Attenuation of a Pathogenic Mycoplasma Strain by Modification of the obg Gene by Using Synthetic Biology Approaches.

Mycoplasma species are responsible for several economically significant livestock diseases for which there is a need for new and improved vaccines. Most of the existing mycoplasma vaccines are attenuated strains that have been empirically obtained by serial passages or by chemical mutagenesis. The recent development of synthetic biology approaches has opened the way for the engineering of live mycoplasma vaccines. Using these tools, the essential GTPase-encoding gene obg was modified directly on the Mycoplasma mycoides subsp. capri genome cloned in yeast, reproducing mutations suspected to induce a temperature-sensitive (TS+) phenotype. After transplantation of modified genomes into a recipient cell, the phenotype of the resulting M. mycoides subsp. capri mutants was characterized. Single-point obg mutations did not result in a strong TS+ phenotype in M. mycoides subsp. capri, but a clone presenting three obg mutations was shown to grow with difficulty at temperatures of ≥40°C. This particular mutant was then tested in a caprine septicemia model of M. mycoides subsp. capri infection. Five out of eight goats infected with the parental strain had to be euthanized, in contrast to one out of eight goats infected with the obg mutant, demonstrating an attenuation of virulence in the mutant. Moreover, the strain isolated from the euthanized animal in the group infected with the obg mutant was shown to carry a reversion in the obg gene associated with the loss of the TS+ phenotype. This study demonstrates the feasibility of building attenuated strains of mycoplasma that could contribute to the design of novel vaccines with improved safety.IMPORTANCE Animal diseases due to mycoplasmas are a major cause of morbidity and mortality associated with economic losses for farmers all over the world. Currently used mycoplasma vaccines exhibit several drawbacks, including low efficacy, short time of protection, adverse reactions, and difficulty in differentiating infected from vaccinated animals. Therefore, there is a need for improved vaccines to control animal mycoplasmoses. Here, we used genome engineering tools derived from synthetic biology approaches to produce targeted mutations in the essential GTPase-encoding obg gene of Mycoplasma mycoides subsp. capri Some of the resulting mutants exhibited a marked temperature-sensitive phenotype. The virulence of one of the obg mutants was evaluated in a caprine septicemia model and found to be strongly reduced. Although the obg mutant reverted to a virulent phenotype in one infected animal, we believe that these results contribute to a strategy that should help in building new vaccines against animal mycoplasmoses.

In vivo role of capsular polysaccharide in Mycoplasma mycoides.

Capsular polysaccharides have been confirmed to be an important virulence trait in many gram-positive and gram-negative bacteria. Similarly, they are proposed to be virulence traits in minimal Mycoplasma that cause disease in humans and animals. In the current study, goats were infected with the caprine pathogen Mycoplasma mycoides subsp. capri or an engineered mutant lacking the capsular polysaccharide, galactofuranose. Goats infected with the mutant strain showed only transient fever. In contrast, 5 of 8 goats infected with the parental strain reached end-point criteria after infection. These findings confirm that galactofuranose is a virulence factor in M. mycoides.

Analysis of immune responses to recombinant proteins from strains of Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia.

Current contagious bovine pleuropneumonia (CBPP) vaccines are based on live-attenuated strains of Mycoplasma mycoides subsp. mycoides (Mmm). These vaccines have shortcomings in terms of efficacy, duration of immunity and in some cases show severe side effects at the inoculation site; hence the need to develop new vaccines to combat the disease. Reverse vaccinology approaches were used and identified 66 candidate Mycoplasma proteins using available Mmm genome data. These proteins were ranked by their ability to be recognized by serum from CBPP-positive cattle and thereafter used to inoculate naïve cattle. We report here the inoculation of cattle with recombinant proteins and the subsequent humoral and T-cell-mediated immune responses to these proteins and conclude that a subset of these proteins are candidate molecules for recombinant protein-based subunit vaccines for CBPP control.

Comparison of in vivo and in vitro properties of capsulated and noncapsulated variants of Mycoplasma mycoides subsp. mycoides strain Afadé: a potential new insight into the biology of contagious bovine pleuropneumonia.

