RESUMEN
There is a worldwide problem of disease caused by Mycoplasma (M.) bovis in cattle; it has a significant detrimental economic and animal welfare impact on cattle rearing. Infection can manifest as a plethora of clinical signs including mastitis, pneumonia, arthritis, keratoconjunctivitis, otitis media and genital disorders that may result in infertility and abortion. Current diagnosis and control information are reviewed and analysed to identify gaps in knowledge of the causative organism in respect of the disease pathology, diagnosis and control methods. The main considerations are as follows: no vaccines are commercially available; antimicrobial resistance is increasing; diagnostic and antimicrobial sensitivity testing needs to be improved; and a pen-side test would facilitate more rapid diagnosis and implementation of treatment with antimicrobials. More data on host susceptibility, stress factors, immune response and infectious dose levels are required. The impact of asymptomatic carriers, M. bovis survival in the environment and the role of wildlife in transmitting the disease also needs investigation. To facilitate development of vaccines, further analysis of more M. bovis genomes, its pathogenic mechanisms, including variable surface proteins, is required, along with reproducible disease models.
Asunto(s)
Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/prevención & control , Control de Enfermedades Transmisibles/métodos , Infecciones por Mycoplasma/veterinaria , Mycoplasma bovis/aislamiento & purificación , Animales , Antibacterianos/uso terapéutico , Vacunas Bacterianas/administración & dosificación , Bovinos , Enfermedades de los Bovinos/microbiología , Femenino , Infecciones por Mycoplasma/diagnóstico , Infecciones por Mycoplasma/microbiología , Infecciones por Mycoplasma/prevención & control , Mycoplasma bovis/patogenicidadRESUMEN
Mycoplasma bovis is a major bovine pathogen associated with bovine respiratory disease complex and is responsible for substantial economic losses worldwide. M. bovis is also associated with other clinical presentations in cattle, including mastitis, otitis, arthritis, and reproductive disorders. To gain a better understanding of the genetic diversity of this pathogen, a multilocus sequence typing (MLST) scheme was developed and applied to the characterization of 137 M. bovis isolates from diverse geographical origins, obtained from healthy or clinically infected cattle. After in silico analysis, a final set of 7 housekeeping genes was selected (dnaA, metS, recA, tufA, atpA, rpoD, and tkt). MLST analysis demonstrated the presence of 35 different sequence types (STs) distributed in two main clonal complexes (CCs), defined at the double-locus variant level, namely, CC1, which included most of the British and German isolates, and CC2, which was a more heterogeneous and geographically distant group of isolates, including European, Asian, and Australian samples. Index of association analysis confirmed the clonal nature of the investigated M. bovis population, based on MLST data. This scheme has demonstrated high discriminatory power, with the analysis showing the presence of genetically distant and divergent clusters of isolates predominantly associated with geographical origins.
Asunto(s)
Enfermedades de los Bovinos/microbiología , Análisis por Conglomerados , Variación Genética , Tipificación de Secuencias Multilocus , Infecciones por Mycoplasma/veterinaria , Mycoplasma bovis/clasificación , Mycoplasma bovis/aislamiento & purificación , Animales , Bovinos , Enfermedades de los Bovinos/epidemiología , Genes Bacterianos , Genes Esenciales , Genotipo , Salud Global , Epidemiología Molecular , Datos de Secuencia Molecular , Infecciones por Mycoplasma/epidemiología , Infecciones por Mycoplasma/microbiología , Mycoplasma bovis/genética , FilogeografíaRESUMEN
Mycoplasma bovis isolates with decreased susceptibilities to tetracyclines are increasingly reported worldwide. The acquired molecular mechanisms associated with this phenomenon were investigated in 70 clinical isolates of M. bovis. Sequence analysis of the two 16S rRNA-encoding genes (rrs3 and rrs4 alleles) containing the primary binding pocket for tetracycline (Tet-1 site) was performed on isolates with tetracycline hydrochloride MICs of 0.125 to 16 µg/ml. Mutations at positions A965T, A967T/C (Escherichia coli numbering) of helix 31, U1199C of helix 34, and G1058A/C were identified. Decreased susceptibilities to tetracycline (MICs, ≥2 µg/ml) were associated with mutations present at two (A965 and A967) or three positions (A965, A967, and G1058) of the two rrs alleles. No tet(M), tet(O), or tet(L) determinants were found in the genome of any of the 70 M. bovis isolates. The data presented correlate (P<0.0001) the mutations identified in the Tet-1 site of clinical isolates of M. bovis with decreased susceptibility to tetracycline.
