants. Population diversity was characterized by genomic macrorestriction and by examining the capacity of different strains to produce capsular polysaccharides, a feature now known to vary amongst mycoplasma species pathogenic to ruminants. This is the first report of M. feriruminatoris isolation from Alpine ibex in France. Phylogenetic analyses further suggested that M. feriruminatoris might constitute a 4th species in a genetic cluster that so far contains only important ruminant pathogens, the so-called Mycoplasma mycoides cluster. A PCR assay for specific identification is proposed. These French isolates were not clonal, despite being collected in a restricted region of the Alps, which signifies a considerable diversity of the new species. Strains were able to concomitantly produce two types of capsular polysaccharides, β-(1→6)-galactan and β-(1→6)-glucan, with variation in their respective ratio, a feature never before described in mycoplasmas.

Spiroplasma citri is a plant-pathogenic mollicute. Recently, the so-called nonphytopathogenic S. citri mutant GMT 553 was obtained by insertion of transposon Tn4001 into the first gene of the fructose operon. Additional fructose operon mutants were produced either by gene disruption or selection of spontaneous xylitol-resistant strains. The behavior of these spiroplasma mutants in the periwinkle plants has been studied. Plants infected via leafhoppers with the wild-type strain GII-3 began to show symptoms during the first week following the insect-transmission period, and the symptoms rapidly became severe. With the fructose operon mutants, symptoms appeared only during the fourth week and remained mild, except when reversion to a fructose+ phenotype occurred. In this case, the fructose+ revertants quickly overtook the fructose¯ mutants and the symptoms soon became severe. When mutant GMT 553 was complemented with the fructose operon genes that restore fructose utilization, severe pathogenicity, similar to that of the wild-type strain, was also restored. Finally, plants infected with the wild-type strain and grown at 23°C instead of 30°C showed late symptoms, but these rapidly became severe. These results are discussed in light of the role of fructose in plants. Fructose utilization by the spiroplasmas could impair sucrose loading into the sieve tubes by the companion cells and result in accumulation of carbohydrates in source leaves and depletion of carbon sources in sink tissues.

Two hundred and fifty-seven transposon Tn4001 mutants of Spiroplasma citri strain GII3 were used for transmission assays by the leafhopper vector Circulifer haematoceps into periwinkle (Catharanthus roseus) plants. Multiplication of the mutants in the two hosts, the leafhopper and the plant, as well as the symptom expression in the plant were studied. Two mutants, GMT 470 and GMT 553, caused no symptoms on plants. Tn4001 is inserted as a single copy in the genome of these mutants. Mutant GMT 470 did not multiply, or multiplied only poorly, in the leaf-hopper and was not transmitted by the insect to the plant, nor to culture medium through Parafilm membrane. The growth rate of GMT 470 in SP4 medium was twice as slow as that of wild-type strain GII3. Mutant GMT 553 multiplied in the leafhopper as well as the wild-type spiro-plasma, and was transmitted by the leafhoppers into the plants, where it reached the same titers as the wild-type strain but in approximately twice as much time. The plants containing high titers of mutant GMT 553 remained symptomless for several weeks. However, symptoms began to develop at a time when revertants that had lost the transposon were detected.

Mycoplasmas are wall-less bacteria with many species spread across various animal hosts in which they can be pathogenic. Despite their reduced anabolic capacity, some mycoplasmas are known to secrete hetero- and homopolysaccharides, which play a role in host colonization through biofilm formation or immune evasion, for instance. This study explores how widespread the phenomenon of capsular homopolysaccharide secretion is within mycoplasmas, and investigates the diversity of both the molecules produced and the synthase-type glycosyltransferases responsible for their production. Fourteen strains representing 14 (sub)species from four types of hosts were tested in vitro for their polysaccharide secretion using both specific (immunodetection) and nonspecific (sugar dosage) assays. We evidenced a new, atypical homopolymer of β-(1 → 6)-glucofuranose (named glucofuranan) in the human pathogen Mycoplasma (M.) fermentans, as well as a β-(1 → 6)-glucopyranose polymer for the turkey pathogen M. iowae and galactan (β-(1 → 6)-galactofuranose) and β-(1 → 2)-glucopyranose for M. bovigenitalium infecting ruminants. Sequence and phylogenetic analyses revealed a huge diversity of synthases from varied Mycoplasma species. The clustering of these membrane-embedded glycosyltransferases into three main groups was only partially correlated to the structure of the produced homopolysaccharides., (© 2024 The Author(s). Molecular Microbiology published by John Wiley & Sons Ltd.)