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.

Development of an improved vaccine for contagious bovine pleuropneumonia: an African perspective on challenges and proposed actions.

Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides (Mmm) is an economically very important cattle disease in sub-Saharan Africa. CBPP impacts animal health and poverty of livestock-dependent people through decreased animal productivity, reduced food supply, and the cost of control measures. CBPP is a barrier to trade in many African countries and this reduces the value of livestock and the income of many value chain stakeholders. The presence of CBPP also poses a constant threat to CBPP-free countries and creates costs in terms of the measures necessary to ensure the exclusion of disease. This opinion focuses on the biomedical research needed to foster the development of better control measures for CBPP. We suggest that different vaccine development approaches are followed in parallel. Basic immunology studies and systematic OMICs studies will be necessary in order to identify the protective arms of immunity and to shed more light on the pathogenicity mechanisms in CBPP. Moreover a robust challenge model and a close collaboration with African research units will be crucial to foster and implement a new vaccine for the progressive control of this cattle plague.

Cattle immunized against the pathogenic L-α-glycerol-3-phosphate oxidase of Mycoplasma mycoides subs. mycoides fail to generate neutralizing antibodies and succumb to disease on challenge.

The membrane-associated enzyme L-α-glycerol-3-phosphate oxidase (GlpO) of Mycoplasma mycoides subs. mycoides (Mmm), the causal agent of contagious bovine pleuropneumonia (CBPP) has been identified as a virulence factor responsible for the release of toxic by-products such as H2O2 that mediate host cell injury. Since CBPP pathogenesis is based on host inflammatory reactions, we have determined the capacity of recombinant GlpO to generate in vivo protective responses against challenge in immunized cattle. We also investigated whether sera raised against recombinant GlpO in cattle and mice inhibit production of H2O2 by Mmm. Immunization of cattle with recombinant GlpO did not protect against challenge with a virulent strain of Mmm. Further, although both murine and bovine antisera raised against recombinant GlpO detected recombinant and native forms of GlpO in immunoblot assays with similar titres, only murine antibodies could neutralize GlpO enzymatic function. The data raise the possibility that Mmm has adapted to evade potential detrimental antibody responses in its definitive host.

Evaluation of antimicrobial activity against Mycoplasma mycoides subsp. mycoides Small Colony using an in vitro dynamic dilution pharmacokinetic/pharmacodynamic model.

The objectives of this study were to assess the activity of oxytetracycline (OTC), danofloxacin and tulathromycin against Mycoplasma mycoides subsp. mycoides Small Colony, the causative agent of contagious bovine pleuropneumonia, in an in vitro dynamic concentration model and to determine the concentration and/or time dependence of such activity. Time-kill assays that simulated elimination of antimicrobials from the body were performed. Initial antimicrobial concentrations corresponded to various multiples of the MIC and cultures were diluted in a stepwise fashion with either drug-free or drug-containing artificial medium to mimic administration by single-release bolus or infusion, respectively. Where appropriate, data were fitted to sigmoidal E(max) models. OTC produced no change in mycoplasma titre from the initial inoculum size, regardless of the concentration or means of drug exposure. Both danofloxacin and tulathromycin resulted in a decrease in mycoplasma titre but neither was bactericidal (99.9 % kill) over 12 h. A greater antimycoplasmal effect, defined as the change in log(10) (c.f.u. ml(-1)) over 12 h, was achieved when danofloxacin was administered as a single-release bolus, suggesting concentration-dependent activity, whereas the antimycoplasmal effect of tulathromycin was comparable following administration by single-release bolus or infusion, owing to its long half-life.

Disruption of the S41 peptidase gene in mycoplasma mycoides capri impacts proteome profile, H(2)O(2) production, and sensitivity to heat shock.