Asunto(s)
Antibacterianos/farmacología , Mycoplasma bovis/genética , Resistencia a la Tetraciclina/genética , Tetraciclina/farmacología , Pruebas de Sensibilidad Microbiana , Mutación , Mycoplasma bovis/efectos de los fármacos , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
The in vitro activity of enrofloxacin against 73 Mycoplasma synoviae field strains isolated in Israel and Europe was determined by broth microdilution. Decreased susceptibility to enrofloxacin was identified in 59% of strains, with the MICs ranging from 1 to >16 µg/ml. The estimated MIC50 and MIC90 values for enrofloxacin were 2 and 8 µg/ml, respectively. Moreover, this study showed that 92% of recent Israeli field isolates (2009 to 2011) of M. synoviae have MICs of ≥ 2 µg/ml to enrofloxacin. Comparison of the quinolone resistance-determining regions (QRDRs) in M. synoviae isolates revealed a clear correlation between the presence of one of the amino acid substitutions Asp79-Asn, Thr80-Ala/Ile, Ser81-Pro, and Asp84-Asn/Tyr/His of the ParC QRDR and decreased susceptibility to enrofloxacin (MIC, ≥ 1 µg/ml). Amino acid substitutions at positions GyrA 87, GyrB 401/402, and ParE 420/454 were also identified, but there was no clear-cut correlation with susceptibility to enrofloxacin. Comparison of vlhA molecular profiles revealed the presence of 9 different genotypes in the Israeli M. synoviae field isolates and 10 genotypes in the European isolates; only one vlhA genotype (type 4) was identified in both cohorts. Based on results of vlhA molecular typing, several mechanisms for emergence and dissemination of Israeli enrofloxacin-resistant M. synoviae isolates are suggested.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Fluoroquinolonas/farmacología , Lectinas/genética , Mycoplasma synoviae/efectos de los fármacos , Mycoplasma synoviae/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Girasa de ADN/genética , Topoisomerasa de ADN IV/genética , Enrofloxacina , Europa (Continente) , Genotipo , Israel , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Tipificación Molecular , Infecciones por Mycoplasma , Mycoplasma synoviae/aislamiento & purificación , Quinolonas/farmacología , Análisis de Secuencia de ADNRESUMEN
Molecular characterization of the quinolone-resistance determining regions (QRDRs) of DNA gyrase and topoisomerase IV in 93 Mycoplasma gallisepticum field strains isolated in different geographic regions revealed discrepancies between minimal inhibitory concentration values and presence of amino acid substitutions within the QRDRs of GyrA and ParC in 9/93 (10%) strains. This may delimitate applicability of a gene-based assay to detect fluoroquinolone resistance in this avian pathogen.
