Identification Of Mycoplasmas Research Articles

First molecular investigation to detect avian Mycoplasma species in clinical samples from laying-hen farms in Tunisia

BackgroundAvian mycoplasmas are known pathogens, which cause severe economic losses in poultry flocks. PCR is a rapid, sensitive, and less expensive diagnostic tool than culture for the identification of mycoplasmas in poultry farms. The objective of this study was to determine by PCR the presence of Mycoplasma spp., Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), and Mycoplasma pullorum (MP) in laying hens located in the Sfax region, in the South of Tunisia.ResultsA total of 781 tracheal swabs were collected from 13 laying-hen farms without clinical signs at the date of sampling. MP was detected by a newly described specific PCR assay. The prevalence calculated from PCR results at the flock level was 100% for Mycoplasma spp., 0% for MG, 84.6% for MS and 61.5% for MP. The overall prevalence at the animal level was 38.7% for Mycoplasma spp., 0% for MG, 25% for MS and 6.4% for MP. The overall prevalence of 100% of avian mycoplasmas in laying-hen farms (38.7% prevalence at the animal level) shows an alarming situation.ConclusionsThese results underline the importance of monitoring the emergence and spread of Mycoplasma strains in farms in order to decrease economic losses due to mycoplasmoses.

Antibodies Specific to Membrane Proteins Are Effective in Complement-Mediated Killing of Mycoplasma bovis.

The metabolic inhibition (MI) test is a classic test for the identification of mycoplasmas, used for measuring the growth-inhibiting antibodies directed against acid-producing mycoplasmas, although their mechanism still remains obscure. To determine the major antigens involved in the immune killing of Mycoplasma bovis, we used a pulldown assay with anti-M. bovis antibodies as bait and identified nine major antigens. Among these antigens, we performed the MI test and determined that the growth of M. bovis could be inhibited effectively in the presence of complement by antibodies against specifically membrane protein P81 or UgpB in the presence of complement. Using a complement killing assay, we demonstrated that M. bovis can be killed directly by complement and that antibody-dependent complement-mediated killing is more effective than that by complement alone. Complement lysis and scanning electron microscopy results revealed M. bovis rupture in the presence of complement. Together, these results suggest that the metabolic inhibition of M. bovis is antibody-dependent complement-mediated killing. This study provides new insights into mycoplasma killing by the complement system and may guide future vaccine development studies for the treatment of mycoplasma infection. Furthermore, our findings also indicate that mycoplasmas may be an appropriate new model for studying the lytic activity of membrane attack complex (MAC).

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Is a Superior Diagnostic Tool for the Identification and Differentiation of Mycoplasmas Isolated from Animals.

In veterinary diagnostic laboratories, identification of mycoplasmas is achieved by demanding, cost-intensive, and time-consuming methods that rely on antigenic or genetic identification. Since matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) seems to represent a promising alternative to the currently practiced cumbersome diagnostics, we assessed its applicability for the identification of almost all mycoplasma species isolated from vertebrate animals so far. For generating main spectrum profiles (MSPs), the type strains of 98 Mycoplasma, 11 Acholeplasma, and 5 Ureaplasma species and, in the case of 69 species, 1 to 7 clinical isolates were used. To complete the database, 3 to 7 representatives of 23 undescribed Mycoplasma species isolated from livestock, companion animals, and wildlife were also analyzed. A large in-house library containing 530 MSPs was generated, and the diversity of spectra within a species was assessed by constructing dendrograms based on a similarity matrix. All strains of a given species formed cohesive clusters clearly distinct from all other species. In addition, phylogenetically closely related species also clustered closely but were separated accurately, indicating that the established database was highly robust, reproducible, and reliable. Further validation of the in-house mycoplasma library using 335 independent clinical isolates of 32 mycoplasma species confirmed the robustness of the established database by achieving reliable species identification with log scores of ≥1.80. In summary, MALDI-TOF MS proved to be an excellent method for the identification and differentiation of animal mycoplasmas, combining convenience, ease, speed, precision, and low running costs. Furthermore, this method is a powerful and supportive tool for the taxonomic resolution of animal mycoplasmas.