Members of the Mycoplasma mycoides cluster are among the most virulent of the mycoplasmas, causing worldwide economically significant diseases of cattle and goats. A distinguishing phenotype among the members of the cluster is the ability to degrade casein. The MMCAP2_0241 gene, an S41 peptidase, confers the proteolytic phenotype in Mycoplasma mycoides subsp. capri GM12. In order to determine the impact of disruption of the gene, we used differential proteome profiling to compare the M. mycoides subsp. capri wild type with a mutant lacking the proteolytic phenotype. Disruption of MMCAP2_0241 resulted in altered phenotypes reminiscent of M. mycoides subsp. mycoides SC and had significant impacts on the proteome profile of the microbe. The mutant exhibited increased production of hydrogen peroxide, decreased lactate dehydrogenase activity, and increased sensitivity to heat shock.

Detecção do grupo mycoplasma mycoides por imunoperoxidase indireta (IPI) e PCR-REA em conduto auditivo de bovinos / Detection of mycoplasma mycoides cluster by indirect immunoperoxidase (IPI) and PCR-REA in the ear canal of bovines

O Grupo Mycoplasma mycoides (GMM) foi diagnosticado por PCR-REA e imunoperoxidase indireta (IPI) em amostras de lavados de conduto auditivo de bovinos no Estado do Rio de Janeiro, Brasil. 60 bovinos foram selecionados aleatoriamente. As lavagens foram feitas com uso de seringas estéreis contendo um volume de 60 mL de solução salina tamponada (PBS pH 7.2). As amostras obtidas foram estocadas em glicerol (1:2) e congeladas a 20ºC até uso. Estas amostras foram diluídas até 10-5 e repicadas em meio Hayflick modificado, sólido e líquido, sendo incubados a 37ºC por 48-72 horas. As placas foram mantidas em microaerofilia e observadas diariamente, para visualização das colônias típicas em ôovo-fritoõ. Das 60 amostras cultivadas, 48 (80,00 por cento) foram positivas para Mycoplasma spp. A prevalência obtida para o GMM na IPI foi de 20,0 por cento (12/60) enquanto na PCR-REA foi de 41,7 por cento (25/60). Das cepas tipificadas pela IPI 58,3 por cento (7/12) foram M. mycoides subsp. mycoides LC e 41,7 por cento (5/12) foram M. capricolum. Na PCR-REA o grupo M. mycoides foi confirmado pela visualização de um amplicon de 785bp, compatível com este grupo. O valor encontrado no teste Kappa para associação entre estes testes foi de 0,14 (P>0,05. Na clivagem do produto da PCR com a enzima de restrição AluI, de cepas de referências e dos isolados de ouvido os fragmentos obtidos foram de 81, 98, 186 e 236pb, mas não de 370pb, que é específica para o agente da Pleuropneumonia Contagiosa Bovina. A presença de espécies de micoplasmas no conduto auditivo de bovinos assintomáticos representa um risco para propagação de Mycoplasma spp. entre rebanhos bovinos no Brasil.

Mycoplasma mycoides cluster (MMC) was diagnosed by polimerase chain reaction-restriction endonuclease analysis (PCR-REA) and indirect immunoperoxidase (IPI), both, carried out in flushing from external ear canal, collected from bovine at slaughter time in the State of Rio de Janeiro, southeastern, Brazil. A total of 60 bovines were randomly selected. Sterile syringes (60mL) loaded with buffer solution (PBS, pH 7.2) were used for the ear canal flushing. The obtained samples were stored in glycerol (1:2) and frozen at -20ºC until use. These specimens were diluted up to 10-5, inoculated in liquid and solid modified Hayflickïs media and incubated at 37ºC for 2-3 days. The plates were kept in a microaerophilia condition and examined every two days under a stereomicroscope for the presence of typical colonies ôfried-eggõ. In this study, 35 strains selected in agreement with their biochemistry and physiologic proprieties, were used. From the 60 cultivated samples, 48 (80.00 percent) were positive for Mycoplasma spp. Under IPI the prevalence obtained for MMC was 20.0 percent (12/60) while by PCR-REA it was 41.7 percent (25/60). The IPI typing of these isolates resulted in 58.3 percent (7/12) for M.mycoides mycoides LC and 41.7 percent (5/12) for M. capricolum. PCR-REA for MMC was confirmed by the amplicon size of 785bp, compatible with this group. The Kappa value for the association between these two tests was 0.14 (p>0.05). After restriction analysis with AluI in all MMC strains the fragments size obtained were of 81, 98, 186 and 236bp, but not of 370bp that is compatible with Mycoides mycoides mycoides SC of bovine type. The presence of mycoplasmas species in the ear canal of asymptomatic bovines represent a risk of subsequent propagation of Mycoplasma spp. among bovine herds in Brazil.

