Search

Mycoplasma gallisepticum (MG) is an economically important pathogen of poultry worldwide, causing chronic respiratory disease in chickens and turkeys. Differentiation of MG strains is critical, especially in countries where poultry flocks are vaccinated with live vaccines, In this study, oligonucleotide primers were designed based on a region preceding the trinucleotide repeat of a member of the vIhA gene family, and amplicons of 145-352 bp were generated from cultures of 10 different MG strains, including the ts-11, F and 6/85 vaccine strains. High-resolution melting (HRM) curve analysis of the resultant amplicons could differentiate all MG drains. Analysis of the nucleotide sequences of the amplicons from each strain revealed that each melting curve profile related to a unique DNA sequence. The HRM curve profiles (for ts-11) remained consistent after at least five passages under laboratory conditions. PCR-HRM curve analysis of 33 DNA extracts derived from respirator), swabs, or mycoplasma cultures grown from respiratory swabs, of ts-11-vaccinated commercial or specific pathogen-free chickens identified all these specimens, according to their sequences, as ts-11. The potential of the PCR-HRM curve analysis was also shown in the genotyping of 30 additional MG isolates from Europe, the USA and Israel. The results presented in this study indicate that PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping of MG isolates/strains using both MG cultures and clinical swabs. DOI 10.1099/mic.0.031351-0

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia, which affects pig farms worldwide, causing heavy economic losses. In the infection process, this bacterium is exposed to reactive oxygen species (ROS) from its own metabolism or generated by the host as one of the strategies used to neutralize the pathogen. Although the presence of classical antioxidant enzymes would be expected in M. hyopneumoniae, important genes directly related to protection against ROS, such as superoxide dismutase, catalases and glutathione peroxidase, have not been identified by sequence homology in the genome sequence annotation. Among the few identified M. hyopneumoniae genes coding for proteins possibly involved with suppression of ROS-mediated damage, one (tpx) coding for a peroxiredoxin (MhPrx) has been recognized. The sequence and phylogenetic analyses perfomed in this study indicate that MhPrx is closely related to the atypical 2-Cys peroxiredoxin subfamily, although it has only one cysteine in its sequence. The MhPrx coding DNA sequence was cloned and expressed in Escherichia coli to produce a recombinant MhPrx (rMhPrx), which was purified and used to immunize mice and produce an anti-MhPrx polyclonal antiserum. Probing of M. hyopneumoniae extracts with this antiserum demonstrated that MhPrx is expressed in all three tested strains (J, 7422 and 7448). Cross-linking assays and size-exclusion chromatography indicate that rMhPrx forms dimers, as has been established for atypical 2-Cys peroxiredoxins. Furthermore, a metal-catalysed oxidation system was used to assay the activity of rMhPrx, showing that it can protect DNA from ROS-mediated damage and may play an essential role during infection. DOI 10.1099/mic.0.030643-0

Mycoplasma penetrans has the ability to change its surface lipoprotein profiles frequently. The P35 family lipoproteins encoded by the mpl genes are key players in this profile variation. The M. penetrans HF-2 genome has 38 mpl genes that form three gene clusters. Most of these mpl genes have an invertible promoter sequence that is responsible for the ON/OFF switching of individual mpl gene expression. Here, we identified the recombinase that catalyses inversions of the mpl gene promoters. We focused on two open reading frames of the M. penetrans HF-2 genome, namely MYPE2900 and MYPE8180, which show significant homology to the tyrosine site-specific recombinase (Tsr) family proteins. Since genetic tools for M. penetrans are still not developed, we cloned the MYPE2900 and MYPE8180 genes and expressed them in Mycoplasma pneumoniae and Escherichia coll. The promoter regions of the mpl genes [p35 (MYPE6810) or p42 (MYPE6630) genes] were also introduced into M. pneumoniae and E. coil cells expressing MYPE2900 or MYPE8180. Inversion of these promoters occurred in the presence of the MYPE2900 gene but not in the presence of the MYPE8180 gene, indicating that the MYPE2900 gene product is the recombinase that catalyses mpl gene promoter inversions. We used a PCR-based method to detect mpl promoter inversion. This method also enabled us to detect inversions of 10 mpl gene promoters in M. penetrans HF-2 cells. All these promoter inversions occurred at the 12 bp inverted repeat (IR) sequences flanking the promoter sequence. The consensus sequence of these IRs was proposed as TAAYNNNDATTA (Y=C or T; D=A, G or T).

