RÉSUMÉ
Possessing only essential genes, a minimal cell can reveal mechanisms and processes that are critical for the persistence and stability of life1,2. Here we report on how an engineered minimal cell3,4 contends with the forces of evolution compared with the Mycoplasma mycoides non-minimal cell from which it was synthetically derived. Mutation rates were the highest among all reported bacteria, but were not affected by genome minimization. Genome streamlining was costly, leading to a decrease in fitness of greater than 50%, but this deficit was regained during 2,000 generations of evolution. Despite selection acting on distinct genetic targets, increases in the maximum growth rate of the synthetic cells were comparable. Moreover, when performance was assessed by relative fitness, the minimal cell evolved 39% faster than the non-minimal cell. The only apparent constraint involved the evolution of cell size. The size of the non-minimal cell increased by 80%, whereas the minimal cell remained the same. This pattern reflected epistatic effects of mutations in ftsZ, which encodes a tubulin-homologue protein that regulates cell division and morphology5,6. Our findings demonstrate that natural selection can rapidly increase the fitness of one of the simplest autonomously growing organisms. Understanding how species with small genomes overcome evolutionary challenges provides critical insights into the persistence of host-associated endosymbionts, the stability of streamlined chassis for biotechnology and the targeted refinement of synthetically engineered cells2,7-9.
Sujet(s)
Évolution moléculaire , Gènes essentiels , Génome bactérien , Mycoplasma mycoïdes , Biologie synthétique , Biotechnologie/méthodes , Biotechnologie/tendances , Division cellulaire , Génome bactérien/génétique , Mutation , Mycoplasma mycoïdes/cytologie , Mycoplasma mycoïdes/génétique , Mycoplasma mycoïdes/croissance et développement , Biologie synthétique/méthodes , Taille de la cellule , Épistasie , Sélection génétique , Aptitude génétique , Symbiose , Tubuline/composition chimiqueRÉSUMÉ
Mycoplasma mycoides subspecies mycoides (Mmm) adhesion is tissue and host specific. Inhibition of adhesion will prevent Mmm from binding to lung cells and hence prevent colonization and disease. The aim of this study was to develop a panel of Mmm monoclonal antibodies against Mmm and use these antibodies to investigate their inhibitory effect on the adherence of Mmm to bovine lung epithelial cells (BoLEC), and to further identify an antigen to any of the inhibitory antibodies. Thirteen anti-Mycoplasma mycoides subsp. mycoides (AMMY) monoclonal antibodies (mAbs) inhibited adhesion by at least 30% and ten of the mAbs bound to multiple bands on Western blots suggesting that the antibodies bound to proteins of variable sizes. AMMY 10, a previously characterized Mmm- capsular polysaccharide (CPS) specific antibody, inhibited growth of Mmm in vitro and also caused agglutination of Mmm total cell lysate. AMMY 5, a 2-oxo acid dehydrogenase acyltransferase (Catalytic domain) (MSC_0267) specific antibody, was identified and polyclonal rabbit serum against recombinant MSC_0267 blocked adhesion of Mmm to BoLEC by 41%. Antigens recognized by these antibodies could be vaccine candidate(s) and should be subsequently tested for their ability to induce a protective immune response in vivo.