Asunto(s)
Fluoroquinolonas/farmacología , Mutación , Mycoplasma gallisepticum/efectos de los fármacos , Mycoplasma gallisepticum/genética , Sustitución de Aminoácidos , Animales , Aves , Girasa de ADN/genética , Topoisomerasa de ADN IV/química , Topoisomerasa de ADN IV/genética , Enrofloxacina , Pruebas de Sensibilidad Microbiana , Infecciones por Mycoplasma/microbiología , Mycoplasma gallisepticum/aislamiento & purificación , Enfermedades de las Aves de Corral/microbiología , Quinolonas/farmacologíaRESUMEN
Monitoring of the susceptibility of Mycoplasma bovis field isolates to antibiotics is important for the appropriate choice of treatment. However, in vitro susceptibility testing of mycoplasmas is technically demanding and time-consuming, especially for clinical isolates, and is rarely performed in mycoplasma diagnostic laboratories. Thus, the development of methods allowing rapid real-time detection of resistant strains of M. bovis in clinical samples is a high priority for successful treatment. In this study, a novel TaqMan single-nucleotide-polymorphism (SNP) real-time PCR assay, which enables the rapid identification of M. bovis strains with different susceptibilities to fluoroquinolones, was developed and evaluated. The TaqMan SNP real-time PCR assay is based on the amplification of a 97-bp fragment of the parC quinolone resistance-determining region (QRDR) and allows the specific detection of four possible genotypes: GAC or GAT (susceptible to fluoroquinolones) and AAC or AAT (resistant to fluoroquinolones). Four TaqMan minor groove binder (MGB) probes identifying 1-base mismatches were designed and applied in a dual-probe assay with two reaction tubes. The TaqMan SNP real-time PCRs developed are highly specific for M. bovis, with a detection limit of 5 fg/microl (about 5 M. bovis genomes). In addition, all four SNP real-time PCR tests have almost the same efficiency (97.7% [GAC], 94% [AAC], 99.99% [GAT], and 98% [AAT]). Taken together, the data suggest that this SNP real-time PCR assay has potential as a routine diagnostic test for the detection of decreased susceptibility of M. bovis to fluoroquinolones.
Asunto(s)
Antibacterianos/farmacología , ADN Bacteriano/genética , Fluoroquinolonas/farmacología , Mycoplasma bovis/efectos de los fármacos , Mycoplasma bovis/genética , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple , Animales , Proteínas Bacterianas/genética , Bovinos , Enfermedades de los Bovinos/microbiología , Topoisomerasa de ADN IV/genética , Genotipo , Pruebas de Sensibilidad Microbiana/métodos , Infecciones por Mycoplasma/microbiología , Infecciones por Mycoplasma/veterinaria , Mycoplasma bovis/aislamiento & purificación , Sondas de Oligonucleótidos/genética , Sensibilidad y EspecificidadRESUMEN
A syndrome in which white foci and granulopustular lesions appeared on the vaginal mucous membranes of Holstein cows in several dairy herds in Israel is described. During clinical and diagnostic investigations, Mycoplasma bovigenitalium was isolated from 11 of 20 clinical cases. Vaginal swabs taken from the same cows yielded three isolates of Mycoplasma canadense, which were all associated with the M bovigenitalium infection. Two isolates of small, round, non-enveloped viral particles were approximately 25 nm in diameter and characteristic of enteroviruses on negative-staining electron microscopy.
Asunto(s)
Enfermedades de los Bovinos/microbiología , Infecciones por Mycoplasma/veterinaria , Mycoplasma/aislamiento & purificación , Vulvovaginitis/veterinaria , Feto Abortado/microbiología , Feto Abortado/patología , Aborto Veterinario , Animales , Biopsia , Bovinos , Enfermedades de los Bovinos/epidemiología , Enfermedades de los Bovinos/patología , Industria Lechera , Femenino , Israel/epidemiología , Mycoplasma/clasificación , Infecciones por Mycoplasma/epidemiología , Infecciones por Mycoplasma/patología , Vulvovaginitis/microbiología , Vulvovaginitis/patologíaRESUMEN
Comparison of the quinolone resistance-determining regions (QRDRs) in 42 Mycoplasma bovis clinical isolates revealed amino acid substitutions at both GyrA (position 83) and ParC (position 84) in 10/11 enrofloxacin-resistant strains. The mutation present in the parC QRDR was discriminative for enrofloxacin resistance by parC PCR-restriction fragment length polymorphism. Comparison of molecular profiles by insertion sequence typing suggests that the currently prevalent enrofloxacin-resistant M. bovis strain evolved by selection under field conditions from one of the susceptible strains.