The terminal organelle is a differentiated structure that plays a key role in mycoplasma cytadherence and locomotion. For this reason, the analysis of Mycoplasma genitalium mutants displaying anomalous terminal organelles could improve our knowledge regarding the structural elements required for proper locomotion. In this study, we isolated several M. genitalium mutants having transposon insertions within the mg218 or mg317 genes, which encode the orthologues of Mycoplasma pneumoniae HMW2 and HMW3 cytoskeletal proteins, respectively. As expected, [mg218.sup.-] and [mg31.sup.7-] mutants exhibit a reduced gliding motility, although their ability to attach to solid surfaces was not completely abolished. Interestingly, most of the [mg218.sup.-] mutants expressed N-terminal MG218 derivatives and showed the presence of short terminal organelles retaining many of the functions displayed by this structure in the wild-type strain, suggesting that the N-terminal region of this protein is an essential element in the architecture of the terminal organelle. Separately, the analysis of [mg317.sup.-] mutants indicates that MG317 protein is involved in the formation of the terminal button and contributes to anchoring the electron-dense core to the cell membrane. The results presented here clearly show that MG218 and MG317 proteins are implicated in the maintenance of gliding motility and cytadherence in M. genitalium.

Mycoplasma hyopneumoniae, a component of the porcine respiratory disease complex, colonizes the respiratory tract of swine by binding to the cilia of the bronchial epithelial cells. Mechanisms of pathogenesis are poorly understood for M. hyopneumoniae, but previous work has indicated that it responds to the environmental stressors heat shock, iron deprivation and oxidative compounds. For successful infection, M. hyopneumoniae must effectively resist host responses to the colonization of the respiratory tract. Among these are changes in hormonal levels in the mucosal secretions. Recent work in the stress responses of other bacteria has included the response to the catecholamine norepinephrine. The idea that M. hyopneumoniae can respond to a host hormone, however, is novel and has not previously been demonstrated. To test this, organisms in the early exponential phase of growth were exposed to 100 [micro]M norepinephrine for 4 h, and RNA samples from these cultures were collected and compared to RNA samples from control cultures using two-colour PCR-based M. hyopneumoniae microarrays. The M. hyopneumoniae response included slowed growth and changes in mRNA transcript levels of 84 genes, 53 of which were upregulated in response to norepinephrine. A larger proportion of the genes upregulated than those downregulated were involved with transcription and translation. The downregulated genes were mostly involved with metabolism, which correlated with the reduction in growth of the mycoplasma. Approximately 51% of the genes were hypothetical with no known function. Thus, in response to norepinephrine, M. hyopneumoniae appears to upregulate protein expression while downregulating general metabolism.

The genome of Mycoplasma gallisepticum strain [R.sub.low] has been sequenced completely, but subsequent genetic studies have been limited by the lack of a replicable vector system. In this study, replicable plasmids were constructed for M. gallisepticum and Mycoplasma imitans using the oriC region upstream from the soj gene. The oriC plasmids of M. gallisepticum (pGTLori) and M. imitans (pMlori) replicated in both species, but Mycoplasma pneumoniae could not support replication of pGTLori. A 180 bp section of the oriC region of M. gallisepticum was found to be the minimal region required for plasmid replication in M. gallisepticum strain S6, the shortest oriC region defined for mycoplasmas. Targeted gene disruption of vlhA1.1 of M. gallisepticum S6 was attempted using these oriC plasmids. Constructs made in pPLoriC7 integrated into the M. gallisepticum genomic oriC region, not into the targeted gene, whereas those made in pMlori disrupted the vlhA1.2 gene, which has 97% DNA sequence identity with the vlhA1.1 gene. During in vitro passages, antimicrobial selection pressure did not influence the rate of chromosomal integration. These oriC plasmids will thus be useful for genetic studies, including inactivation or expression of selected genes, in M. gallisepticum and M. imitans, and will lead to a better understanding of their molecular biology. They are, to our knowledge, the first replicable plasmids developed for the Pneumoniae phylogenetic group of mycoplasmas.

