Assessment of PCR for routine identification 
of species of the <i>Mycoplasma mycoides</i> cluster 
in ruminants

Grand, Dominique Le; Saras, Estelle; Blond, Denis; Solsona, Michel; Poumarat, François

doi:10.1051/vetres:2004037

Cited by 23 publications

(22 citation statements)

References 31 publications

(36 reference statements)

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“…Consequently, to identify these species easily, important efforts have been made over the past decades to develop diagnostic tools that rely on the antigenic (9, 10) and molecular identification of isolates, mainly based on PCR assays (7,(11)(12)(13)(14). Nevertheless, the identification of certain mycoplasmas, such as subspecies belonging to the Mycoplasma mycoides cluster, comprising several important ruminant pathogens that are closely related both antigenically and genetically, has remained problematic (15,16). Although genes encoding 16S rRNA have long been the usual targets for universal PCRs followed by sequence or amplicon analysis (17,18), species-specific PCRs targeting 16S rRNA or other housekeeping genes are often favored because the techniques are more rapid and do not require a specialized laboratory (for reviews, see references 12 and 7).…”

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confidence: 99%

Identification and Subtyping of Clinically Relevant Human and Ruminant Mycoplasmas by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

et al. 2013

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g Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) recently emerged as a technology for the identification of bacteria. In this study, we aimed to evaluate its applicability to human and ruminant mycoplasmal identification, which can be demanding and time-consuming when using phenotypic or molecular methods. In addition, MALDI-TOF MS was tested as a subtyping tool for certain species. A total of 29 main spectra (MSP) from 10 human and 13 ruminant mycoplasma (sub)species were included in a mycoplasma MSP database to complete the Bruker MALDI Biotyper database. After broth culture and protein extraction, MALDI-TOF MS was applied for the identification of 119 human and 143 ruminant clinical isolates that were previously identified by antigenic or molecular methods and for subcultures of 73 ruminant clinical specimens that potentially contained several mycoplasma species. MALDI-TOF MS resulted in accurate (sub)species-level identification with a score of >1.700 for 96% (251/262) of the isolates. The phylogenetically closest (sub)species were unequivocally distinguished. Although mixtures of the strains were reliably detected up to a certain cellular ratio, only the predominant species was identified from the cultures of polymicrobial clinical specimens. For typing purposes, MALDI-TOF MS proved to cluster Mycoplasma bovis and Mycoplasma agalactiae isolates by their year of isolation and genome profiles, respectively, and Mycoplasma pneumoniae isolates by their adhesin P1 type. In conclusion, MALDI-TOF MS is a rapid, reliable, and cost-effective method for the routine identification of high-density growing mycoplasmal species and shows promising prospects for its capacity for strain typing.

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confidence: 99%

Identification and Subtyping of Clinically Relevant Human and Ruminant Mycoplasmas by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

et al. 2013

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“…This cluster includes six taxa, which are phylogenetically closely related based on their 16S rRNA gene sequences (21); present similar serological patterns associated with important intertaxon cross-reactivity (12); and, consequently, are difficult to differentiate. Four taxa contain pathogens for small ruminants, with three, namely, M. mycoides subsp.…”

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confidence: 99%

“…mycoides biotype Small Colony (20,28) and M. capricolum subsp. capripneumoniae (27) are available, but the accurate identification of the remaining taxa of the M. mycoides cluster is more problematic because of intertaxon cross-reactivity and high intrataxon variability (12). Two direct PCR assays currently are proposed in the literature for the specific identification of M. capricolum subsp.…”

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confidence: 99%

Suppression-Subtractive Hybridization as a Strategy To Identify Taxon-Specific Sequences within the Mycoplasma mycoides Cluster: Design and Validation of an M. capricolum subsp. capricolum -Specific PCR Assay