Asunto(s)
Antiinfecciosos/farmacología , Topoisomerasa de ADN IV/genética , Fluoroquinolonas/farmacología , Mycoplasma bovis/efectos de los fármacos , Mycoplasma bovis/genética , Mutación Puntual , Enrofloxacina , Pruebas de Sensibilidad Microbiana , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Emergence of resistance to fluoroquinolones is mainly due to chromosomal mutations in genes encoding the subunits of the drug's target enzymes, DNA gyrase and topoisomerase IV, which are essential for DNA replication. The quinolone resistance-determining regions (QRDRs) of these genes were characterized in 25 Mycoplasma gallisepticum strains isolated from commercial poultry flocks during 1997-2007, which exhibited different levels of susceptibility to fluoroquinolones. All enrofloxacin-resistant isolates harbored amino acid substitutions in the QRDRs of each of three proteins (GyrA, GyrB, and ParC). Molecular typing of those strains by random amplification of polymorphic DNA and gene-targeted sequencing supports ongoing, stepwise selection of resistant strains from the existing reservoir of susceptible M. gallisepticum strains.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Fluoroquinolonas/farmacología , Mycoplasma gallisepticum/efectos de los fármacos , Mycoplasma gallisepticum/genética , Sustitución de Aminoácidos/genética , Animales , Secuencia de Bases , Pollos , Girasa de ADN/genética , Topoisomerasa de ADN IV/genética , ADN Bacteriano/química , Enrofloxacina , Genes Bacterianos , Datos de Secuencia Molecular , Mutación , Mycoplasma gallisepticum/clasificación , Técnica del ADN Polimorfo Amplificado Aleatorio/veterinaria , PavosRESUMEN
Mycoplasma bovis, the most important etiological agent of bovine mycoplasmosis, undergoes extensive antigenic variation of major and highly immunogenic surface lipoprotein antigens (Vsps). A family of 13 related but divergent vsp genes, which occur as single chromosomal copies, was recently found in the chromosome of M. bovis. In the present study, the molecular mechanism mediating the high-frequency phase variation of two Vsps (VspA and VspC) as representatives of the Vsp family was investigated. Analysis of clonal isolates exhibiting phase transitions of VspA or of VspC (i.e., ON-->OFF-->ON) has shown that DNA inversions occur during Vsp phase variation. The upstream region of each vsp gene contains two sequence cassettes. The first (cassette no. 1), a 71-bp region upstream of the ATG initiation codon, exhibits 98% homology among all vsp genes, while the second (cassette no. 2), upstream of cassette no. 1, ranges in size from 50 to 180 bp and is more divergent. Examination of the ends of the inverted fragments during VspA or VspC phase variation revealed that in both cases, a change in the organization of vsp upstream cassettes involving three vsp genes had occurred. Primer extension and Northern blot analysis have shown that a specific cassette no. 2, designated A(2), is an active promoter and that juxtaposition of this regulatory element to a silent vsp gene by DNA inversions allows transcription initiation of the recipient gene. Further genetic analysis revealed that phase variation of VspA or of VspC involves two site-specific DNA inversions occurring between inverted copies of a specific 35-bp sequence present within the conserved cassette no. 1. A model for the control of Vsp phase variation is proposed.
Asunto(s)
Variación Antigénica , Antígenos Bacterianos , Antígenos de Superficie/genética , Proteínas de la Membrana Bacteriana Externa , Lipoproteínas/genética , Proteínas de la Membrana/genética , Mycoplasma/genética , Regiones Promotoras Genéticas/genética , Antígenos de Superficie/metabolismo , Secuencia de Bases , Inversión Cromosómica , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica , Lipoproteínas/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mycoplasma/crecimiento & desarrollo , Mycoplasma/inmunología , Fenotipo , Recombinación Genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
A family of 13 related but divergent vsp genes was recently found in the chromosome of the bovine pathogen Mycoplasma bovis. The vsp genomic locus was shown to undergo high-frequency rearrangements and to mediate phenotypic switching of variable lipoprotein antigens (Vsps) on the mycoplasma cell surface. Here we report that the vsp gene repertoire is subject to changes. Genetic analysis of M. bovis clonal isolates displaying distinct Vsp phenotypes showed that an intergenic recombination event between two closely related members of the vsp gene family, the formerly expressed vspA gene and the vspO gene, led to the formation of a new chimeric and functional vsp gene, vspC. The 5' end of the recombination event was identified within the highly conserved vsp-upstream region, while the 3' end was localized within the first repetitive domain (R(A)1) present in both vspA and vspO structural genes. As a result, the vspC gene is an embodiment of the following domains: an N-terminus-encoding region linked to the highly conserved vsp-upstream region provided by the vspO gene; and a C-terminus-encoding region and the more distal and divergent vsp-upstream region acquired from the vspA gene. The generation of chimeric genes encoding surface antigens may provide an important element of genetic variation and an additional source of antigenic diversification within the mycoplasma population.