Sujet(s)
Anticorps antibactériens/immunologie , Antigènes bactériens/immunologie , Adhérence bactérienne/immunologie , Mycoplasma mycoïdes/immunologie , Animaux , Anticorps monoclonaux/immunologie , Antigènes bactériens/isolement et purification , Technique de Western , Bovins , Électrophorèse sur gel de polyacrylamide , Tests au latex , Poumon/immunologie , Poumon/microbiologie , Spectrométrie de masse , Mycoplasma mycoïdes/croissance et développement , Péripneumonie contagieuse/immunologie , Péripneumonie contagieuse/microbiologieRÉSUMÉ
BACKGROUND: Mycoplasmosis is a common infection in human and veterinary medicine, and is associated with chronic inflammation and high morbidity. Mycoplasma species are often intrinsically resistant to many conventional antimicrobial therapies, and the resistance patterns of pathogenic mycoplasmas to commonly used medicinal (antimicrobial) plant extracts are currently unknown. METHODS: Aqueous extracts, ethanol extracts, or oils of the targeted plant species and colloidal silver were prepared or purchased. Activity against the wall-less bacterial pathogen Mycoplasma mycoides subsp. capri was determined and compared to activities measured against Escherichia coli and Bacillus subtilis. Antimicrobial susceptibility testing was performed by broth microdilution assays. The lethal or inhibitory nature of each extract was determined by subculture into neat growth medium. RESULTS: Growth of M. mycoides capri, E. coli, and B. subtilis was inhibited by elderberry extract, oregano oil, ethanol extract of oregano leaves, and ethanol extract of goldenseal root. No inhibition was seen with aqueous extract of astragalus or calendula oil. Growth of M. mycoides capri and B. subtilis was inhibited by ethanol extract of astragalus, whereas growth of E. coli was not. Similarly, M. mycoides capri and E. coli were inhibited by aqueous extract of thyme, but B. subtilis was unaffected. Only B. subtilis was inhibited by colloidal silver. Measured MICs ranged from 0.0003 mg/mL to 3.8 mg/mL. Bacteriostatic and bactericidal effects differed by species and extract. CONCLUSIONS: The atypical pathogen M. mycoides capri was sensitive to extracts from many medicinal plants commonly used as antimicrobials in states of preparation and concentrations currently available for purchase in the United States and Europe. Variation in bacteriostatic and bactericidal activities between species and extracts indicates that multiple effecter compounds are present in these plant species.
Sujet(s)
Antibactériens/pharmacologie , Magnoliopsida , Infections à Mycoplasma/microbiologie , Mycoplasma mycoïdes/effets des médicaments et des substances chimiques , Extraits de plantes/pharmacologie , Plantes médicinales , Animaux , Antibactériens/usage thérapeutique , Bacillus subtilis/effets des médicaments et des substances chimiques , Bacillus subtilis/génétique , Escherichia coli/effets des médicaments et des substances chimiques , Escherichia coli/croissance et développement , Humains , Tests de sensibilité microbienne , Infections à Mycoplasma/traitement médicamenteux , Mycoplasma mycoïdes/croissance et développement , Phytothérapie , Extraits de plantes/usage thérapeutique , Ruminants , Argent/pharmacologieRÉSUMÉ
BACKGROUND: Mycoplasma mycoides subspecies mycoides Small Colony (MmmSC) is the causative agent of Contagious Bovine Pleuropneumonia (CBPP), a disease of substantial economic importance in sub-Saharan Africa. Failure of vaccination to curtail spread of this disease has led to calls for evaluation of the role of antimicrobials in CBPP control. Three major classes of antimicrobial are effective against mycoplasmas, namely tetracyclines, fluoroquinolones and macrolides. Therefore, the objectives of this study were to determine the effector kinetics of oxytetracycline, danofloxacin and tulathromycin against two MmmSC field strains in artificial medium and adult bovine serum. METHODS: Minimum inhibitory concentrations (MIC) were determined for oxytetracycline, danofloxacin and tulathromycin against MmmSC strains B237 and Tan8 using a macrodilution technique, and time-kill curves were constructed for various multiples of the MIC over a 24 hour period in artificial medium and serum. Data were fitted to sigmoid E(max) models to obtain 24 hour-area under curve/MIC ratios for mycoplasmastasis and, where appropriate, for mycoplasmacidal activity and virtual mycoplasmal elimination. RESULTS: Minimum inhibitory concentrations against B237 were 20-fold higher, 2-fold higher and approximately 330-fold lower in serum than in artificial medium for oxytetracycline, danofloxacin and tulathromycin, respectively. Such differences were mirrored in experiments using Tan8. Oxytetracycline was mycoplasmastatic against both strains in both matrices. Danofloxacin elicited mycoplasmacidal activity against B237 and virtual elimination of Tan8; similar maximum antimycoplasmal effects were observed in artificial medium and serum. Tulathromycin effected virtual elimination of B237 but was mycoplasmastatic against Tan8 in artificial medium. However, this drug was mycoplasmastatic against both strains in the more physiologically relevant matrix of serum. CONCLUSIONS: Oxytetracycline, danofloxacin and tulathromycin are all suitable candidates for further investigation as potential treatments for CBPP. This study also highlights the importance of testing drug activity in biological matrices as well as artificial media.
