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PLoS One ; 11(3): e0152171, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27010566


Mycoplasma meleagridis infection is widespread in turkeys, causing poor growth and feathering, airsacculitis, osteodystrophy, and reduction in hatchability. Like most mycoplasma species, M. meleagridis is characterized by its inability to synthesize purine and pyrimidine nucleotides de novo. Consistent with this intrinsic deficiency, we here report the cloning, expression, and characterization of a M. meleagridis gene sequence encoding a major surface nuclease, referred to as Mm19. Mm19 consists of a 1941-bp ORF encoding a 646-amino-acid polypeptide with a predicted molecular mass of 74,825 kDa. BLASTP analysis revealed a significant match with the catalytic/dimerization domain of type II restriction enzymes of the RE_AlwI superfamily. This finding is consistent with the genomic location of Mm19 sequence, which dispalys characteristics of a typical type II restriction-modification locus. Like intact M. meleagridis cells, the E. coli-expressed Mm19 fusion product was found to exhibit a nuclease activity against plasmid DNA, double-stranded DNA, single-stranded DNA, and RNA. The Mm19-associated nuclease activity was consistently enhanced with Mg2+ divalent cations, a hallmark of type II restriction enzymes. A rabbit hyperimmune antiserum raised against the bacterially expressed Mm19 strongly reacted with M. meleagridis intact cells and fully neutralized the surface-bound nuclease activity. Collectively, the results show that M. meleagridis expresses a strong surface-bound nuclease activity, which is the product of a single gene sequence that is related to the RE_AlwI superfamily of endonucleases.

Proteínas de Bactérias/metabolismo , Endonucleases/metabolismo , Mycoplasma meleagridis/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Escherichia coli/genética , Soros Imunes , Mycoplasma meleagridis/enzimologia , Coelhos
Rev. investig. vet. Perú (Online) ; 22(3): 274-277, jul.-sept 2011. ilus
Artigo em Espanhol | LIPECS | ID: biblio-1110790


Se determinó la prevalencia de anticuerpos a Mycoplasma meleagridis en 20 lotes de pavos reproductores del departamento de Lima durante el año 2007. Se tomaron 400 muestras de sangre de pavos machos y hembras, entre 8 y 57 semanas de edad, en los distritos de Asia y Chilca. Las muestras se analizaron mediante el ensayo de inmunoabsorción ligado a la enzima (ELISA) a fin de determinar la presencia de anticuerpos contra M. meleagridis. Se encontraron tres muestras positivas (una por lote), resultando una prevalencia de 0.75 ± 0.04%. La seropositividad por lote afectado fue de 5%, sin que se mostrasen signos clínicos compatibles con la enfermedad. Se concluye que los 20 lotes examinados no fueron expuestos al microorganismo y que estos resultados se deben a reacciones falsas positivas.

A field study was conducted to determine the prevalence of antibodies against Mycoplasma meleagridis in 20 turkey breeder flocks located in the Lima region during 2007. A total of 400 blood samples were taken from male and female birds of 8 to 57 weeks old, from Asia and Chilca districts. The indirect enzyme linked immunosorbent assay (ELISA) was used to measure serum antibodies against M. meleagridis. Three positive samples were found (one per flock) representing a prevalence of 0.75 ± 0.04%. The seropositive reactivity per affected flock was 5%, and without showing clinical signs compatible to those observed in the disease. According to these findings, it is concluded that the 20 flocks were not exposed to M. meleagridis and that the positive results may be due to false positive seroreactions.

Animais , Anticorpos , Ensaio de Imunoadsorção Enzimática , Mycoplasma meleagridis , Perus , Prevalência , Peru
Avian Dis ; 55(1): 8-12, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21500629


Mycoplasma meleagridis (MM) is a major cause of disease and economic loss in turkeys. Formerly it was thought that this species was very host specific and only restricted to turkey. In this study, we report on the recovery of MM from breeding flocks of chickens located near a turkey breeding unit. Ten MM field strains were isolated (by culture on Frey broth medium) from tracheal swabs of chickens displaying clinical signs of mycoplasmosis-essentially respiratory symptoms and poor performance. Assignment of the isolated field strains to MM was confirmed by a growth inhibition assay using MM-specific polyclonal antiserum and by PCR amplification targeting the 16S rRNA sequence as well as the Mm14 sequence, a MM-species-specific DNA fragment previously identified and characterized in our laboratory. The nucleotide sequence of Mm14 proved to be highly conserved among the 10 MM field strains, indicating a common source of infection. However, on the basis of slight differences in sodium dodecyl sulfate-polyacrylamide gel electrophoresis whole-cell proteins and western blot profiles, two groups of the isolated MM field strains could be distinguished. Evidence of MM infection of chickens was further provided by serology, since 13.77% (35/254) of sera proved positive to MM by either rapid serum agglutination or recombinant antigen-based enzyme-linked immunosorbent assay. In addition, sera of all chickens from which MM was isolated were positive for antibodies to MM. Collectively, the data unambiguously show that MM could infect chickens; thus, MM warrants further exploration to determine its pathogenicity in this unusual host.