Культивирование микоплазм ― ретроспектива и перспективы

Isolation and identification of mycoplasmas from different species of domestic animals have recently become increasingly important. This prompted us to carry out research work to simplify the methods of sampling, cultivation, cloning, and storage of mycoplasmas living on the mucous membranes of dogs and cats. However, the unique properties of these bacteria, actually occupying an intermediate position between extracellular and intracellular parasites, often lead to confusion and misunderstanding. Indeed, even highly qualified veterinarians often have the wrong judgment that mycoplasmas are obligate intracellular bacteria similar Chlamydia or Rickettsia. Therefore, before presenting the main results of our studies, we gave a brief description of these unusual bacteria in this small review, with putting a focus on the properties which prevent to effective laboratory diagnostics.

Mycoplasma mycoidessubsp.capriandMycoplasma mycoidessubsp.mycoidesLC can be grouped into a single subspecies

Mycoplasma mycoides subsp. capri and Mycoplasma mycoides subsp. mycoides LC can be combined into one taxon on the basis of several contributions on both DNA sequence and protein analyses reported in the literature. Moreover, for the differentiation and identification of mycoplasmas of the "mycoides cluster", we investigated the rpoB gene, encoding the beta-subunit of the RNA polymerase. A segment of 527 bp of the rpoB gene was amplified from 31 strains of ruminant mycoplasmas by PCR. The nucleotide sequences were determined and aligned, and accurate genetic relationships were calculated. Cluster analysis of rpoB DNA allowed species differentiation within the "mycoides cluster" and confirmed that M. mycoides subsp. capri and M. mycoides subsp. mycoides LC cannot be distinguished from each other. "Mycoplasma mycoides subsp. capri" is proposed as a common name for both subspecies.

Quantitative detection and phylogeny-based identification of mycoplasmas and ureaplasmas from human immunodeficiency virus type 1-positive patients

More than 20 years ago, Mycoplasma genitalium was first isolated from men with nongonococcal urethritis (NGU). Some strains of Mycoplasma and Ureaplasma are associated with NGU or other genitourinary infections. It is reasonable to suggest that mycoplasmas and ureaplasmas may increase the development of acquired immunodeficiency syndrome in human immunodeficiency virus type 1 (HIV-1)-infected patients. This study investigated a rapid and reliable diagnostic method, based on a LightCycler SYBER green I real-time PCR assay and phylogenetic analysis, for detecting mycoplasmas and ureaplasmas in urine from HIV-1-infected patients. Primers were designed to amplify a 711-bp fragment of the 16S rRNA gene. Positive PCR-amplified products were further sequenced, and a phylogenetic tree was constructed by neighbor joining of the V4 and V5 sequences from 15 prototypes strains. PCR-positive samples were detected in 17 (9.1%) urine samples, their prototypes being as follows: 2/17 (11.2%) M. genitalium, 1/17 (5.9%) M. hominis, 12/17 (70.6%) Ureaplasma urealyticum, 1/17 (5.9%) mixed M. genitalium and M. hominis, and 1/17 (5.9%) M. hominis and U. urealyticum, respectively. Mycoplasma and Ureaplasma load for each specimen was 3.8 x 10 copies/ml to 7.2 x 10(3) copies/ml. Overall, this study demonstrates that real-time PCR and phylogenetic analysis are the fastest available methods for the detection of mycoplasmas in the urogenital tract.

Biochemical characterization and serological identification of mycoplasmas isolated from pneumonic lungs of goats slaughtered in abattoirs in Northern Nigeria

Forty-two (2.65%) pneumonic lungs were seen in 1580 goats slaughtered at three different locations in Northern Nigeria. The pneumonic lungs were screened to identify the possible Mycoplasma species in the animals. Twenty-seven isolates of Mycoplasma isolated from the pneumonic lungs of goats, either alone or in association with other bacteria, were characterized biochemically and identified serologically. The organisms were categorized into six groups videlicet, Group A were identified as Mycoplasma arginini (10); Group B as Mycoplasma capricolum (3); Group C as Mycoplasma mycoides subsp . mycoides LC (2); Group D as Mycoplasma capricolum subsp. capripneumoniae (1); Group E as Mycoplasma bovis (1) and Group F as Mycoplasma V (10). M. arginini and Mycoplasma V were seen in goats ≤3 years and ≥7 years while M. capricolum was consistently identified in goats between 4 and 5 years of age.