Mycoplasma mycoides subspecies rnycoides small colony (SC) is the aetiologic agent of contagious bovine pleuropneumonia (CBPP), a respiratory disease causing important losses in cattle production. The publication of the genome sequence of M. mycoides subsp, mycoides SC should facilitate the identification of putative virulence factors. However, real progress in the study of molecular mechanisms of pathogenicity also requires efficient molecular tools for gene inactivation. In the present study, we have developed a transposon-based approach for the random mutagenesis of M. mycoides subsp, mycoides SC. A PCR-based screening assay enabled the characterization of several mutants with knockouts of genes potentially involved in pathogenicity. The initial transposon was further improved by combining it with the transposon [gamma][delta] TnpR/res recombination system to allow the production of unmarked mutations. Using this approach, we isolated a mutant free of antibiotic-resistance genes, in which the gene encoding the main lipoprotein LppQ was disrupted. The mutant was found to express only residual amounts of the truncated N-terminal end of LppQ. This approach opens the way to study virulence factors and pathogen-host interactions of M. mycoides subsp, mycoides SC and to develop new, genetically defined vaccine strains.

Mycoplasma synoviae is an economically important pathogen of poultry worldwide, causing respiratory infection and synovitis in chickens and turkeys. Identification of M. synoviae isolates is of critical importance, particularly in countries in which poultry flocks are vaccinated with the live attenuated M. synoviae strain MS-H. Using oligonucleotide primers complementary to the single-copy conserved 5' end of the variable lipoprotein and haemagglutinin gene (vlhA), amplicons of ~400 bp were generated from 35 different M. synoviae strains/isolates from chickens and subjected to mutation scanning analysis. Analysis of the amplicons by single-strand conformation polymorphism (SSCP) revealed 10 distinct profiles (A-J). Sequencing of the amplicons representing these profiles revealed that each profile related to a unique sequence, some differing from each other by only one base-pair substitution. Comparative high-resolution melting (HRM) curve analysis of the amplicons using SYTO 9 green fluorescent dye also displayed profiles which were concordant with the same 10 SSCP profiles (A-J) and their sequences. For both mutation detection methods, the Australian M. synoviae strains represented one of the A, B, C or D profiles, while the USA strains represented one of the E, F, G, H, I or J profiles. The results presented in this study show that the PCR-based SSCP or HRM curve analyses of vlhA provide high-resolution mutation detection tools for the detection and identification of M. synoviae strains. In particular, the HRM curve analysis is a rapid and effective technique which can be performed in a single test tube in less than 2 h.

Despite their small size and reduced genomes, many mycoplasma cells have complex structures involved in virulence. Mycoplasma pneumoniae has served as a model for the study of virulence factors of a variety of mycoplasma species that cause disease in humans and animals. These cells feature an attachment organelle, which mediates cytadherence and gliding motility and is required for virulence. An essential component of the architecture of the attachment organelle is an internal detergent-insoluble structure, the electron-dense core. Little information is known regarding its underlying mechanisms. Mycoplasma amphoriforme, a close relative of both M. pneumoniae and the avian pathogen Mycoplasma gallisepticum, is a recently discovered organism associated with chronic bronchitis in immunosuppressed individuals. This work describes both the ultrastructure of M. amphoriforrne strain A3[9.sup.T] as visualized by scanning electron microscopy and the gliding motility characteristics of this organism on glass. Though externally resembling M. gallisepticum, M. amphoriforme cells were found to have a Triton X-100-insoluble structure similar to the M. pneumoniae electron-dense core but with different dimensions. M. amphoriforme also exhibited gliding motility using time-lapse microcinematography; its movement was slower than that of either M. pneumoniae or M. gallisepticum.

Mycoplasma genitalium has been proposed as a suitable model for an in-depth understanding of the biology of a free-living organism. This paper reports that the expression of the aminoglycoside resistance gene aac(6')-aph(2'), the only selectable marker hitherto available for M. genitalium genetic studies, correlates with a growth impairment of the resistant strains. In light of this finding, a tetM438 construction based on the tetracycline resistance gene tetM was developed; it can be used efficiently in M. genitalium and confers multiple advantages when compared to aac(6')-aph(2'). The use of tetM438 significantly improves transformation efficiency and generates visible colonies faster. Finally, the improvements in the pMTnTetM438 construction made it possible to obtain insertions in genes which have not been previously considered to be dispensable under laboratory growth conditions.