Mycoplasma feriruminatoris is a fast-growing Mycoplasma species isolated from wild Caprinae and first described in 2013. M. feriruminatoris isolates have been associated with arthritis, kerato conjunctivitis, pneumonia and septicemia, but were also recovered from apparently healthy animals. To better understand what defines this species, we performed a genomic survey on 14 strains collected from free-ranging or zoo-housed animals between 1987 and 2017, mostly in Europe. The average chromosome size of the M. feriruminatoris strains was 1,040±0,024 kbp, with 24 % G+C and 852±31 CDS. The core genome and pan-genome of the M. feriruminatoris species contained 628 and 1312 protein families, respectively. The M. feriruminatoris strains displayed a relatively closed pan-genome, with many features and putative virulence factors shared with species from the M. mycoides cluster, including the MIB-MIP Ig cleavage system, a repertoire of DUF285 surface proteins and a complete biosynthetic pathway for galactan. M. feriruminatoris genomes were found to be mostly syntenic, although repertoires of mobile genetic elements, including Mycoplasma Integrative and Conjugative Elements, insertion sequences, and a single plasmid varied. Phylogenetic- and gene content analyses confirmed that M. feriruminatoris was closer to the M. mycoides cluster than to the ruminant species M. yeatsii and M. putrefaciens . Ancestral genome reconstruction showed that the emergence of the M. feriruminatoris species was associated with the gain of 17 gene families, some of which encode defence enzymes and surface proteins, and the loss of 25 others, some of which are involved in sugar transport and metabolism. This comparative study suggests that the M. mycoides cluster could be extended to include M. feriruminatoris . We also find evidence that the specific organization and structure of the DnaA boxes around the oriC of M. feriruminatoris may contribute to drive the remarkable fast growth of this minimal bacterium.

The bacterial secretome comprises polypeptides expressed at the cell surface or released into the extracellular environment as well as the corresponding secretion machineries. Despite their reduced coding capacities, Mycoplasma spp. are able to produce and release several components into their environment, including polypeptides, exopolysaccharides and extracellular vesicles. Technical difficulties in purifying these elements from the complex broth media used to grow mycoplasmas have recently been overcome by optimizing growth conditions and switching to chemically defined culture media. However, the secretion pathways responsible for the release of these structurally varied elements are still poorly described in mycoplasmas. We propose the use of the term 'releasome,' instead of secretome, to refer to molecules released by mycoplasmas into their environment. The aim of this review is to more precisely delineate the elements that should be considered part of the mycoplasmal releasome and their role in the interplay of mycoplasmas with host cells and tissues., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer GB is currently organizing a Research Topic with the author FT., (Copyright © 2022 Gaurivaud and Tardy.)

Contagious agalactia is associated with mastitis, keratoconjunctivitis, arthritis, pneumonia, and septicemia in small ruminants in countries with large dairy industries worldwide. The causative agents belong to four (sub)species of the Mycoplasma genus that have remained essentially susceptible to antimicrobials, including to the widely-used tetracycline family. However, some clinical isolates have been detected that show increased minimum inhibitory concentrations of tetracyclines, although they do not harbor the mutation in the 16SrRNA gene usually associated with resistance. The present work aimed to assess whether efflux pumps, infrequently described in mycoplasmas, could participate in the observed moderate loss of susceptibility. General efflux mechanisms were measured (i) using the fluorescence property of ethidium bromide when accumulated intracellularly and intercalated in the mycoplasma genomes, its active extrusion resulting in a temperature-dependent decrease in fluorescence and (ii) monitoring the growth inhibition of mycoplasmas by subinhibitory concentrations of tetracycline with or without reserpine, a known inhibitor of efflux in other bacteria. Both methods revealed non-specific efflux phenomena in most of the isolates tested, although their efficacy was difficult to quantify. This property could contribute to the acquisition of mutations conferring resistance by maintaining intracellular concentrations of tetracyclines at subinhibitory levels.