Asunto(s)
Antígenos de Superficie/genética , Proteínas de la Membrana Bacteriana Externa/genética , Lipoproteínas/genética , Mycoplasma/genética , Mycoplasma/inmunología , Proteínas Recombinantes de Fusión , Recombinación Genética , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Antígenos de Superficie/química , Antígenos de Superficie/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Secuencia de Bases , Cromosomas Bacterianos , Clonación Molecular , Variación Genética/genética , Lipoproteínas/química , Lipoproteínas/inmunología , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Análisis de Secuencia de ADNRESUMEN
A Mycoplasma agalactiae genomic fragment carrying four vsp-related genes (designated avg: agalactiae variable genes) was cloned, sequenced and compared to the vspA gene of Mycoplasma bovis. The following features were revealed: (i) the presence of a highly conserved vsp 5' upstream region; (ii) a highly homologous vsp N-terminal end encoding a putative lipoprotein signal sequence; (iii) sequence divergence of the rest of the mature proteins. By using avg specific probes in Southern blot analysis of genomic DNAs of M. agalactiae strains as well as of isolates from infected animals, marked DNA polymorphism of avg fragments was demonstrated. In addition, the avg genomic fingerprints were monitored for a period of 7 months, in isolates of M. agalactiae from an individual chronically infected animal. The results provided evidence that a chromosomal region of M. agalactiae, carrying vsp-related genes, undergoes rearrangements in vivo in the natural animal host during the course of infection.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Cromosomas Bacterianos/genética , Reordenamiento Génico , Infecciones por Mycoplasma/veterinaria , Mycoplasma/genética , Animales , Antígenos Bacterianos , Secuencia de Bases , Genes Bacterianos , Datos de Secuencia Molecular , Mycoplasma/aislamiento & purificación , Infecciones por Mycoplasma/microbiología , Análisis de Secuencia de ADN , Ovinos , Enfermedades de las Ovejas/microbiologíaRESUMEN
A putative cytadhesin-related protein (PvpA) undergoing variation in its expression was identified in the avian pathogen Mycoplasma gallisepticum. The pvpA gene was cloned, expressed in Escherichia coli, and sequenced. It exhibits 54 and 52% homology with the P30 and P32 cytadhesin proteins of the human pathogens Mycoplasma pneumoniae and Mycoplasma genitalium, respectively. In addition, 50% homology was found with the MGC2 cytadhesin of M. gallisepticum and 49% homology was found with a stretch of 205 amino acids of the cytadherence accessory protein HMW3 of M. pneumoniae. The PvpA molecule possesses a proline-rich carboxy-terminal region (28%) containing two identical directly repeated sequences of 52 amino acids and a tetrapeptide motif (Pro-Arg-Pro-X) which is repeated 14 times. Genetic analysis of several clonal isolates representing different expression states of the PvpA product ruled out chromosomal rearrangement as the mechanism for PvpA phase variation. The molecular basis of PvpA variation was revealed in a short tract of repeated GAA codons, encoding five successive glutamate resides, located in the N-terminal region and subject to frequent mutation generating an in-frame UAA stop codon. Size variation of the PvpA protein was observed among M. gallisepticum strains, ranging from 48 to 55 kDa and caused by several types of deletions occurring at the PvpA C-terminal end and within the two directly repeated sequences. By immunoelectron microscopy, the PvpA protein was localized on the mycoplasma cell surface, in particular on the terminal tip structure. Collectively, these findings suggest that PvpA is a newly identified variable surface cytadhesin protein of M. gallisepticum.