Mycoplasma leachii sp. nov. as a new species designation for Mycoplasma sp. bovine group 7 of Leach, and reclassification of Mycoplasma mycoides subsp. mycoides LC as a serovar of Mycoplasma mycoides subsp. capri.

The Mycoplasma mycoides cluster consists of six pathogenic mycoplasmas causing disease in ruminants, which share many genotypic and phenotypic traits. The M. mycoides cluster comprises five recognized taxa: Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), M. mycoides subsp. mycoides Large Colony (MmmLC), M. mycoides subsp. capri (Mmc), Mycoplasma capricolum subsp. capricolum (Mcc) and M. capricolum subsp. capripneumoniae (Mccp). The group of strains known as Mycoplasma sp. bovine group 7 of Leach (MBG7) has remained unassigned, due to conflicting data obtained by different classification methods. In the present paper, all available data, including recent phylogenetic analyses, have been reviewed, resulting in a proposal for an emended taxonomy of this cluster: (i) the MBG7 strains, although related phylogenetically to M. capricolum, hold sufficient characteristic traits to be assigned as a separate species, i.e. Mycoplasma leachii sp. nov. (type strain, PG50(T) = N29(T) = NCTC 10133(T) = DSM 21131(T)); (ii) MmmLC and Mmc, which can only be distinguished by serological methods and are related more distantly to MmmSC, should be combined into a single subspecies, i.e. Mycoplasma mycoides subsp. capri, leaving M. mycoides subsp. mycoides (MmmSC) as the exclusive designation for the agent of contagious bovine pleuropneumonia. A taxonomic description of M. leachii sp. nov. and emended descriptions of M. mycoides subsp. mycoides and M. mycoides subsp. capri are presented. As a result of these emendments, the M. mycoides cluster will hereafter be composed of five taxa comprising three subclusters, which correspond to the M. mycoides subspecies, the M. capricolum subspecies and the novel species M. leachii.

Experimental infection of goats with an unusual strain of Mycoplasma mycoides subsp. capri isolated in Jordan.

Goats were infected experimentally with a mycoplasma (the "Irbid" strain) isolated previously from a goat with contagious agalactia in northern Jordan. The strain was unusual in that, although it had been identified by molecular methods as Mycoplasma mycoides subsp. mycoides LC/Mycoplasma mycoides subsp. capri, it showed no inhibition of growth by any of the hyperimmune rabbit antisera conventionally used to speciate members of the Mycoplasma mycoides cluster. Animals were infected either intratracheally or by aerosol and placed "in-contact" with other goats. After 2 weeks, those infected intratracheally became febrile, showing a nasal discharge and slight conjunctivitis, followed a week later by respiratory distress and polyarthritis; lesions seen at necropsy included coagulative necrotic pneumonia, fibrinous pleurisy with pleural exudate, and inflammatory exudates, necrosis and fibrosis in the joints. Animals infected by aerosol showed much milder clinical signs, including nasal discharge and occasional swollen joints. In the "in-contact" goats, seroconversion was first seen after 7 weeks, accompanied by coughing and laboured respiration; lesions in this group consisted of fibrinous pneumonia with focal areas of necrosis and abundant pleural exudate.

Analysis of the immunoproteome of Mycoplasma mycoides subsp. mycoides small colony type reveals immunogenic homologues to other known virulence traits in related Mycoplasma species.

Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) has been eradicated in the developed world, but it is still present in many countries of sub-Saharan Africa. After initially successful control measures in the 1960s it has been spreading due to a lack of money, fragmentation of veterinary services, uncontrolled cattle movement, insufficient vaccine efficacy and sensitivity of current diagnostic tests. In this study we used two-dimensional polyacrylamide gel electrophoresis followed by immunoblot with sera from MmmSC-infected animals and MALDI-ToF mass spectrometry to identify novel immunogenic proteins as candidate molecules for improved diagnostics and vaccines. We identified 24 immunogens recognized by pooled sera from experimentally infected cattle. Furthermore, a serum from an animal with acute clinical disease as well as severe pathomorphological lesions recognized 13 additional immunogens indicating variation in the antibody responses to CBPP amongst cattle. Most immunogens showed compelling similarity to protein/gene sequences in the two ruminant pathogens M. capricolum subsp. capricolum and M. mycoides subsp. mycoides large colony type both belonging to the mycoides cluster. Three of these proteins, namely glycerol-3-phosphate oxidase, adenylosuccinate synthase, and glyceraldehyde-3-phosphate dehydrogenase, had no compelling homologue in the other distantly related bovine pathogen M. agalactiae. In addition, translation elongation factor Tu, heat shock protein 70, pyruvate dehydrogenase, and FKBP-type peptidyl-prolyl isomerase, which have been found to mediate adhesion to host tissue in other mycoplasmas were shown to be expressed and recognized by sera. These proteins have potential for the development of improved diagnostic tests and possibly vaccines.

Cytotoxicity of Mycoplasma mycoides subsp. mycoides small colony type to bovine epithelial cells.

The cytotoxicities of various strains of Mycoplasma mycoides subsp. mycoides small colony type (SC), the agent of contagious bovine pleuropneumonia (CBPP), were measured in vitro using embryonic calf nasal epithelial (ECaNEp) cells. Strains isolated from acute cases of CBPP induced high cytotoxicity in the presence of glycerol, concomitant with the release of large amounts of toxic H2O2 that were found to be translocated into the cytoplasms of the host cells by close contact of the Mycoplasma strains with the host cells. Currently used vaccine strains also showed high cytotoxicity and high H2O2 release, indicating that they are attenuated in another virulence attribute. Strains isolated from recent European outbreaks of CBPP with mild clinical signs, which are characterized by a defect in the glycerol uptake system, released small amounts of H2O2 and showed low cytotoxicity to ECaNEp cells. M. mycoides subsp. mycoides SC strain PG1 released large amounts of H2O2 but was only slightly cytotoxic. PG1 was found to have a reduced capacity to bind to ECaNEp cells and was unable to translocate H2O2 into the bovine cells, in contrast to virulent strains that release large amounts of H2O2. Thus, an efficient translocation of H2O2 into host cells is a prerequisite for the cytotoxic effect and requires an intact adhesion mechanism to ensure a close contact between mycoplasmas and host cells.

Beta-D-glucoside utilization by Mycoplasma mycoides subsp. mycoides SC: possible involvement in the control of cytotoxicity towards bovine lung cells.

BACKGROUND: Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC) is among the most serious threats for livestock producers in Africa. Glycerol metabolism-associated H2O2 production seems to play a crucial role in virulence of this mycoplasma. A wide number of attenuated strains of M. mycoides subsp. mycoides SC are currently used in Africa as live vaccines. Glycerol metabolism is not affected in these vaccine strains and therefore it does not seem to be the determinant of their attenuation. A non-synonymous single nucleotide polymorphism (SNP) in the bgl gene coding for the 6-phospho-beta-glucosidase (Bgl) has been described recently. The SNP differentiates virulent African strains isolated from outbreaks with severe CBPP, which express the Bgl isoform Val204, from strains to be considered less virulent isolated from CBPP outbreaks with low mortality and vaccine strains, which express the Bgl isoform Ala204. RESULTS: Strains of M. mycoides subsp. mycoides SC considered virulent and possessing the Bgl isoform Val204, but not strains with the Bgl isoform Ala204, do trigger elevated levels of damage to embryonic bovine lung (EBL) cells upon incubation with the disaccharides (i.e., beta-D-glucosides) sucrose and lactose. However, strains expressing the Bgl isoform Val204 show a lower hydrolysing activity on the chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside (pNPbG) when compared to strains that possess the Bgl isoform Ala204. Defective activity of Bgl in M. mycoides subsp. mycoides SC does not lead to H2O2 production. Rather, the viability during addition of beta-D-glucosides in medium-free buffers is higher for strains harbouring the Bgl isoform Val204 than for those with the isoform Ala204. CONCLUSION: Our results indicate that the studied SNP in the bgl gene is one possible cause of the difference in bacterial virulence among strains of M. mycoides subsp. mycoides SC. Bgl does not act as a direct virulence factor, but strains possessing the Bgl isoform Val204 with low hydrolysing activity are more prone to survive in environments that contain high levels of beta-D-glucosides, thus contributing in some extent to mycoplasmaemia.