A total of 67 Mycoplasma gallisepticum field isolates from the USA, Israel and Australia, and 10 reference strains, were characterized by gene-targeted sequencing (GTS) analysis of portions of the putative cytadhesin pvpA gene, the cytadhesin gapA gene, the cytadhesin mgc2 gene, and an uncharacterized hypothetical surface lipoprotein-encoding gene designated genome coding DNA sequence (CDS) MGA_0319. The regions of the surface-protein-encoding genes targeted in this analysis were found to be stable within a strain, after sequencing different in vitro passages of M. gallisepticum reference strains. Gene sequences were first analysed on the basis of gene size polymorphism. The pvpA and mgc2 genes are characterized by the presence of different nucleotide insertions/deletions. However, differentiation of isolates based solely on pvpA/mgc2 [P.sub.c]R size polymorphism was not found to be a reliable method to differentiate among M. gallisepticum isolates. On the other hand, GTS analysis based on the nucleotide sequence identities of individual and multiple genes correlated with epidemiologically linked isolates and with random amplified polymorphic DNA (RAPD) analysis. GTS analysis of individual genes, gapA, MGA_0319, mgc2 and pvpA, identified 17, 16, 20 and 22 sequence types, respectively. GTS analysis using multiple gene sequences mgc2/pvpa and gapA/MGA_0319/mgc2/pvpA identified 38 and 40 sequence types, respectively. GTS of multiple surface-protein-encoding genes showed better discriminatory power than RAPD analysis, which identified 36 pattern types from the same panel of M. gallisepticum strains. These results are believed to provide the first evidence that typing of M. gallisepticum isolates by GTS analysis of surface-protein genes is a sensitive and reproducible typing method and will allow rapid global comparisons between laboratories.

Mycoplasma synoviae, a major pathogen of poultry, contains a single expressed, full-length vlhA gene encoding its haemagglutinin, and a large number of vlhA pseudogenes that can be recruited by multiple site-specific recombination events to generate chimaeric variants of the expressed gene. The position and distribution of the vlhA pseudogene regions, and their relationship with the expressed gene, have not been investigated. To determine the relationship between these regions, a physical map of the M. synoviae genome was constructed using the restriction endonucleases Smal, I-Ceul, BsiWl, Apal and Xhol and radiolabelled probes for rrnA, recA and tufA. A cloned fragment encoding the unique portion of the expressed vlhA gene and two PCR products containing conserved regions of the ORF 3 and ORF 6 vlhA pseudogenes were used to locate the regions containing these genes on the map. The chromosome of M. synoviae was found to be 890.4 kb and the two rRNA operons were in the same orientation. Both the expressed vlhA gene and the vlhA pseudogenes were confined to the same 114 kb region of the chromosome. These findings indicate that, unlike Mycoplasma gallisepticum, in which the vlhA genes are located in several loci around the chromosome and in which antigenic variation is generated by alternating transcription of over 40 translationally competent genes, M. synoviae has all of the vlhA sequences clustered together, suggesting that close proximity is needed to facilitate the site-specific recombinations used to generate diversity in the expressed vlhA gene.

The phylogenically related Mycoplasma agalactiae and Mycoplasma bovis species are two ruminant pathogens difficult to differentiate and for which a limited amount of sequence data are available. To assess the degree of genomic diversity existing between and within these mycoplasma species, sets of DNA fragments specific for M. bovis type-strain PG45 or for M. agalactiae type-strain PG2 were isolated by suppression subtractive hybridization and used as probes on a panel of M. agalactiae and M. bovis field isolates. Results indicated that approximately 70% of the DNA fragments specific to one or the other type strain are represented in all field isolates of the corresponding species. Only one M. bovis isolate, which was first classified as M. agalactiae, reacted with 15% of the PG2-specific probes, while several M. agalactiae isolates reacted with 15% of the PG45-specific probes. Sequence analyses indicated that most of the genomic diversity observed within one species is related to ORFs with (i) no homologies to proteins recorded in the databases or (ii) homologies to proteins encoded by restriction modification systems. Reminiscent of gene transfer as a means for genomic diversity, a PG45-specific DNA fragment with significant homologies to a central protein of an integrative conjugative element of Mycoplasma fermentans (ICEF) was found in most M. bovis field isolates and in a few M. agalactiae isolates. Finally, sequences encoding part of DNA polymerase III were found in both sets of M. agalactiae- and M. bovis-specific DNA fragments and were used to design a species-specific PCR assay for the identification and differentiation of M. agalactiae and M. bovis.