Sujet(s)
Anti-infectieux/pharmacologie , Anti-infectieux/usage thérapeutique , Mycoplasma mycoïdes/effets des médicaments et des substances chimiques , Mycoplasma mycoïdes/croissance et développement , Péripneumonie contagieuse/traitement médicamenteux , Péripneumonie contagieuse/microbiologie , Animaux , Bovins , Numération de colonies microbiennes , Diholoside/pharmacologie , Diholoside/usage thérapeutique , Fluoroquinolones/pharmacologie , Fluoroquinolones/usage thérapeutique , Composés hétérocycliques/pharmacologie , Composés hétérocycliques/usage thérapeutique , Tests de sensibilité microbienne , Modèles biologiques , Oxytétracycline/pharmacologie , Oxytétracycline/usage thérapeutique , Facteurs tempsRÉSUMÉ
A 'synthetic' microbe has been created by introducing the artificially produced genome of one species into the cytoplasm of another. The technology allows the introduction of easily transferable adaptive units, as well as sets of genes that have likely never been transferred successfully.
Sujet(s)
Bioingénierie , Génie génétique , Génome bactérien , Mycoplasma mycoïdes/génétique , Protéines bactériennes/analyse , ADN bactérien/synthèse chimique , Mycoplasma capricolum/génétique , Mycoplasma mycoïdes/croissance et développement , Mycoplasma mycoïdes/physiologie , PhénotypeRÉSUMÉ
Genes of the Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC) coding for proteins capable of eliciting protective T-cell memory responses have potential for incorporation into a recombinant subunit vaccine against contagious bovine pleuropneumonia (CBPP). Here we used lymphocytes from cattle that had completely recovered from infection to screen products of MmmSC genes for recognition by CD4(+) effector memory (Tem) and central memory (Tcm) T lymphocytes. Six MmmSC genes (abc, gapN, glpO, lppA, lppB, and ptsG) were expressed as histidine-tagged recombinant polypeptides, or synthetic overlapping peptides, before inclusion in proliferation and gamma interferon (IFN-gamma) assays. Only two MmmSC antigens, LppA and PtsG, consistently induced recall proliferation from immune CD4(+) T cells and IFN-gamma production in all animals tested. Moreover, LppA and PtsG were shown to possess epitopes recognized by both short-lived CD4(+) Tem and long-lived CD4(+) Tcm cells.
Sujet(s)
Antigènes bactériens/immunologie , Protéines bactériennes/immunologie , Lymphocytes T CD4+/immunologie , Maladies des bovins/immunologie , Mycoplasma mycoïdes/immunologie , Péripneumonie contagieuse/immunologie , Animaux , Antigènes bactériens/génétique , Protéines bactériennes/génétique , Bovins , Maladies des bovins/microbiologie , Prolifération cellulaire , Cartographie épitopique , Mémoire immunologique , Interféron gamma/métabolisme , Mycoplasma mycoïdes/génétique , Mycoplasma mycoïdes/croissance et développement , Péripneumonie contagieuse/microbiologieRÉSUMÉ
We report the design, synthesis, and assembly of the 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including "watermark" sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication.
Sujet(s)
Bioingénierie , Génie génétique , Génome bactérien , Mycoplasma capricolum/génétique , Mycoplasma mycoïdes/génétique , Protéines bactériennes/analyse , Séquence nucléotidique , Clonage moléculaire , ADN bactérien/synthèse chimique , ADN bactérien/génétique , Escherichia coli/génétique , Délétion de gène , Gènes bactériens , Données de séquences moléculaires , Mycoplasma mycoïdes/croissance et développement , Mycoplasma mycoïdes/physiologie , Mycoplasma mycoïdes/ultrastructure , Phénotype , Plasmides , Réaction de polymérisation en chaîne , Polymorphisme génétique , Saccharomyces cerevisiae/génétique , Transformation bactérienneRÉSUMÉ
This study was designed to evaluate the validity of PCR for the direct detection of Mycoplasma (M.) agalactiae and Mycoplasma mycoides subsp. capri (Mmc), as the two species most frequently causing contagious agalactia (CA) in goats. The PCR method was compared with the traditional culture technique to determine which method was most efficient at identifying all auricular carriers present in herds. The samples analyzed were 307 ear swabs taken from goats reared in a CA endemic area. We assessed the validity of each technique to detect each species and agreement between both methods. For each species, the result was taken as true-positive when at least one of the two tests was positive. Of the swabs tested, 246 were scored positive by PCR (235 and 11 for Mmc and M. agalactiae, respectively) and 117 showed a positive culture result (113 for Mmc and 4 for M. agalactiae). 133 of the PCR-positive samples (124 and 9 for Mmc and M. agalactiae, respectively) yielded negative culture results and 4 culture-positive samples tested negative using PCR (2 for each species). Sensitivity and negative predictive values for PCR were 84.62 and 99.32 (for M. agalactiae) and 99.16 and 97.22% (for Mmc) respectively, and for culture were 30.77 and 97.03 (for M. agalactiae) and 47.08 and 36.08% (for Mmc), respectively. PCR proved to be a rapid and sensitive method for the detection of mycoplasmas in the external ear of asymptomatic carriers. Tools such as this are needed to adopt efficient control measures against CA.