Asunto(s)
Adhesinas Bacterianas/genética , Genes Bacterianos , Mycoplasma/genética , Adhesinas Bacterianas/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Codón sin Sentido/genética , Cartilla de ADN/genética , ADN Bacteriano/genética , Escherichia coli/genética , Expresión Génica , Variación Genética , Humanos , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Peso Molecular , Mutación , Mycoplasma/química , Mycoplasma/ultraestructura , Mycoplasma pneumoniae/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Eliminación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The family of variable surface lipoproteins (Vsps) of the bovine pathogen Mycoplasma bovis includes some of the most immunogenic antigens of this microorganism. Vsps were shown to undergo high-frequency phase and size variations and to possess extensive reiterated coding sequences extending from the N-terminal end to the C-terminal end of the Vsp molecule. In the present study, mapping experiments were conducted to detect regions with immunogenicity and/or adhesion sites in repetitive domains of four Vsp antigens of M. bovis, VspA, VspB, VspE, and VspF. In enzyme-linked immunosorbent assay experiments, sera obtained from naturally infected cattle showed antibodies to different repeating peptide units of the Vsps, particularly to units R(A)1, R(A)2, R(A)4.1, R(B)2.1, R(E)1, and R(F)1, all of which were found to contain immunodominant epitopes of three to seven amino acids. Competitive adherence trials revealed that a number of oligopeptides derived from various repeating units of VspA, VspB, VspE, and VspF partially inhibited cytoadhesion of M. bovis PG45 to embryonic bovine lung cells. Consequently, putative adherence sites were identified in the same repeating units (R(A)1, R(A)2, R(A)4.1, R(B)2.1, R(E)1, and R(F)1) and in R(F)2. The positions and lengths of the antigenic determinants were mostly identical to those of adhesion-mediating sites in all short repeating units, whereas in the considerably longer R(F)1 unit (84 amino acid residues), there was only one case of identity among four immunogenic epitopes and six adherence sites. The identification of epitopes and adhesive structures in repetitive domains of Vsp molecules is consistent with the highly immunogenic nature observed for several members of the Vsp family and suggests a possible function for these Vsp molecules as complex adherence-mediating regions in pathogenesis.
Asunto(s)
Proteínas Bacterianas/inmunología , Mapeo Epitopo , Lipoproteínas/inmunología , Mycoplasma/química , Secuencias Repetitivas de Aminoácido , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Monoclonales/inmunología , Adhesión Bacteriana , Bovinos , Ensayo de Inmunoadsorción Enzimática , Femenino , Proteínas de la Membrana/inmunología , Datos de Secuencia Molecular , Mycoplasma/inmunologíaRESUMEN
Major lipoprotein antigens, known as variable membrane surface lipoproteins (Vsps), on the surface of the bovine pathogen Mycoplasma bovis were shown to spontaneously undergo noncoordinate phase variation between ON and OFF expression states. The high rate of Vsp phenotypic switching was also shown to be linked with DNA rearrangements that occur at high frequency in the M. bovis chromosome (I. Lysnyansky, R. Rosengarten, and D. Yogev, J. Bacteriol. 178:5395-5401, 1996). In the present study, 13 single-copy vsp genes organized in a chromosomal cluster were identified and characterized. All vsp genes encode highly conserved N-terminal domains for membrane insertion and lipoprotein processing but divergent mature Vsp proteins. About 80% of each vsp coding region is composed of reiterated coding sequences that create a periodic polypeptide structure. Eighteen distinct repetitive domains of different lengths and amino acid sequences are distributed within the products of the various vsp genes that are subject to size variation due to spontaneous insertions or deletions of these periodic units. Some of these repeats were found to be present in only one Vsp family member, whereas other repeats recurred at variable locations in several Vsps. Each vsp gene is also 5' linked to a highly homologous upstream region composed of two internal cassettes. The findings that rearrangement events are associated with Vsp phenotypic switching and that multiple regions of high sequence similarity are present upstream of the vsp genes and within the vsp coding regions suggest that modulation of the Vsp antigenic repertoire is determined by recombination processes that occur at a high frequency within the vsp locus of M. bovis.