122. The use of risk pathways and HACCP to assess CBPP control by use of veterinary cordon fences in Botswana

Rapid and specific detection of organisms belonging to the Mycoplasma mycoides cluster, among them Mycoplasma (M.) mycoides subsp. mycoides SC, the agent of contagious bovine pleuropneumonia, is an important condition for effective control of the respective animal diseases. In an effort to improve diagnosis, a PCR identification scheme for these mycoplasmas was developed. A set of primer combinations derived from the CAP-21 genomic regions of member organisms of the Mycoplasma mycoides cluster was selected by means of which complete differentiation within the cluster can be accomplished. Nested PCR involving cluster-specific amplification at the first stage and group-specific amplification using internal primers at the second stage was shown to be applicable for identification of all six groups forming the cluster. For example, external primers P1/P2 and internal primers P6/P7 were used to distinguish M. mycoides subsp. mycoides SC from LC strains. Using the present PCR procedure, identification of mycoplasmas could be considerably accelerated in comparison to conventional methods (5 h vs. one week) and specificity was also improved.

MOLECULAR IDENTIFICATION AND CHARACTERIZATION OF AVIAN MYCOPLASMAS

Serological techniques are recommended procedures for the identification of Mycoplasma species. They enable separation of mycoplasmas at the species level and are widely used for practical laboratory identification of the isolated microorganisms. However, complex growth media are required for the isolation of Mycoplasma species and some strains of pathogenic Mycoplasma species grow very fastidious and can not be cultured. Introduction of DNA based diagnostic methods in mycoplasma research enabled survey of the whole genome in a single assay and reveal also information about non-coding regions of the genome. Restriction enzyme analysis is a powerful method for differentiation of close related strains but requires high quality of DNA which can only be obtained from cultured mycoplasmas. The increasing number of cloned mycoplasma genomic fragments are successfully used for identification and differentiation of mycoplasma strains and species in hybridization studies. The polymerase chain reaction enables identification and characterization of mycoplasmas without culture directly from clinical material. Large genomic sequencing projects produced complete nucleotide sequence from two Mycoplasma species and several more are in progress. This data will open the possibility to understand better the biology of mycoplasmas.

Identification of mycoplasmas by dot immunobinding on membrane filtration (MF dot).

AbstractThe definitive identification of mycoplasmas is usually based on serological procedures, including growth inhibition (1,2), metabolic inhibition (3,4), immunofluorescence (5–7), and immunobinding assays (8–14). The technique most commonly adopted for the routine identification of mycoplasma species isolated from clinical material is, at present, the immunobinding assay involving either broth culture (12,14), mycoplasma colonies on agar plates, or the imprints of colonies (8–13). Several procedures with polyclonal or monoclonal antibodies (MAb) have been described. All these assay systems are based on the detection of mycoplasma surface antigens, which are believed to be highly specific.KeywordsPlate BottomMycoplasma SpeciesSlow AgitationSurface Antigenic VariationHyperimmune SeronThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Application of Chicken Immune Sera to Identification of Mycoplasmas by the Filter Immunobinding Method.

Application of chicken immune sera to identification of Mycoplasma species by the filter immunobinding (FIB) method was investigated. Immune sera against type strains of 15 Mycoplasma species were prepared in chickens by inoculation with concentrated Mycoplasma organisms. Chicken immune sera, when absorbed in a broth medium and diluted in a blocking solution, showed a definite specificity with homologous Mycoplasma antigens by FIB. The identification results of field isolates of Mycoplasma gallisepticum and M. synoviae by FIB using absorbed chicken immune sera were consistent with those obtained by conventional tests based on biochemical properties and growth inhibition using rabbit immune sera. Chicken immune sera were evidenced to be useful for identification of Mycoplasma species when applied to FIB although they were not applicable in growth and metabolism inhibition tests.