Galinhas , Infecções por Mycoplasma/veterinária , Mycoplasma meleagridis/isolamento & purificação , Doenças das Aves Domésticas/microbiologia , Animais , Infecções por Mycoplasma/microbiologia , Mycoplasma meleagridis/genética , RNA Ribossômico 16S/genética , Testes Sorológicos/veterinária
J Microbiol Methods ; 82(1): 11-8, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20346992


Novel analytical methods for a next generation of diagnostic devices combine attributes from sensitive, accurate, fast, simple and multiplexed analysis methods. Here, we describe a possible contribution to these by the application of a lateral flow microarray where a panel of recombinant protein antigens was used to differentiate bovine serum samples in the context of the lung disease contagious bovine pleuropneumonia (CBPP). Lateral flow arrays were produced by attaching nitrocellulose onto microscopic slides and spotting of the recombinant proteins onto the membranes. The developed assay included evaluations of substrate matrix and detection reagents to allow for short assay times and convenient read-out options, and to yield a total assay time from sample application to data acquisition of less than ten minutes. It was found that healthy and disease-affected animals could be discriminated (AUC=97%), and we suggest that the use of an antigen panel in combination with the lateral flow device offers an emerging analytical tool towards a simplified but accurate on-site diagnosis.

Proteínas de Bactérias/sangue , Doenças dos Bovinos/diagnóstico , Mycoplasma meleagridis/química , Pleuropneumonia Contagiosa/diagnóstico , Análise Serial de Proteínas/métodos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bovinos , Doenças dos Bovinos/sangue , Doenças dos Bovinos/genética , Doenças dos Bovinos/metabolismo , Mycoplasma meleagridis/genética , Mycoplasma meleagridis/metabolismo , Pleuropneumonia Contagiosa/sangue , Pleuropneumonia Contagiosa/genética , Pleuropneumonia Contagiosa/metabolismo
Avian Dis ; 52(2): 214-21, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18646449


We have previously identified species-specific DNA fragments, referred to as MS2/28 and Mm14, of Mycoplasma synoviae and Mycoplasma meleagridis, respectively. In the present study, we extended our analysis of the MS2/28 fragment that was found to encode a species-specific antigenic site, and we demonstrated the specificity of the Mycoplasma gallisepticum hemagglutinin protein encoded by pMGA1.2 (a member of the vlhA gene family). Then, we combined the Escherichia coli-expressed products of MS2/28, Mm14, and pMGA1.2, to develop a recombinant antigen-based enzyme-linked immunosorbent assay (recELISA), for the simultaneous and specific detection of antibodies to the three aforementioned major avian mycoplasma species. For comparative purposes, a novel in-house crude antigen capture ELISA (capELISA) was developed in parallel. In the latter protocol, the microtiter wells were enriched in species-specific antigens by capturing sonicated crude antigens on coated rabbit polyclonal antibodies that had been extensively adsorbed with the whole antigen of the heterologous species. With regard to rapid serum agglutination, both ELISA tests were highly specific, and they showed a significant correlation when field sera from naturally infected birds were tested. recELISA proved to be highly specific because absorbance values, with the heterologous species, were significantly lower (P < 0.001) than those obtained with capELISA. Given its cost-effectiveness and simplicity, the recombinant antigen-based ELISA seems to represent a valid tool for the specific screening of the three major avian mycoplasma species. recELISA will be particularly useful with regard to trade control because a large number of samples from various fields could be rapidly processed.

Ensaio de Imunoadsorção Enzimática/veterinária , Infecções por Mycoplasma/veterinária , Mycoplasma gallisepticum , Mycoplasma meleagridis , Mycoplasma synoviae , Doenças das Aves Domésticas/diagnóstico , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Sequência de Bases , Galinhas , DNA Bacteriano/genética , Diagnóstico Diferencial , Ensaio de Imunoadsorção Enzimática/métodos , Dados de Sequência Molecular , Infecções por Mycoplasma/diagnóstico , Mycoplasma gallisepticum/genética , Mycoplasma gallisepticum/imunologia , Mycoplasma meleagridis/genética , Mycoplasma meleagridis/imunologia , Mycoplasma synoviae/genética , Mycoplasma synoviae/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Homologia de Sequência de Aminoácidos , Testes Sorológicos/métodos , Testes Sorológicos/veterinária , Especificidade da Espécie , Perus
Vet Microbiol ; 119(1): 31-41, 2007 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-16973309


A recombinant phage library harbouring Mycoplasma meleagridis (MM) genomic DNA fragments was generated in the bacteriophage lambda gt11 expression vector. The library was screened for expression of MM specific antigens with a polyclonal antiserum that had been preadsorbed with antigens of the most common unrelated avian mycoplasma species. A 49-amino acid antigenic domain unique to MM was isolated, expressed in Escherichia coli, and its serodiagnostic potential was demonstrated. An antiserum raised against this MM-specific antigenic domain recognized a cluster of seven membrane-associated MM proteins with molecular masses ranging from 34 to 75 kDa. Overall, this study resulted in the identification of a potent serodiagnostic tool and revealed the complex antigenic nature of MM.

