RESUMO
The membrane-protein 81 gene (mb-mp81) of Mycoplasma bovis was cloned, sequenced and compared to membrane-protein 81 gene (ma-mp81) of Mycoplasma agalactiae. After alignment of both sequences, specific primers pairs were designed from variable or unchanging nucleotide segments. In this study, we describe the development and optimization of a multiplex-PCR (MPCR) for the rapid detection of M. agalactiae and M. bovis strains. In addition, a simple and rapid PCR-restriction fragment length polymorphism (RFLP) assay, using the restriction enzymes AluI, DraI, RsaI and XbaI, is described to distinguish between both species. The results suggest that MPCR and PCR-RFLP assays could be used as an alternative method in routine diagnosis for rapid and specific simultaneous detection of M. agalactiae and M. bovis.
Assuntos
Mycoplasma agalactiae/genética , Mycoplasma agalactiae/isolamento & purificação , Mycoplasma bovis/genética , Mycoplasma bovis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clonagem Molecular , Diagnóstico Diferencial , Proteínas de Membrana/química , Proteínas de Membrana/genética , Peso MolecularRESUMO
Mycoplasma agalactiae, the causative agent of contagious agalactia in small ruminants, produces a protein, named P80, that is detectable in all wild-type isolates examined to date and that appears expressed during the early phase of infection. We describe here the identification, cloning and expression of the gene encoding P80 (ma-mp81). The deduced amino acid sequence is consistent with a hydrophobic and basic protein that possesses a lipoprotein signal peptide. Sequence analysis of gene ma-mp81 suggests that P80 is a membrane lipoprotein that shows significant homology with other putative lipoproteins of M. pneumoniae. An internal 1-kb fragment of ma-mp81 was expressed in Escherichia coli as a 6xHis-tagged protein. The purified recombinant protein greatly reacted with polyclonal anti-P80 sera raised in lamb.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mycoplasma/genética , Sequência de Aminoácidos , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Proteínas de Transporte/isolamento & purificação , Clonagem Molecular , Escherichia coli/genética , Genes Bacterianos/genética , Proteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Proteínas Recombinantes , Análise de Sequência de DNARESUMO
We have constructed a physical map of the Mycoplasma agalactiae strain PG2 chromosome analyzing it by pulsed field gel electrophoresis in a contour-clamped homogeneous electric-field system. We mapped 33 cleavage sites generated with SmaI, XhoI, SalI, EclXI and BsiWI restriction endonucleases using double digestions, one- and two-dimensional pulsed electrophoresis, cross-hybridization and linking clones. We have also mapped the loci of some genes by Southern hybridization.
Assuntos
Genoma Bacteriano , Mycoplasma/genética , Southern Blotting , Mapeamento Cromossômico , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Eletroforese em Gel de Campo Pulsado , Mapeamento por RestriçãoRESUMO
Five sets of vaccines were prepared using Mycoplasma agalactiae washed cultures inactivated with phenol (1), formalin (2), heat-treatment (3), sodium hypochlorite (4) and saponin (5). All sets, except for saponin-vaccine, were adjuvated with aluminium hydroxide. Five groups of six sarda ewes were inoculated twice before pregnancy, once during pregnancy and challenged during the lactation period. Monthly blood samples were taken from the vaccinated sheep and from 12 controls: sera were assayed by immunoblotting, ELISA and growth inhibition tests. Four control sheep were infected by intracanalicular route with pooled mycoplasmas at concentrations of 10(4), 10(5), 10(6) and 10(7) CCU. The challenge involved using infected milk to contaminate the remaining sheep. All the controls and some ewes from groups 2, 3 and 4 developed contagious agalactia. Ewes vaccinated with phenol- and saponin-inactivated mycoplasmas resisted experimental challenge. These results suggest that these two vaccines are effective and that their use could limit the diffusion of M. agalactiae infection.
Assuntos
Vacinas Bacterianas/uso terapêutico , Infecções por Mycoplasma/prevenção & controle , Mycoplasma/imunologia , Animais , Anticorpos Antibacterianos/biossíntese , Modelos Animais de Doenças , Feminino , Mycoplasma/crescimento & desenvolvimento , Gravidez , Ovinos , Vacinas de Produtos Inativados/uso terapêuticoRESUMO
Mycoplasma agalactiae and Mycoplasma bovis are closely related species in phylogenetic terms. Pulsed Field Gel Electrophoresis (PFGE) of SmaI, EclXI, Bsi WI, MluI, BssHII, SalI, XhoI, NruI and ApaI digested DNAs were used to analyse and to compare restriction fragment length polymorphism between M. agalactiae and M. bovis and to estimate their genome sizes. SmaI, EclXI and Bsi WI enzymes cleaved DNAs of both microrganisms. MluI, BssHII, SalI, XhoI and NruI digested only M. agalactiae DNA whereas ApaI cut only M. bovis DNA. The total DNA length was established to be 945 +/- 8.4 Kb for M. agalactiae and 961 +/- 18.9 Kb for M. bovis.