Mycoplasma mobile has a flask-shaped cell morphology and glides toward its tapered end at a rate of 3-7 cell lengths per s (2.0-4.5 [micro]m [s.sup.-1]) by an unknown mechanism. Gliding requires that the surface of the cell is in contact with a solid substrate, such as glass or plastic. In order to characterize the nature of the outer surface of M. mobile, monoclonal antibodies were raised against intact cells and screened for their ability to recognize surface proteins. Four antibodies were identified and their protein targets were determined. One antibody recognized the Gli349 protein, which is known to be involved in glass binding and gliding. This antibody was also able to displace attached M. mobile cells from glass, suggesting that Gli349 is the major adhesion protein in M. mobile. The other three antibodies recognized members of the Mvsp family of proteins, which are presumably the major surface antigens of M. mobile. Immunofluorescence studies were performed to localize these proteins on the surface of M. mobile cells. Gli349 localized to the proximal region of the tapered part of the cell (the 'neck'), while the various Mvsp family members showed several distinct patterns of subcellular localization. MvspN and MvspO localized to the distal end of the tapered part of the cell (the 'head'), MvspK localized to the main part of the cell (the 'body'), and Mvspl localized to both the head and body but not the neck. This analysis shows that M. mobile surprisingly expresses multiple versions of its major surface antigen at once but differentiates its surface by differential localization of the various paralogues.

The genes P1 (MPN141) and ORF6 (MPN142) are essential for the successful colonization of the human respiratory tract by Mycoplasma pneumoniae. Both genes are located in the P1 operon, which consists of three genes. The P1 gene is the second gene in the operon, followed by the ORF6 gene. The P1 gene contains two (RepMP2/3, RepMP4) and the ORF6 gene one (RepMP5) specific repetitive DNA sequence, of which seven to nine similar but not identical copies are dispersed on the genome. Despite this large potential pool for genetic variation, M. pneumoniae isolates from patients contain only one of two distinct combinations of the genes P1 and ORF6. To analyse the functions of the repetitive DNA sequences, two 'new' combinations of the genes P1 and ORF6 were constructed, keeping the P1 gene constant but exchanging RepMP5 copies of the ORF6 gene. M. pneumoniae was transformed with these constructs and the transformants were tested for their ability to grow and survive under in vitro conditions and in guinea pigs. The two transformants colonized the respiratory tract of guinea pigs and showed no obvious differences in their growth behaviour compared to M. pneumoniae isolates from patients. The results indicate that the subtype-specific combinations of the repetitive elements in the P1 and ORF6 genes are not essential for the successful adherence of M. pneumoniae to host cells and the colonization of the respiratory tract of guinea pigs.

Examination of the nucleotide sequences of the 16S-23S intergenic transcribed spacer (ITS) region of Mycoplasma imitans and Mycoplasma gallisepticum identified a putative transposase gene located only in the ITS of M. imitans, which can be used as a genetic marker to distinguish these two species. The relative size of the PCR products of the ITS region allowed a clear distinction to be made between strains of M. imitans and M. gallisepticum, both of which could be readily discriminated from the type strains of all the other recognized avian Mycoplasma species. In addition, the putative transposase gene assigned in the ITS of M. imitans was shown to include a sequence homologous to that of the P75 gene of M. gallisepticum. This is believed to be the first description of an insertion element in the rRNA operon region of a mycoplasma species.

Researchers describe the proteins that complex to the P1 adhesion of Mycoplasma pneumoniae. They include the cytoskeleton-associated 65 kDa protein, the cytoskeleton-forming proteins HMW1 and HMW3, the chaperone DnaK, and the E1alpha subunit of pyruvate dehydrogenase.

Research has been conducted on the aetiological agent of contagious bovine pleuropneumonia. The cloning and characterization of a genetic locus containing difference between Mycoplasma mycoides subsp. mycoides clusters are described.

Research was conducted to determine the most antigenic region of MSPB and to evaluate the potential of a serological test based on the antigen, in comparison to the RSA. A comparison of the RU values of the sera taken from chickens collected at various time points after inoculation with Mycoplasma synoviae revealed that MSPB antibodies could be detected by day 7 post-inoculation. Results indicate that the MSPB-ELISA could be sensitive as the RSA in its ability to detect M. synoviae antibodies in chicken sera early in infection.