Many mycoplasma species are isolated from the ruminant lungs as either saprophytes or true pathogens. These wall-less bacteria possess a minimal genome and reduced metabolic capabilities. Accordingly, they rely heavily on their hosts for the supply of essential metabolites and, notably, peptides. Seven of 13 ruminant lung-associated Mycoplasma (sub)species were shown to possess caseinolytic activity when grown in rich media and assessed with a quantitative fluorescence test. For some species, this activity was detected in spent medium, an indication that proteases were secreted outside the mycoplasma cells. To identify these proteases, we incubated concentrated washed cell pellets in a defined medium and analyzed the supernatants by tandem mass spectrometry. Secreted-protease activity was detected mostly in the species belonging to the Mycoplasma mycoides cluster (MMC) and, to a lesser extent, in Mycoplasma bovirhinis Analyzing a Mycoplasma mycoides subsp. capri strain, chosen as a model, we identified 35 expressed proteases among 55 predicted coding genes, of which 5 were preferentially found in the supernatant. Serine protease S41, acquired by horizontal gene transfer, was responsible for the caseinolytic activity, as demonstrated by zymography and mutant analysis. In an M. capricolum mutant, inactivation of the S41 protease resulted in marked modification of the expression or secretion of 17 predicted surface-exposed proteins. This is an indication that the S41 protease could have a role in posttranslational cleavage of surface-exposed proteins and ectodomain shedding, whose physiological impacts still need to be explored. IMPORTANCE Few studies pertaining to proteases in ruminant mycoplasmas have been reported. Here, we focus on proteases that are secreted outside the mycoplasma cell using a mass spectrometry approach. The most striking result is the identification, within the Mycoplasma mycoides cluster, of a serine protease that is exclusively detected outside the mycoplasma cells and is responsible for casein digestion. This protease may also be involved in the posttranslational processing of surface proteins, as suggested by analysis of mutants showing a marked reduction in the secretion of extracellular proteins. By analogy, this finding may help increase understanding of the mechanisms underlying this ectodomain shedding in other mycoplasma species. The gene encoding this protease is likely to have been acquired via horizontal gene transfer from Gram-positive bacteria and sortase-associated surface proteases. Whether this protease and the associated ectodomain shedding are related to virulence has yet to be ascertained., (Copyright © 2019 American Society for Microbiology.)

Release of extracellular vesicles (EV) by Gram-negative and positive bacteria is being frequently reported. EV are nano-sized, membrane-derived, non-self-replicating, spherical structures shed into the extracellular environment that could play a role in bacteria-host interactions. Evidence of EV production in bacteria belonging to the class Mollicutes, which are wall-less, is mainly restricted to the genus Acholeplasma and is scanty for the Mycoplasma genus that comprises major human and animal pathogens. Here EV release by six Mycoplasma (sub)species of clinical importance was investigated. EV were obtained under nutritional stress conditions, purified by ultracentrifugation and observed by electron microscopy. The membrane proteins of EV from three different species were further identified by mass spectrometry as a preliminary approach to determining their potential role in host-pathogen interactions. EV were shown to be released by all six (sub)species although their quantities and sizes (30-220 nm) were very variable. EV purification was complicated by the minute size of viable mycoplasmal cells. The proteins of EV-membranes from three (sub)species included major components of host-pathogen interactions, suggesting that EV could contribute to make the host-pathogen interplay more complex. The process behind EV release has yet to be deciphered, although several observations demonstrated their active release from the plasma membrane of living cells. This work shed new light on old concepts of "elementary bodies" and "not-cell bound antigens"., Competing Interests: The authors have declared that no competing interests exist.