[Contagious bovine pleuropneumonia--a nearly forgotten disease?]. / Die Lungenseuche der Rinder-- eine fast vergessene Erkrankung?

Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) is a notifiable disease and has to be reported to the World Organisation for Animal Health (Office International des Epizooties, OIE, http://www.oie.int). Despite the fact that the last reported cases in Germany date back to 1926, the risk of introduction through clinically inconspicuous animals from countries where the disease is still endemic is rising. This is due mainly to an increase in international trade of live cattle and the failure to contain CBPP in many parts of Africa and elsewhere. To detect and eliminate this highly contagious infectious disease of the bovine respiratory tract, it is necessary to recognize matching clinical symptoms as soon as possible, as well as to have efficient methods for its detection on hand. In the present paper, we describe clinical manifestations and review state-of-the-art research, as well as currently used detection methods.

Molecular mechanisms of pathogenicity of Mycoplasma mycoides subsp. mycoides SC.

Mycoplasma mycoides subsp. mycoides SC, the aetiological agent of contagious bovine pleuropneumonia (CBPP), is considered the most pathogenic of the Mycoplasma species. Its virulence is probably the result of a coordinated action of various components of an antigenically and functionally dynamic surface architecture. The different virulence attributes allow the pathogen to evade the host's immune defence, adhere tightly to the host cell surface, persist and disseminate in the host causing mycoplasmaemia, efficiently import energetically valuable nutrients present in the environment, and release and simultaneously translocate toxic metabolic pathway products to the host cell where they cause cytotoxic effects that are known to induce inflammatory processes and disease. This strategy enables the mycoplasma to exploit the minimal genetic information in its small genome, not only to fulfil the basic functions for its replication but also to damage host cells in intimate proximity thereby acquiring the necessary bio-molecules, such as amino acids and nucleic acid precursors, for its own biosynthesis and survival.

Viable Mycoplasma mycoides ssp. mycoides small colony-mediated depression of the bovine cell responsiveness to the mitogen concanavalin A.

Mycoplasma mycoides ssp. mycoides biotype Small Colony (MmmSC) is the causative agent of contagious bovine pleuropneumonia (CBPP), which is still a major tropical cattle disease. Development of an efficient vaccine requires an understanding of the immunopathology of CBPP as MmmSC presents a strong ability to escape the host immune response. The objective of this study was to determine whether the presence of MmmSC can modulate the immune response induced by the mitogen Concanavalin A (ConA) on bovine immune cells [peripheral blood mononuclear cells (PBMC) and lymph node (LN) cells]. Comparative analysis of the immunomodulating properties of viable versus heat-killed MmmSC on ConA-stimulated immune cells revealed that while heat-killed MmmSC had no effect, viable MmmSC strongly depressed, in a concentration-dependent manner, the ConA mitogenic activity (blastogenesis and interferon-gamma production). Both B-cell and T-cell activation were affected with the highest impact on the CD4 T cells. The phenotypic analysis showed that the ConA-induced proliferation of CD25(+) cells was strongly reduced when co-exposed to viable MmmSC, confirming that events associated with ConA-induced cell activation were suppressed by the pathogen. This study thus demonstrated that viable MmmSC is able to inhibit the polyclonal mitogenic activity of the ConA on bovine PBMC and LN cells. This finding strongly suggests that the persistence of viable MmmSC may also thus inhibit the bovine immune response directed towards inactivated MmmSC, whether dead or in the form of antigens, also present during infection. This study confirmed that MmmSC has evolved an efficient mechanism to prevent its elimination from the host.