Transmission electron microscopy revealed the presence of a 11- to 30-nm capsular material surrounding Mycoplasma penetrans. Staining studies suggest that this capsular material is composed of polysaccharides and anchored to the plasma membrane by polypeptides. Electrophoretic analysis revealed the presence of high- and low-molecular mass glycosylated materials in the capsule with different heat and enzyme sensitivities.

An analysis of the PCR-amplified tuf genes of phytoplasmas coming from various phylogenetic groups was conducted. The methods used in the study were PCR amplification of tuf genes, RFLP analysis and Southern blot hybridization. Findings showed that phytoplasmas possess only one replica of the tuf genes, compared with other gram-positive bacteria. Furthermore, the nucleotide sequencing showed two pairs of primers responsible for the amplification of the majority of phylogenic groups.

Mycoplasma capricolum and Mycoplasma pneumoniae were isolated and purified to understand the thioredoxin reductase system (NTS) in mycoplasmas. Results reveal that NTS activity on these mycoplasmas had an optimal ph of 6.5-7.5 and was dependent on NADPH as an electron donor. Furthermore, their cysteine dithiol active regions have isoleucine rather than threonine. Results also suggest that mycoplasmas may have evolved the NTS to protect themselves from the consequences of their self-generated oxidative challenge.

Mycoplasma hominis 7488 encodes a 120 kDa polypeptide which has a hypervariable part that is recognized by the human humoral immune response. This domain may be essential in evading the host's immune response and helps in the survival of the pathogen. The heterogeneity of the hypervariable domain was confirmed by the gene fragments that are amplified from M. hominis isolates via PCR.

Immunogold electron microscopy shows variable surface proteins VspA, VspB, VspC and VspD, labelled with gold particles, to be located on the surface of single Mycoplasma bovis cells unmasked by other structures. Clones expressing different Vsps as well as individual clones of M. bovis show the same distribution patterns of strongly-, weakly- and un-labelled cells. No correlations are found between the number of labelled colonies in immunobinding assay and those in immunoelectron microscopy. M. bovis clones differ in their surface antigens showing high M. bovis variable potential.

The strains of the Mycoplasma mycoides cluster vary in their patterns of substrate use and the range of substrates used, enabling the identification of various strains. The strains of M. mycoides subsp. mycoides differ in their ability to use mannose, maltose, glucosamine and trehalose. While the type strain F38 of M. capricolum subsp. capricopneumoniae does not oxidize sugars such as glucose, like other strains, it uses lactate, ethanol and 2-oxobutyrate at high concentrations. The substrate utilization pattern is a true parameter of tissue specificity and pathogenicity.

Comparative studies on the operons of Mycoplasma flocculare and M. hyopneumoniae reveal 96.7% homology, similar to that detected for 16s rRNA, and reinforce the concept of phylogenetic inter-relationships. The structural gene sequence elements may form a stem essential for mature 16S and 23S rRNA. The 5S rRNA gene exhibits high homology with the corresponding M. hyopneumoniae structural gene.

The sensitivity of different types of polymerase chain reaction (PCR) primers was evaluated, and their specificity was studied. PCR technology was used to identify Mycoplasma pneumoniae infections. In a triplex polymerase method, the presence of smaller fragments on the primers prevented their annealing or polymerization.

Extensive research on mycoplasma adhesion may eventually lead to the development of a vaccine for M. pneumonia. Mycoplasmas are organisms that lack a cell wall and cause infections by adhering to specific receptors on the host cell membrane. This attachment is due to the presence of adhesins that are incorporated into their cell membrane.Research efforts on M. pneumoniae and M. genitalium has led to the identification of the chemical and molecular properties of the major adhesins P1 and MgPa. These findings pave the way for the development of a vaccine for M. pneumonia based on the P1 molecule.

A study was conducted to analyze the antigenic features and cytolocalization of the Mycoplasma penetrans antigen, P35. Immunoelectron microscopy was carried out to show that P35 is exposed at the cell surface and distributed all over the membrane. Linear B-epitopes were then mapped using the ELISA approach based on a set of overlapping peptides. Results indicated a number of research implications concerning lipoprotein antigenicity during mycoplasma infections.