Sujet(s)
Techniques bactériologiques/médecine vétérinaire , Maladies des oreilles/médecine vétérinaire , Infections à Mycoplasma/médecine vétérinaire , Mycoplasma agalactiae/physiologie , Mycoplasma mycoïdes/physiologie , Réaction de polymérisation en chaîne/médecine vétérinaire , Animaux , Techniques bactériologiques/méthodes , Numération de colonies microbiennes/méthodes , Oreille/microbiologie , Maladies des oreilles/diagnostic , Maladies des oreilles/microbiologie , Maladies des chèvres/diagnostic , Maladies des chèvres/microbiologie , Capra , Infections à Mycoplasma/diagnostic , Infections à Mycoplasma/microbiologie , Mycoplasma agalactiae/génétique , Mycoplasma agalactiae/croissance et développement , Mycoplasma agalactiae/isolement et purification , Mycoplasma mycoïdes/génétique , Mycoplasma mycoïdes/croissance et développement , Mycoplasma mycoïdes/isolement et purification , Reproductibilité des résultats , Sensibilité et spécificitéRÉSUMÉ
We recently reported the chemical synthesis, assembly, and cloning of a bacterial genome in yeast. To produce a synthetic cell, the genome must be transferred from yeast to a receptive cytoplasm. Here we describe methods to accomplish this. We cloned a Mycoplasma mycoides genome as a yeast centromeric plasmid and then transplanted it into Mycoplasma capricolum to produce a viable M. mycoides cell. While in yeast, the genome was altered by using yeast genetic systems and then transplanted to produce a new strain of M. mycoides. These methods allow the construction of strains that could not be produced with genetic tools available for this bacterium.
Sujet(s)
Clonage moléculaire , Techniques de transfert de gènes , Génie génétique , Génome bactérien , Mycoplasma capricolum/génétique , Mycoplasma mycoïdes/génétique , Saccharomyces cerevisiae/génétique , Centromère , Méthylation de l'ADN , DNA restriction enzymes/génétique , DNA restriction enzymes/métabolisme , Type III site-specific deoxyribonuclease/génétique , Mycoplasma mycoïdes/croissance et développement , Mycoplasma mycoïdes/isolement et purification , Plasmides , Analyse de séquence d'ADN , Délétion de séquence , Transformation bactérienneRÉSUMÉ
Two vaccines against Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (LC type) were developed using inactivated strains selected in previous characterization studies. The vaccines differed in terms of the adjuvants used: aluminium hydroxide (vaccine A) or aluminium hydroxide plus purified saponin (vaccine B). These vaccines were tested on 60 pregnant goats and 60 seronegative kids that were challenged by placing in a herd with a history of caprine contagious agalactia (CCA). Our findings indicate the effectiveness of the vaccines in preventing the appearance of new clinical signs such as mastitis, abortion, pneumonia and polyarthritis in CCA affected herds.
Sujet(s)
Vaccins antibactériens/immunologie , Infections à Mycoplasma/immunologie , Mycoplasma agalactiae/immunologie , Mycoplasma mycoïdes/immunologie , Adjuvants immunologiques/administration et posologie , Animaux , Anticorps antibactériens/sang , Vaccins antibactériens/administration et posologie , ADN bactérien/analyse , Femelle , Capra , Lait/microbiologie , Infections à Mycoplasma/sang , Infections à Mycoplasma/prévention et contrôle , Mycoplasma agalactiae/génétique , Mycoplasma agalactiae/croissance et développement , Mycoplasma mycoïdes/génétique , Mycoplasma mycoïdes/croissance et développement , Réaction de polymérisation en chaîne , Grossesse , Vaccination/méthodesRÉSUMÉ
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.