Genomic differences between type strain PG1 and field strains of Mycoplasma mycoides subsp. mycoides small-colony type.

The recently accomplished complete genomic sequence analysis of the type strain PG1 of Mycoplasma mycoides subsp. mycoides small-colony type revealed four large repeated segments of 24, 13, 12, and 8 kb that are flanked by insertion sequence (IS) elements. Genetic analysis of type strain PG1 and African, European, and Australian field and vaccine strains revealed that the 24-kb genetic locus is repeated only in PG1 and not in other M. mycoides subsp. mycoides SC strains. In contrast, the 13-kb genetic locus was found duplicated in some strains originating from Africa and Australia but not in strains that were isolated from the European outbreaks. The 12- and 8-kb genetic loci were found in two and three copies, respectively, in all 28 strains analyzed. The flanking IS elements are assumed to lead to these tandem duplications, thus contributing to genomic plasticity. This aspect must be considered when designing novel diagnostic approaches and recombinant vaccines.

Mycoplasma mycoides ssp. mycoides biotype small colony-secreted components induce apoptotic cell death in bovine leucocytes.

Contagious bovine pleuropneumonia, caused by Mycoplasma mycoides ssp. mycoides biotype small colony (MmmSC), is one of the most serious cattle diseases in Africa. Several observations suggested that MmmSC had evolved an efficient way to escape the bovine immune responses by triggering host-cell cytotoxicity. This study was implemented to determine whether the cytotoxic effect was due to apoptotic cell death. To that end, bovine blood cells were cultured for up to 3 days in the presence of viable or heat-killed MmmSC compared to unstimulated cultures. The findings provided evidence for a viable MmmSC-induced, time-dependent apoptosis in bovine blood leucocytes, whereas heat-killed MmmSC had no effect. Morphological and physiological changes (evidenced by TUNEL and annexin V staining) typical of apoptosis were observed in response to viable MmmSC. All the lymphocyte subsets as well as the monocyte/granulocyte subset exhibited extensive apoptosis after exposure to viable MmmSC. Our results demonstrated a potential role for MmmSC-secreted components as pathogenic factors able to induce programmed cell death in bovine blood leucocytes.

A metabolic enzyme as a primary virulence factor of Mycoplasma mycoides subsp. mycoides small colony.

During evolution, pathogenic bacteria have developed complex interactions with their hosts. This has frequently involved the acquisition of virulence factors on pathogenicity islands, plasmids, transposons, or prophages, allowing them to colonize, survive, and replicate within the host. In contrast, Mycoplasma species, the smallest self-replicating organisms, have regressively evolved from gram-positive bacteria by reduction of the genome to a minimal size, with the consequence that they have economized their genetic resources. Hence, pathogenic Mycoplasma species lack typical primary virulence factors such as toxins, cytolysins, and invasins. Consequently, little is known how pathogenic Mycoplasma species cause host cell damage, inflammation, and disease. Here we identify a novel primary virulence determinant in Mycoplasma mycoides subsp. mycoides Small Colony (SC), which causes host cell injury. This virulence factor, released in significant amounts in the presence of glycerol in the growth medium, consists of toxic by-products such as H2O2 formed by l-alpha-glycerophosphate oxidase (GlpO), a membrane-located enzyme that is involved in the metabolism of glycerol. When embryonic calf nasal epithelial cells are infected with M. mycoides subsp. mycoides SC in the presence of physiological amounts of glycerol, H2O2 is released inside the cells prior to cell death. This process can be inhibited with monospecific anti-GlpO antibodies.