The genus Mycoplasma , a group of free-living, wall-less prokaryotes includes more than 100 species of which dozens are primary pathogens of humans and domesticated animals. Mycoplasma species isolated from wildlife are rarely investigated but could provide a fuller picture of the evolutionary history and diversity of this genus. In 2013 several isolates from wild Caprinae were tentatively assigned to a new species, Mycoplasma ( M.) feriruminatoris sp. nov., characterized by an unusually rapid growth in vitro and close genetic proximity to ruminant pathogenic species. We suspected that atypical isolates recently collected from Alpine ibex in France belonged to this new species. The present study was undertaken to verify this hypothesis and to further characterize the French ibex isolates. Phylogenetic analyses were performed to identify the isolates and position them in trees containing several other mycoplasma species pathogenic to domesticated ruminants. Population diversity was characterized by genomic macrorestriction and by examining the capacity of different strains to produce capsular polysaccharides, a feature now known to vary amongst mycoplasma species pathogenic to ruminants. This is the first report of M. feriruminatoris isolation from Alpine ibex in France. Phylogenetic analyses further suggested that M. feriruminatoris might constitute a 4th species in a genetic cluster that so far contains only important ruminant pathogens, the so-called Mycoplasma mycoides cluster. A PCR assay for specific identification is proposed. These French isolates were not clonal, despite being collected in a restricted region of the Alps, which signifies a considerable diversity of the new species. Strains were able to concomitantly produce two types of capsular polysaccharides, β-(1→6)-galactan and β-(1→6)-glucan, with variation in their respective ratio, a feature never before described in mycoplasmas.

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Mycoplasma mycoides subsp. mycoides (Mmm) strain Afadé had previously been shown to undergo spontaneous phase variations between an opaque capsulated variant and a translucent (TR) variant devoid of a capsule but able to secrete cell-free exopolysaccharides. This phase variation is associated with an ON/OFF genetic switch in a glucose permease gene. In this study, in vivo and in vitro assays were conducted to compare the virulence of the two variants and their abilities to resist host defence. Capsulated variants were shown, in a mouse model, to induce longer bacteraemia that was correlated with better serum resistance in vitro. In contrast, TR variants displayed better ability to adhere to an inert support, linked to the absence of a capsule, changes in cell surface hydrophobicity and increased resistance to antimicrobial peptide and hydrogen peroxide. The switch from one variant population to another, which was observed both in vivo and in vitro under stress conditions, is further discussed as a means for Mmm to modulate its interactions with animal hosts during different stages of the disease., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Contagious bovine pleuropneumonia is a severe respiratory disease of cattle that is caused by a bacterium of the Mycoplasma genus, namely Mycoplasma mycoides subsp. mycoides (Mmm). In the absence of classical virulence determinants, the pathogenicity of Mmm is thought to rely on intrinsic metabolic functions and specific components of the outer cell surface. One of these latter, the capsular polysaccharide galactan has been notably demonstrated to play a role in Mmm persistence and dissemination. The free exopolysaccharides (EPS), also produced by Mmm and shown to circulate in the blood stream of infected cattle, have received little attention so far. Indeed, their characterization has been hindered by the presence of polysaccharide contaminants in the complex mycoplasma culture medium. In this study, we developed a method to produce large quantities of EPS by transfer of mycoplasma cells from their complex broth to a chemically defined medium and subsequent purification. NMR analyses revealed that the purified, free EPS had an identical β(1->6)-galactofuranosyl structure to that of capsular galactan. We then analyzed intraclonal Mmm variants that produce opaque/translucent colonies on agar. First, we demonstrated that colony opacity was related to the production of a capsule, as observed by electron microscopy. We then compared the EPS extracts and showed that the non-capsulated, translucent colony variants produced higher amounts of free EPS than the capsulated, opaque colony variants. This phenotypic variation was associated with an antigenic variation of a specific glucose phosphotransferase permease. Finally, we conducted in silico analyses of candidate polysaccharide biosynthetic pathways in order to decipher the potential link between glucose phosphotransferase permease activity and attachment/release of galactan. The co-existence of variants producing alternative forms of galactan (capsular versus free extracellular galactan) and associated with an antigenic switch constitutes a finely tuned mechanism that may be involved in virulence.

We report here the draft genome sequences of Mycoplasma auris and Mycoplasma yeatsii, two species commonly isolated from the external ear canal of Caprinae.

We report here the draft genome sequences of Mycoplasma alkalescens, Mycoplasma arginini, and Mycoplasma bovigenitalium. These three species are regularly isolated from bovine clinical specimens, although their role in disease is unclear.

Mycoplasma putrefaciens is one of the etiologic agents of contagious agalactia in goats. We report herein the complete genome sequence of Mycoplasma putrefaciens strain 9231.

