A PCR for the detection of mycoplasmas belonging to the Mycoplasma mycoides cluster: Application to the diagnosis of contagious agalactia

Woubit, Salah; Manso-Silván, Lucía; Lorenzon, Sophie; Gaurivaud, Patrice; Poumarat, François; M, Pellet; Singh, Vijendra Pal; Thiaucourt, François

doi:10.1016/j.mcp.2007.05.008

Cited by 21 publications

(16 citation statements)

References 29 publications

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“…Because of the economic importance of CA, control strategies have been developed that are based mainly on the eradication of the infection by an accurate detection of the CA causative agents. This goal can be achieved by two recently developed PCRs, one specific for M. agalactiae (14) and the other one globally targeting the members of the M. mycoides cluster (28). However, the identification of an isolate to the taxon level is important in order to survey the individual prevalence of taxa and the emergence of new molecular types (18).…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

mentioning

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).…”

mentioning

confidence: 99%

“…capri) and M. capricolum subsp. capricolum would be particularly valuable, as both mycoplasma species induce CA in goats; CA is a disease of economic importance with a worldwide distribution (1,28). M. mycoides subsp.…”

mentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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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 resulting, typical "fried-egg" Mycoplasma colonies were examined by light microscopy at 10ϫ magnification. In order to confirm that the isolated colonies were M. agalactiae, we tested the enrichment broth cultures for the presence of strains of Mycoplasma species involved in contagious agalactia in small ruminants (i.e., M. mycoides cluster and M. putrefaciens), using PCR methods previously published (18,29).…”

Section: Capri M Mycoides Subsp Mycoides Lc and M Putrefaciens)mentioning

confidence: 99%

Mycoplasma agalactiae p40 Gene, a Novel Marker for Diagnosis of Contagious Agalactia in Sheep by Real-Time PCR: Assessment of Analytical Performance and In-House Validation Using Naturally Contaminated Milk Samples

et al. 2009

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We evaluated the capacity of the Mycoplasma agalactiae p40 gene as a diagnostic marker for contagious agalactia in sheep by quantitative real-time PCR. The p40 gene encodes an immunodominant adhesin that plays a key role in cytoadhesion of M. agalactiae. The assay was 100% specific, with an analytical sensitivity of 1 genome equivalent (GE), a quantification that is highly linear (R 2 > 0.992) and efficient (PCR efficiency, >0.992) over a 6-log dynamic range, down to 10 GE. We evaluated the capacity of the assay to detect Mycoplasma agalactiae in 797 milk samples (373 raw sheep milk samples from refrigerated tanks of different farms and 424 milk samples from individual sheep of a flock positive for M. agalactiae). In parallel, we also tested the samples by using microbiological isolation coupled with microscopy identification and by a PCR method recommended by the World Organization for Animal Health. While our assay was able to detect 57 (15.28%) positive samples of the 373 milk samples from different farms, identification by microbiological isolation coupled with microscopy detected only 36 (9.65%) samples, and the conventional PCR detected 31 (8.31%) samples. These findings showed that our assay based on the p40 gene is more specific and sensitive for the detection of M. agalactiae in actual natural samples and, thus, can be a promising alternative tool for diagnosis and epidemiological studies of M. agalactiae infection.

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Rapid and sensitive detection of Mollicutes in cell culture by polymerase chain reaction

Shahhosseiny

Hosseiny

Khoramkhorshid

et al. 2010

J. Basic Microbiol.

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Infections with Mollicutes species (such as Mycoplasma, Acholeplasma, and Ureaplasma) can induce a variety of problems in living organisms and laboratory cell cultures. Therefore, it is necessary to establish a routine diagnostic protocol for Mycoplasma infection in order to ensure reliable research results, as well as the safety of commercial biological products. For that purpose a novel PCR-based procedure using specific designed primers complementary to 16S rRNA genome region of mollicute species was evaluated. PCR was optimized and sensitivity and specificity was evaluated by defined cell count concentrations (2-31250 CFU/ml) of different strains of Mycoplasma, Acholeplasma and Ureaplasma. Amplicon (272 bp) was cloned by PCR-cloning and sequenced by dideoxy chain termination. PCR, was found to be able to detect 10 copies of mollicute target DNA. No cross-reactivity with genomic DNA of non-mollicute bacteria or human cell lines was observed. Forty seven human and animal cell lines were evaluated for mollicute contamination. Twenty five cell lines (53%) were correctly identified as contaminated by this molecular approach. The results of this study demonstrated that this PCR-based method is not only fast and reproducible, but also highly sensitive and specific for detecting contaminant mycoplasmas in cell cultures.

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A PCR for the detection of mycoplasmas belonging to the Mycoplasma mycoides cluster: Application to the diagnosis of contagious agalactia

Cited by 21 publications

References 29 publications

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

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

Mycoplasma agalactiae p40 Gene, a Novel Marker for Diagnosis of Contagious Agalactia in Sheep by Real-Time PCR: Assessment of Analytical Performance and In-House Validation Using Naturally Contaminated Milk Samples

Rapid and sensitive detection of Mollicutes in cell culture by polymerase chain reaction

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