Identification of mycoplasmas by immunofluorescence.

Immunofluorescence has been used as a diagnostic tool for identification of mycoplasmas cultured on artificial medium and also for detection of the organisms in situ in infected hosts and for detection of contaminated cell cultures. The technique has been used in research investigations to locate mycoplasmas in pathogenicity studies in both animals and in organ cultures, and it is also one of the recommended serological tests for the characterization of new species of Mollicutes (). Another diagnostic application of immunofluorescence is the detection of mycoplasma antibodies (), but the topic falls outside the scope of this chapter.

Recovery of mycoplasmas from animals.

Of the 30 or so mollicute species that have been isolated from small and large ruminants, only a handful have been shown to cause disease in their own right. This chapter will concentrate on methods for sampling, transporting, and isolating those pathogenic mycoplasmas, including Mycoplasma mycoides subsp. mycoides small colony (SC), Mycoplasma capricolum subsp. capripneumoniae, Mycoplasma bovis, Mycoplasma agalactiae, and M. m. mycoides large colony (LC), which cause economically important diseases, such as contagious bovine pleuropneumonia (CBPP), contagious caprine pleuropneumonia (CCPP), and contagious agalactia. Although DNA amplification techniques are being used with ever-increasing frequency for the detection and identification of mycoplasmas (see Chapters 18– 20), the isolation of these organisms by conventional techniques is still required by most national and international authorities to confirm disease outbreaks.

Biochemical characteristics in mycoplasma identification.

Mycoplasmas are the smallest known free-living organisms, and adapted to a special mode of life as commensal organisms or opportunistic pathogens. Because of their small genome size, they have limited biosynthetic capacity, which means they lack many biochemical pathways found in the Eubacteria. They are highly adapted to their host, which provides most of its nutritional requirements for growth. For this reason, there are only a few biochemical properties that can be investigated in the diagnostic laboratory. Consequently, identification of mycoplasmas is greatly reliant on serological tests based on the recognition of structural membrane proteins by specific antiserum. However, preliminary biochemical characterization can reduce the battery of sera required for final serological identification.

Serological identification of mycoplasmas by growth and metabolic inhibition tests.

Serological identification of mycoplasmas by growth and metabolic inhibition tests.

Polymerase chain reaction and restriction endonuclease digestion for selected members of the "Mycoplasma mycoides cluster" and Mycoplasma putrefaciens.

A specific diagnostic method using the polymerase chain reaction, together with restriction endonuclease digestion patterns, was developed for members of the "Mycoplasma mycoides cluster" that normally occur in the United States (i.e., Mycoplasma mycoides subsp. mycoides Large Colony and Mycoplasma capricolum subsp. capricolum in addition to "cluster" mycoplasma, bovine serogroup 7, and Mycoplasma putrefaciens. The digestion of "cluster" polymerase chain reaction DNA (1,225 bp) amplification products with restriction enzymes AseI and SspI gave mycoplasma species-specific patterns for all strains of M. mycoides subsp. mycoides Large Colony, M. capricolum subsp. capricolum, and bovine group 7 tested. Moreover, we found a nonspecific amplification product for M. putrefaciens that occurred with the oligonucleotide primers used for the "M. mycoides cluster" reaction. However, the restriction endonuclease digestion patterns observed with the restriction enzymes AluI, AseI, and SspI for M. putrefaciens were different than the digestion patterns obtained for the other "cluster" mycoplasmas. This report confirms the usefulness of polymerase chain reaction DNA amplification allied with restriction enzyme digestion profile analysis for the rapid and specific identification of mycoplasmas belonging to the "M. mycoides cluster" and M. putrefaciens.

A filter immunobinding technique for the rapid detection and simultaneous identification of avian and bovine mycoplasmas.