Asunto(s)
Proteínas Bacterianas/genética , Cromosomas Bacterianos , Lipoproteínas/genética , Proteínas de la Membrana/genética , Mycoplasma/genética , Regiones no Traducidas 5'/genética , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos , Secuencia de Bases , Bovinos , Clonación Molecular , Sondas de ADN , Genes Bacterianos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
To assess the extent of interstrain variation, 50 isolates of Mycoplasma (M.) bovis including the type strain PG45 were examined for the presence of a family of variable membrane surface lipoproteins (Vsps) and their genes. Southern hybridization using a genomic fragment carrying three distinct vsp genes (vspAEF) revealed a striking heterogeneity, with only 2/50 strains having identical banding patterns. Cluster analysis of the data showed that most isolates from interrelated herds (groups 1, 2 and 3) were combined in a cluster of 50% homology, while isolates from distinct geographical regions (groups 4, 5 and 6) were linked only at 18% homology. Vsp antigen expression was monitored by Western immunoblotting using four specific monoclonal antibodies (MAbs). Resembling the findings at the DNA level, interstrain variation of Vsp expression among groups 1-3 was less pronounced than among non-interrelated isolates from groups 4-6. Ten out of 50 strains did not hybridize with the vspAEF gene probe at high-stringency conditions, 8/50 failed to react with any of the Vsp-related MAbs, and 6/50 proved negative in both assays. Interestingly, most of these isolates produced hybridization signals at low stringency suggesting major distinctions in their vsp gene structure. The extensive evidence obtained on interstrain vsp gene polymorphism and variation in Vsp expression could provide a basis for a future understanding of the pathogenic potential of individual M. bovis strains.
Asunto(s)
Antígenos Bacterianos/genética , Enfermedades de los Bovinos/microbiología , Infecciones por Mycoplasma/veterinaria , Mycoplasma/genética , Polimorfismo Genético , Animales , Anticuerpos Monoclonales , Antígenos Bacterianos/biosíntesis , Secuencia de Bases , Bovinos , Femenino , Genes Bacterianos , Lipoproteínas/biosíntesis , Lipoproteínas/genética , Mastitis Bovina/microbiología , Datos de Secuencia Molecular , Mycoplasma/inmunología , Mycoplasma/aislamiento & purificación , Infecciones por Mycoplasma/microbiología , Filogenia , Biosíntesis de Proteínas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Mycoplasma bovis, an important pathogen of cattle, was recently shown to possess a family of phase- and size-variable membrane surface lipoprotein antigens (Vsps). These proteins spontaneously undergo noncoordinate phase variation between ON and OFF expression states, generating surface antigenic variation. In the present study, we show that the spontaneously high rate of Vsp phenotypic switching involves DNA rearrangements that occur at high frequency in the M. bovis chromosome. A 1.5-kb HindIII genomic fragment carrying the vspA gene from M. bovis PG45 was cloned and sequenced. The deduced VspA amino acid sequence revealed that 80% of the VspA molecule is composed of reiterated intragenic coding sequences, creating a periodic polypeptide structure. Four distinct internal regions of repetitive sequences in the form of in-tandem blocks extending from the N-terminal to the C-terminal portion of the Vsp product were identified. Southern blot analysis of phenotypically switched isogenic lineages representing ON or OFF phase states of Vsp products suggested that changes in the Vsp expression profile were associated with detectable changes at the DNA level. By using a synthetic oligonucleotide representing a sequence complementary to the repetitive vspA gene region as a probe, we could identify the vspA-bearing restriction fragment undergoing high-frequency reversible rearrangements during oscillating phase transition of vspA. The 1.5-kb HindIII fragment carrying the vspA gene (on state) rearranged and produced a 2.3-kb HindIII fragment (OFF state) and vice versa. Two newly discovered vsp genes (vspE and vspF) were localized on two HindIII fragments flanking the vsp gene upstream and downstream. Southern blot hybridization with vspE- and vspF-specific oligonucleotides as probes against genomic DNA of VspA phase variants showed that the organization and size of the fragments adjacent to the vspA gene remained unchanged during VspA ON-OFF switching. The mechanisms regulating the vsp genes are yet unknown; our findings suggest that a recombinative mechanism possibly involving DNA inversions, DNA insertion, or mobile genetic elements may play a role in generating the observed high-frequency DNA rearrangements.