Sujet(s)
Concanavaline A/pharmacologie , Immunosuppression thérapeutique , Agranulocytes/immunologie , Agranulocytes/microbiologie , Mitogènes/pharmacologie , Mycoplasma mycoïdes/croissance et développement , Animaux , Bovins , Séparation cellulaire , Numération de colonies microbiennes , Température élevée , Interféron gamma/antagonistes et inhibiteurs , Interféron gamma/biosynthèse , Activation des lymphocytes/immunologie , Mitogènes/antagonistes et inhibiteurs , Mycoplasma mycoïdes/pathogénicitéRÉSUMÉ
Flow cytometry together with SYBR green I and propidium iodide was used to study the effects of enrofloxacin, ciprofloxacin, gentamicin, chloramphenicol, oxytetracycline, and tylosin on four mycoplasma species. Inhibition of mycoplasma growth could be detected by as early as 3 h after the start of treatment. The strongest effect was observed with enrofloxacin- and ciprofloxacin-treated cells.
Sujet(s)
Antituberculeux/pharmacologie , Cytométrie en flux , Mycoplasma agalactiae/effets des médicaments et des substances chimiques , Mycoplasma capricolum/effets des médicaments et des substances chimiques , Mycoplasma mycoïdes/effets des médicaments et des substances chimiques , Mycoplasma/effets des médicaments et des substances chimiques , Benzothiazoles , Agents colorants/métabolisme , Diamines , Colorants fluorescents/métabolisme , Tests de sensibilité microbienne , Mycoplasma/croissance et développement , Mycoplasma agalactiae/croissance et développement , Mycoplasma capricolum/croissance et développement , Mycoplasma mycoïdes/croissance et développement , Composés chimiques organiques/métabolisme , Propidium/métabolisme , Quinoléines , Reproductibilité des résultatsRÉSUMÉ
The dynamics of contagious bovine pleuropneumonia (CBPP) transmission vary widely between livestock production systems. This paper describes the development of a homogeneous, stochastic, compartmental model for CBPP transmission in pastoral herds of East Africa. The model was built using parameter estimates based on data published in the literature and on observations of livestock owners obtained through participatory research. The basic reproduction number for CBPP in southern Sudan was estimated to range from 3.2 to 4.6. The homogeneous model indicates that the critical community size for the persistence of CBPP falls within the typical herd sizes for pastoral communities in East Africa suggesting that individual isolated herds are capable of maintaining infection indefinitely. Vaccination alone with currently available vaccines was unlikely to eradicate the disease.
Sujet(s)
Maladies des bovins/microbiologie , Maladies des bovins/transmission , Transmission de maladie infectieuse/médecine vétérinaire , Modèles biologiques , Mycoplasma mycoïdes/croissance et développement , Péripneumonie contagieuse/transmission , Afrique de l'Est , Animaux , Bovins , Maladies des bovins/épidémiologie , Contrôle des maladies transmissibles/méthodes , Péripneumonie contagieuse/épidémiologie , Péripneumonie contagieuse/microbiologie , Population rurale , Processus stochastiquesRÉSUMÉ
The study was conducted with the aim of evaluating the xerovac process as a method for preparing contagious bovine pleuropneumonia (CBPP) vaccine with increased heat resistance. The thermo-protective effects of various concentrations of trehalose in mycoplasma growth medium, various concentrations of trehalose in the dehydration stabilizer and the importance of some divalent cations were assessed. The results obtained indicate that a rapid dehydration of CBPP vaccine following the xerovac method and in an excipient composed of a high concentration of trehalose, renders the product more heat tolerant than a similar vaccine prepared using a regular or an extended freeze drying regime. It was also demonstrated that the addition of chitosan as a mycoplasma precipitating agent conferred additional heat resistance to the vaccine. It is suggested that the application of the xerovac process in the dehydration of CBPP vaccine offers the advantages of a faster, cheaper and easier process over the conventional dehydration methods like freeze drying.