A filter immunobinding (FIB) method was developed for the detection and identification of mycoplasmas. Type strains of a total of 18 avian and bovine mycoplasma species propagated in broth media were diluted and immobilized on a nitrocellulose membrane as antigens for investigating the specificity with rabbit hyperimmune sera. Non-specific FIB reactions were easily eliminated by the procedure of absorbing rabbit hyperimmune sera in the broth. Absorbed rabbit hyperimmune sera exhibited clear species-specificity with mycoplasma antigens by the FIB. These specific reactions always agreed with the results of identification by tests of biochemical properties and growth inhibition for the isolates of M. bovirhinis, M. bovis, M. columbinum, M. columborale, M. gallisepticum and M. synoviae. Some bovine mycoplasma species, which were impossible to identify by growth inhibition test, because of their strong production of film and spots on the agar, specifically reacted with absorbed rabbit hyperimmune sera against M. bovis in the FIB. The detection limit of mycoplasmas by this method was about 10(4)-10(5) colony-forming units/ml, which is lower than that of colony determination on agar. The FIB seems to be a useful technique for rapid detection and simultaneous identification of mycoplasmas.

Mycoplasmoses in poultry

The most important mycoplasmas isolated from domestic avian species include Mycoplasma gallisepticum (MG), M. synoviae (MS), M. meleagridis (MM) and M. iowae (MI). MG causes chronic respiratory disease of chickens and infectious sinusitis in turkeys, resulting in economic losses. MS causes infectious synovitis or mild upper respiratory disease. MM infects only turkeys, causing airsacculitis and sub-optimal production and hatchability. MI is associated with reduced hatchability in turkey flocks. Transmission is either direct, from bird to bird or through the egg, or indirect. Diagnosis is based on isolation and identification of mycoplasmas, according to biochemical, serological or molecular biology tests, or serological examination of host sera by slide agglutination, haemagglutination inhibition or enzyme-linked immunosorbent assay (ELISA) tests. Antibiotics (i.e. tetracyclines, macrolides, quinolones and tiamulin) may be used for therapeutic treatment or prophylactic medication. The eradication of mycoplasma infection can be achieved through improvements in hygiene and management practices, therapeutic treatment of breeder layers and/or of hatching eggs and better monitoring procedures.

A PCR scheme for differentiation of organisms belonging to the Mycoplasma mycoides cluster

Rapid and specific detection of organisms belonging to the Mycoplasma mycoides cluster, among them Mycoplasma (M.) mycoides subsp. mycoides SC, the agent of contagious bovine pleuropneumonia, is an important condition for effective control of the respective animal diseases. In an effort to improve diagnosis, a PCR identification scheme for these mycoplasmas was developed. A set of primer combinations derived from the CAP-21 genomic regions of member organisms of the Mycoplasma mycoides cluster was selected by means of which complete differentiation within the cluster can be accomplished. Nested PCR involving cluster-specific amplification at the first stage and group-specific amplification using internal primers at the second stage was shown to be applicable for identification of all six groups forming the cluster. For example, external primers P1/P2 and internal primers P6/P7 were used to distinguish M. mycoides subsp. mycoides SC from LC strains. Using the present PCR procedure, identification of mycoplasmas could be considerably accelerated in comparison to conventional methods (5 h vs. one week) and specificity was also improved.

Development of two PCR assays for the identification of mycoplasmas causing contagious agalactia

A new detection test for the mycoplasmas causing contagious agalactia, Mycoplasma agalactiae, M. capricolum subsp. capricolum and M. mycoides subsp. mycoldes L.C., was developed. It was based on two polymerase chain reaction assays: the Ma-PCR for the detection of M. agalactiae and the MYC-PCR for the ‘mycoides cluster’ thus including M. capricolum subsp. capricolum and M. mycoides subsp. mycoides L.C. An M. agalactiae strain was identified by a 933-bp Ma-PCR product and no amplification with the MYC-PCR. In contrast, a 460-bp MYC-PCR product and a negative or a 350-bp Ma-PCR product characterized a ‘mycoides cluster’ strain. M. capricolum subsp. capricolum and M. mycoides subsp. mycoides L.C. were identified by their species-specific Asel pattern of the 460-bp MYC-PCR product.