Assuntos
Mycoplasma/genética , Polimorfismo de Fragmento de Restrição , Animais , Sequência de Bases , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Eletroforese em Gel de Campo Pulsado/métodos , Genoma Bacteriano , Humanos , Mycoplasma/isolamento & purificação , Infecções por Mycoplasma/diagnóstico , Infecções por Mycoplasma/veterinária , Mapeamento por RestriçãoRESUMO
We have analyzed antigenic variation of seven M. agalactiae wild strains using different sera from naturally infected sheep. Only 30 day sera recognized all surface proteins and inhibited the growth of mycoplasmas. Furthermore, we have observed that two strongly immunogenic proteins: 55 and 35 kDa were digested using 500 micrograms/ml of trypsin. These two bands are immunoprecipitated together with four other proteins but only the 35 kDa protein is recognized by eluted antibodies.
Assuntos
Antígenos de Bactérias/análise , Infecções por Mycoplasma/imunologia , Mycoplasma/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Superfície/análise , Peso Molecular , Testes de Precipitina , OvinosRESUMO
We developed a simple and rapid method for DNA extraction from sheep milk to use for polymerase chain reaction (PCR) diagnosis of Mycoplasma agalactiae. We tested 357 samples from 21 newly infected flocks (group 1) and 87 samples from 8 flocks infected in the past (group 2). PCR results were compared with those of conventional culture. By PCR we detected 175 positives in group 1, while by culture we detected only 153. Milk samples from group 2 were negative, both by PCR assay and by culture. Our PCR is much faster than culture and reduces the time required for diagnosis from several days to 5 h. The method could be used for the routine diagnosis of contagious agalactia caused by Mycoplasma agalactiae.
Assuntos
Leite/microbiologia , Infecções por Mycoplasma/veterinária , Mycoplasma/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Doenças dos Ovinos , Animais , Southern Blotting , Primers do DNA , DNA Bacteriano/análise , Feminino , Infecções por Mycoplasma/diagnóstico , Reação em Cadeia da Polimerase/veterinária , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , OvinosRESUMO
We have analyzed 81 isolates of Mycoplasma agalactiae from four different regions of Italy between 1990 and 1995 in order to identify antigenic differences through SDS-PAGE and Western blotting and chromosomal DNA restriction endonuclease cleavage pattern differences. Antigenic variability in M. agalactiae isolates was investigated analyzing hydrophobic membrane protein fractions by immunoblotting using pooled sheep antiserum from naturally infected sheep. Large restriction fragments obtained cleaving genomic DNAs with SmaI, NruI, SalI, XhoI, BssHII and KpnI were analyzed by pulsed field gel electrophoresis. Genetic analysis indicates that isolates are all similar without intraspecific differences. This homogeneity was confirmed by immunoblotting: 80 and 50 kDa antigens are present in all strains analyzed.
Assuntos
Mycoplasma/genética , Mycoplasma/imunologia , Animais , Variação Antigênica , Antígenos de Bactérias/genética , Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/isolamento & purificação , Enzimas de Restrição do DNA , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Eletroforese em Gel de Campo Pulsado , Eletroforese em Gel de Poliacrilamida , Feminino , Immunoblotting , Itália/epidemiologia , Epidemiologia Molecular , Mycoplasma/isolamento & purificação , Infecções por Mycoplasma/epidemiologia , Infecções por Mycoplasma/microbiologia , Infecções por Mycoplasma/veterinária , Ovinos , Doenças dos Ovinos/epidemiologia , Doenças dos Ovinos/microbiologiaRESUMO
A polymerase chain reaction (PCR)-based test was developed for the detection of Mycoplasma agalactiae in sheep milk samples. Two oligonucleotide primers were designed to amplify a 375 bp fragment of M. agalactiae chromosomal DNA. Amplified products were analyzed by agarose gel electrophoresis and Southern blot hybridization using a fluorescein labeled 528 bp probe. The primers allowed the amplification of fragment of M. agalactiae DNA and did not amplify any specific fragment of other mycoplasmal DNAs (M. capricolum, M. mycoides subsp. mycoides, M. mycoides subsp. capri, M. putrefaciens, M. arginini and M. bovis) or other bacterial DNAs (S. aureus, S. epidermidis, P. haemalytica, E. coli, S. agalactiae, S. dysgalactiae, S. uberis, B. cereus, P. aeruginosa, S. durans, L. lactis, L. lactis var. diacetilactis, L. mesenteroides, S. thermophilus, L. bulgaricus and L. casei). The limit of detection of PCR assay was between 2.5 and 25 fg of purified DNA and 10(2) CCU ml-1 on mycoplasma cultures. These results indicate that the PCR technique can be used as a rapid and specific diagnostic method for detection of M. agalactiae.