Contagious bovine pleuropneumonia (CBPP) is a severe respiratory disease of cattle and buffalo caused by Mycoplasma mycoides subsp. mycoides "Small Colony" (MmmSC). The agent of CBPP has been isolated from goats in different countries including CBPP-free areas. Goats can therefore be regarded as a putative MmmSC reservoir. No diagnostic test for CBPP surveillance in goats has been proposed as yet. Furthermore, serological tests could be seriously hampered by a widespread caprine infection due to the subspecies M. mycoides subsp. capri (Mmc), which is antigenically very close to MmmSC and displays high levels of genetic variability. A competition ELISA (cELISA) is currently used to screen for CBPP in cattle at the herd level in infected areas. The aim of this study was to see if the same cELISA would be specific enough to be used to screen goats despite the potential concomitant infection with Mmc. The cELISA titers of goats from Mmc-infected and non-infected herds were comparable and negative using the accepted cutoff for bovine sera. In contrast, seroconversion was observed in goats experimentally inoculated with an Mmc strain that cross-reacted with a monoclonal antibody targeting the same epitope as that used in cELISA. The probability of such false positivity occurring under field conditions is very low since Mmc strains with such an atypical antigenic profile emerge only rarely as a result of random nucleotide variation of the epitope-coding region. In conclusion, the commercially available cELISA can be considered specific enough to be used as a primary test to monitor passage of the CBPP agent in goats, but its sensitivity in goats requires further investigation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The assembly of 20,000 sequencing reads obtained from shotgun and chromosome-specific libraries of the Spiroplasma citri genome yielded 77 chromosomal contigs totaling 1,674 kbp (92%) of the 1,820-kbp chromosome. The largest chromosomal contigs were positioned on the physical and genetic maps constructed from pulsed-field gel electrophoresis and Southern blot hybridizations. Thirty-eight contigs were annotated, resulting in 1,908 predicted coding sequences (CDS) representing an overall coding density of only 74%. Cellular processes, cell metabolism, and structural-element CDS account for 29% of the coding capacity, CDS of external origin such as viruses and mobile elements account for 24% of the coding capacity, and CDS of unknown function account for 47% of the coding capacity. Among these, 21% of the CDS group into 63 paralog families. The organization of these paralogs into conserved blocks suggests that they represent potential mobile units. Phage-related sequences were particularly abundant and include plectrovirus SpV1 and SVGII3 and lambda-like SpV2 sequences. Sixty-nine copies of transposases belonging to four insertion sequence (IS) families (IS30, IS481, IS3, and ISNCY) were detected. Similarity analyses showed that 21% of chromosomal CDS were truncated compared to their bacterial orthologs. Transmembrane domains, including signal peptides, were predicted for 599 CDS, of which 58 were putative lipoproteins. S. citri has a Sec-dependent protein export pathway. Eighty-four CDS were assigned to transport, such as phosphoenolpyruvate phosphotransferase systems (PTS), the ATP binding cassette (ABC), and other transporters. Besides glycolytic and ATP synthesis pathways, it is noteworthy that S. citri possesses a nearly complete pathway for the biosynthesis of a terpenoid.

Xylella fastidiosa causes citrus variegated chlorosis (CVC), a destructive disease of citrus. Xylella fastidiosa forms a biofilm inside plants and insect vectors. Biofilms are complex structures involving X. fastidiosa cells and an extracellular matrix which blocks water and nutrient transport in diseased plants. It is hypothesized that the matrix might be composed of an extracellular polysaccharide (EPS), coded by a cluster of nine genes closely related to the xanthan gum operon of Xanthomonas campestris pv. campestris. To understand the role of X. fastidiosa gum genes on biofilm formation and EPS biosynthesis, we produced gumB and gumF mutants. Xylella fastidiosa mutants were obtained by insertional duplication mutagenesis and recovered after triply cloning the cells. Xylella fastidiosa gumB and gumF mutants exhibited normal cell characteristics; typical colony morphology and EPS biosynthesis were not altered. It was of note that X. fastidiosa mutants showed a reduced capacity to form biofilm when BCYE was used as the sustaining medium, a difference not observed with PW medium. Unlike X. campestris pv. campestris, the expression of the X. fastidiosa gumB or gumF genes was not regulated by glucose.