Sujet(s)
Vaccins antibactériens/immunologie , Préparation de médicament/méthodes , Mycoplasma mycoïdes/immunologie , Péripneumonie contagieuse/immunologie , Péripneumonie contagieuse/prévention et contrôle , Animaux , Cations divalents/pharmacologie , Bovins , Chitosane , Stabilité de médicament , Excipients , Lyophilisation , Température élevée , Indicateurs et réactifs , Mycoplasma mycoïdes/croissance et développement , Contrôle de qualitéRÉSUMÉ
The effect of reconstituting freeze-dried vaccine cultures of Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC) strain T(1)44 grown in standard vaccine medium using variable quantities of un-buffered solutions of 1 M MgSO(4) (the current O.I.E.-recommended procedure) has been investigated. Compared to the culture pH prior to desiccation, a drop of up to 2.2 pH units was observed, dependent upon the volume and pH of 1 M MgSO(4) used (1-30 x original culture volume, using 1 M MgSO(4) in the pH range 5.4-7.6). This pH drop appears to be due to the removal of the HPO(4)(2-) buffer capacity of the medium by the formation of insoluble Mg(3)(PO(4))(2) and the release of free H(+) ions. As a result of this lower pH, markedly reduced culture viability was observed over an 8-h period at 37 degrees C for vaccines re-suspended in 1 M MgSO(4) (ca. 6 log(10) drop in titre) compared to re-suspension in dH(2)O (ca. 1.5 log(10) drop in titre). Re-suspension in 1 M MgSO(4) did exhibit a thermoprotective effect at 46 degrees C, but only when the pH was maintained above pH 7.0 by use of HEPES-buffered growth medium (1 log(10) drop in titre compared to 6 log(10) drop using dH(2)O over an 8-h period). Since all current O.I.E.-recommended growth media for MmmSC are based upon a phosphate buffer system, it is therefore recommended that the use of 1 M MgSO(4) as a reconstitution fluid be discontinued as soon as possible and buffered saline be used instead. The use of this reconstitution procedure with the T(1)44 vaccine strain could be a significant factor in the poor efficacy observed with current freeze-dried vaccines against contagious bovine pleuropneumonia in Africa.
Sujet(s)
Vaccins antibactériens , Sulfate de magnésium/pharmacologie , Mycoplasma mycoïdes/immunologie , Vaccins antibactériens/génétique , Vaccins antibactériens/immunologie , Vaccins antibactériens/métabolisme , Cellules cultivées , Milieux de culture , Lyophilisation , Température élevée , Concentration en ions d'hydrogène , Mycoplasma mycoïdes/génétique , Mycoplasma mycoïdes/croissance et développement , TitrimétrieRÉSUMÉ
The use of a buffer system based on N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), in conjunction with standard Gourlay's culture medium was investigated for the growth and maintenance of Mycoplasma mycoides subsp. mycoides SC vaccine strain T(1)44. When the initial pH of the culture medium was adjusted to 8.0, 0.075 M HEPES-NaOH was found to be sufficient to prevent the pH falling below 7.1 at any stage during the growth cycle, even in the presence of 0.5% glucose. Compared to growth in standard unbuffered Gourlay's medium, the final culture titre was found to be one log(10) higher, at 10(11) colour changing units (CCU) per ml, and considerably extended culture survival was observed at 37 degrees C. The titre remained above 10(10) CCU ml(-1) for 4 days, and above 10(8) CCU ml(-1) in excess of 1 month. After 4 month's storage at 37 degrees C the titre had fallen to 5x10(4) CCU ml(-1). In contrast, no viable bacteria could be detected in standard unbuffered medium 3 days after the onset of stationary phase, at which point the pH had dropped to 5.4. No significant difference in growth rate between the two media was observed. Adoption of a HEPES-NaOH buffer system by African vaccine manufacturers should require minimal changes to current formulations and procedures, and should enhance both the final titre and thermostability of freeze-dried and liquid broth vaccines against contagious bovine pleuropneumonia (CBPP).
Sujet(s)
Vaccins antibactériens/biosynthèse , Substances tampon , HEPES/pharmacologie , Mycoplasma mycoïdes/effets des médicaments et des substances chimiques , Mycoplasma mycoïdes/croissance et développement , Péripneumonie contagieuse , Animaux , Bovins , Milieux de culture , Concentration en ions d'hydrogène , Mycoplasma mycoïdes/cytologie , Péripneumonie contagieuse/thérapie , TempératureRÉSUMÉ
Monoclonal antibody (MAb) PK-2 inhibited the in vitro growth of nine Mycoplasma mycoides subsp. mycoides small-colony strains. In contrast to the results with polyclonal antisera, growth inhibition by MAb PK-2 was specific for M. mycoides subsp. mycoides small-colony strains and constituted a reliable means of distinguishing them from other mycoplasmas.