Maigre¹,

Citti

Marenda

et al. 2008

J Clin Microbiol

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The phylogenetically related Mycoplasma capricolum subsp. capricolum and M. mycoides subsp. mycoides biotype Large Colony are two small-ruminant pathogens involved in contagious agalactia. Their respective contributions to clinical outbreaks are not well documented, because they are difficult to differentiate with the current diagnostic techniques. In order to identify DNA sequences specific to one taxon or the other, a suppression-subtractive hybridization approach was developed. DNA fragments resulting from the reciprocal subtraction of the type strains were used as probes on a panel of M. capricolum subsp. capricolum and M. mycoides subsp. mycoides biotype Large Colony strains to assess their intrataxon specificity. Due to a high intrataxon polymorphism and important cross-reactions between taxa, a single DNA fragment was shown to be specific for M. capricolum subsp. capricolum and to be present in all M. capricolum subsp. capricolum field isolates tested in this study. A PCR assay targeting the corresponding gene (simpA51) was designed that resulted in a 560-bp amplification only in M. capricolum subsp. capricolum and in M. capricolum subsp. capripneumoniae (the etiological agent of contagious caprine pleuropneumonia). simpA51 was further improved to generate a multiplex PCR (multA51) that allows the differentiation of M. capricolum subsp. capripneumoniae from M. capricolum subsp. capricolum. Both the simpA51 and multA51 assays accurately identify M. capricolum subsp. capricolum among other mycoplasmas, including all members of the M. mycoides cluster. simpA51 and multA51 PCRs are proposed as sensitive and robust tools for the specific identification of M. capricolum subsp. capricolum and M. capricolum subsp. capripneumoniae.The Mycoplasma genus is composed of wall-less bacteria with small genomes (0.58 to 1.35 Mb) and includes several species known to cause important diseases in humans and animals (24). Most of these species can be grown under laboratory conditions, although they require complex, undefined media and several days to several weeks of incubation because of their limited biosynthetic capacities (24). So far, mycoplasma identification and diagnosis have been based mainly on serological assays after cultivation, but recent ongoing efforts have been directed toward replacing these assays with more rapid and accurate molecular approaches based on the detection of specific DNA sequences.For the veterinary field, members of the so-called Mycoplasma mycoides cluster are of particular concern, because they are important ruminant pathogens that are responsible for economic losses worldwide (1, 28). This cluster includes six taxa, which are phylogenetically closely related based on their 16S rRNA gene sequences (21); present similar serological patterns associated with important intertaxon cross-reactivity (12); and, consequently, are difficult to differentiate. Four taxa contain pathogens for small ruminants, with three, namely, M. mycoides subsp. mycoides biotype Large Colony, M. mycoides subsp. capri,...

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“…Além disso, a PCR-REA é considerada específica para detectar este grupo em amostras de campo, contudo, a PCR-REA não se presta à diferenciação de subespécies do grupo, exceto a cepa MmmSC, sendo um método de escolha para o diagnóstico da CBPP e triagem do GMM, em áreas com suspeita. Estes achados estão de acordo com estudos prévios de Pettersson et al (1996a) (Le Grand et al 2004). Persson et al (1999a) ressaltaram que a detecção de M.mycoides SC após restrição de amplicons possui uma sensibilidade técnica estimada em 30 cópias de DNA por reação de amplificação.…”

Section: Resultados Isolamento E Identificaçãounclassified

Detecção do Grupo Mycoplasma mycoides por imunoperoxidase indireta (IPI) e PCR-REA em conduto auditivo de bovinos

et al. 2010

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465Pesq. Vet. Bras. 30(5): 465-469, maio 2010 RESUMO.-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 -20 o C até uso. Estas amostras foram diluídas até 10 -5 e repicadas em meio Hayflick modificado, sólido e líquido, Mycoplasma mycoides cluster (MMC) was diagnosed by polimerase chain reactionrestriction 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 -20 o C until use. These specimens were diluted up to 10 -5 , inoculated in liquid and solid modified Hayflick´s media and incubated at 37 o C 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 "fried-egg". In this study, 35 strains selected in agreement with their biochemistry and physiologic proprieties, were used. From the 60 cultivated samples, 48 (80.00%) were positive for Mycoplasma spp. Under IPI the prevalence obtained for MMC was 20.0% (12/60) while by PCR-REA it was 41.7% (25/60). The IPI typing of these isolates resulted in 58.3% (7/12) for M.mycoides mycoides LC and 41.7% (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. Detecção do GrupoINDEX TERMS: Mycoplasma mycoides , flushing ear, cattle, PCR-REA diagnostic.

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Assessment of PCR for routine identification of species of the Mycoplasma mycoides cluster in ruminants

Cited by 23 publications

References 31 publications

Identification and Subtyping of Clinically Relevant Human and Ruminant Mycoplasmas by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Identification and Subtyping of Clinically Relevant Human and Ruminant Mycoplasmas by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Suppression-Subtractive Hybridization as a Strategy To Identify Taxon-Specific Sequences within the Mycoplasma mycoides Cluster: Design and Validation of an M. capricolum subsp. capricolum -Specific PCR Assay

Detecção do Grupo Mycoplasma mycoides por imunoperoxidase indireta (IPI) e PCR-REA em conduto auditivo de bovinos

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