Antígenos de Bactérias/genética , Clonagem Molecular , Mycoplasma meleagridis/genética , Mycoplasma meleagridis/imunologia , Animais , Anticorpos Antibacterianos/metabolismo , Antígenos de Bactérias/biossíntese , Antígenos de Bactérias/imunologia , Sequência de Bases , DNA Bacteriano/química , Escherichia coli/genética , Soros Imunes/metabolismo , Immunoblotting/métodos , Dados de Sequência Molecular , Infecções por Mycoplasma/diagnóstico , Infecções por Mycoplasma/veterinária , Doenças das Aves Domésticas/diagnóstico , Doenças das Aves Domésticas/microbiologia , Especificidade da Espécie , Perus
Avian Dis ; 48(3): 505-11, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15529972


Mycoplasma meleagridis (MM) has the ability to cause bone deformity in turkey poults. However, few pathological lesions have been described and no evidence of MM-induced damage to the bones has been shown. In this study, 17-day-old turkey embryos were inoculated with MM into the allantoic cavity. On the 27th day, eight of the 22 embryos presented with curved toes. Scanning electron microscopy of the tarsometatarsal joints showed fissures in the cartilage. Histological sections of the joints revealed only the infiltration of cells with eosinophilic granules. Immunohistochemical staining (IHS) showed the presence of MM in the aggregates of the bone marrow cells and the cells with eosinophilic granules. Some of these cells were harvested by laser capture microdissection (LCM), lysed, and used as template DNA. With a pair of MM-specific primers in a conventional polymerase chain reaction (PCR), a gene product was amplified, and it comigrated with the MM DNA, which indicates that these captured cells contained MM DNA. Thus, this research shows that inoculation of MM into the turkey embryos produced joint lesions and caused cellular infiltration within the bones.

Infecções por Mycoplasma/veterinária , Mycoplasma meleagridis , Doenças das Aves Domésticas/microbiologia , Doenças das Aves Domésticas/patologia , Articulações Tarsianas/ultraestrutura , Perus/microbiologia , Animais , Primers do DNA , Eosinófilos/patologia , Imuno-Histoquímica/veterinária , Microdissecção/veterinária , Microscopia Eletrônica de Varredura/veterinária , Infecções por Mycoplasma/embriologia , Infecções por Mycoplasma/patologia , Reação em Cadeia da Polimerase/veterinária , Doenças das Aves Domésticas/embriologia , Articulações Tarsianas/embriologia
Avian Dis ; 48(4): 916-20, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15666875


Mycoplasma meleagridis (MM) is a known pathogen for turkeys only. In this study, MM was used to inoculate chicken embryos and tumor cells to assess its pathogenic potential for chickens. In chicken embryos, it caused abnormal-shaped toes and severely denuded tracheae. In chicken tumor cells, MM reduced the cellular capacity to release a chemoattractant that causes the migration of heterophils. MM also caused death and/or a reduced growth rate in chicken HD-11 cells, a macrophage-monocyte-derived cell line. Thus, the data show that MM is a potential pathogen for chicken embryos and chickens cells. Further exploration to determine the pathogenicity in chickens may be warranted.

Infecções por Mycoplasma/veterinária , Mycoplasma meleagridis/patogenicidade , Doenças das Aves Domésticas/microbiologia , Animais , Linhagem Celular Tumoral , Quimiotaxia , Embrião de Galinha/microbiologia , Galinhas/microbiologia , Efeito Citopatogênico Viral , Epitélio/ultraestrutura , Infecções por Mycoplasma/patologia , Doenças das Aves Domésticas/patologia , Traqueia/ultraestrutura
Avian Dis ; 47(3): 611-7, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14562888


We used Myoplsma meleagridis (MM) to infect the RP-9 cells and the eggshell membranes and scanning electron microscopy (SEM) and confocal microscopy to study the interactions between the organisms and the cell surfaces. The surface of the RP-9 cells contained numerous projections. After 24 hr of infection with MM, those projections were either lost or aggregated to the side; MM-like particles could be seen on the surface of the cells, and surface fluorescence could be detected by confocal microscopy. On the surface of MM-infected shell membranes were necrotic fibrous tissues and cells detected by SEM and an intense surface fluorescence detected by confocal microscopy. These results indicate that MM infection of the cell surface can result in cellular damage.

Membrana Celular , Infecções por Mycoplasma/veterinária , Mycoplasma meleagridis/ultraestrutura , Doenças das Aves Domésticas/microbiologia , Perus , Animais , Linhagem Celular , Membrana Celular/microbiologia , Membrana Celular/ultraestrutura , Contagem de Colônia Microbiana/veterinária , Casca de Ovo/microbiologia , Casca de Ovo/ultraestrutura , Gema de Ovo/microbiologia , Microscopia Confocal/veterinária , Microscopia Eletrônica de Varredura/veterinária , Infecções por Mycoplasma/microbiologia , Perus/embriologia , Perus/microbiologia