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More than one million new cancer cases occur worldwide every year. Although many clinical trials are applied and recent diagnostic tools are employed, curing cancer disease is still a great challenge for mankind. Heredity and epigenetics are the main risk factors often related to cancer. Although, the infectious etiological role in carcinogenesis was also theorized. By establishing chronic infection and inflammation in their hosts, several microorganisms were suggested to cause cell transformation. Of these suspicious microorganisms, mycoplasmas were well regarded because of their intimate parasitism with host cells, as well as their silent and insidious role during infections. This assumption has opened many questions about the real role played by mycoplasmas in oncogenesis. Herein, we presented a sum up of many studies among the hundreds which had addressed the Mycoplasma-cancer topic over the past 50 years. Research studies in this field have first started by approving the mycoplasmas malignancy potential. Indeed, using animal models and in vitro experiments in various cell lines from human and other mammalians, many mycoplasmas were proven to cause varied modifications leading to cell transformation. Moreover, many studies have looked upon the Mycoplasma-cancer subject from an epidemiological point of view. Diverse techniques were used to assess the mycoplasmas prevalence in patients with cancer from different countries. Not less than 10 Mycoplasma species were detected in the context of at least 15 cancer types affecting the brain, the breast, the lymphatic system, and different organs in the genitourinary, respiratory, gastrointestinal, and urinary tracts. Based on these revelations, one should concede that detection of mycoplasmas often linked to ‘‘wolf in sheep’s clothing’’ is not a coincidence and might have a role in cancer. Thorough investigations are needed to better elucidate this role. This would have a substantial impact on the improvement of cancer diagnosis and its prevention.

Background The etiology of prostate cancer (PCa) is multiple and complex. Among the causes recently cited are chronic infections engendered by microorganisms that often go unnoticed. A typical illustration of such a case is infection due to mollicutes bacteria. Generally known by their lurking nature, urogenital mollicutes are the most incriminated in PCa. This study was thus carried out in an attempt to establish the presence of these mollicutes by PCR in biopsies of confirmed PCa patients and to evaluate their prevalence. Methods A total of 105 Formalin-Fixed Paraffin-Embedded prostate tissues collected from 50 patients suffering from PCa and 55 with benign prostate hyperplasia were subjected to PCR amplification targeting species-specific genes of 5 urogenital mollicutes species, Mycoplasma genitalium, M. hominis, M. fermentans, Ureaplasma parvum, and U. urealyticum. PCR products were then sequenced to confirm species identification. Results significance was statistically assessed using Chi-square and Odds ratio tests. Results PCR amplification showed no positive results for M. genitalium, M. hominis, and M. fermentans in all tested patients. Strikingly, Ureaplasma spp. were detected among 30% (15/50) of PCa patients. Nucleotide sequencing further confirmed the identified ureaplasma species, which were distributed as follows: 7 individuals with only U. parvum, 5 with only U. urealyticum, and 3 co-infection cases. Association of the two ureaplasma species with PCa cases proved statistically significant (P Ureaplasma spp. were mostly identified in patients aged 60 and above with prostatic specific antigen (PSA) level > 4 ng/ml and an invasive malignant prostate tumor (Gleason score 8–10). Conclusions This study uncovered a significant association of Ureaplasma spp. with PCa arguing in favour of their potential involvement in this condition. Yet, this finding, though statistically supported, warrants a thorough investigation at a much larger scale.

Antimicrobial resistance in a number of bacterial pathogens has been shown to spread clonally. To our knowledge, data about the phylodistribution of drug resistance in Mycoplasma hominis are very scarce. The aims of this study were to assess the antimicrobial susceptibility of Mycoplasma hominis clinical strains in Tunisia, to identify the molecular basis of antibiotic resistance, and to investigate the phylogenetic relationships of resistant strains. This study included 65 molecularly typed Mycoplasma hominis clinical strains recovered from Tunisian patients over 18 years (2000-2018). The antimicrobial susceptibility was tested against nine antibacterial agents using the broth microdilution method. Minimum spanning tree was constructed to establish the phylogenetic relationships among resistant isolates. Fluoroquinolones, doxycycline, and josamycine were found to be the most effective antibacterial agents. However, 22 strains belonging to 11 expanded multilocus sequence types (eSTs) proved resistant to tetracycline. The majority of these eSTs were genetically related, indicative of clonal expansion of tetracycline resistance. The present study provides relevant information on the antibiotic susceptibility of Tunisian M. hominis clinical strains, lending support to a clonal transmission of tetracycline resistance. This is likely to have an important implication in monitoring the spread of drug resistance among M. hominis.

To date, very little is known about avian mycoplasma infections in Tunisia. Mycoplasma gallisepticum is one of the most economically significant pathogens for poultry in Tunisia and worldwide. Based on the paucity of data regarding the genetic profiles and antibacterial behavior of M. gallisepticum strains in Tunisia, the present study was conducted. Genetic typing and phylogenetic relationships of 40 M. gallisepticum strains (20 Tunisian isolates, 19 international strains collection, and the S6 reference strain) were investigated by gene-targeted sequencing (GTS) using four loci (pvpA, mgc2, vlhA, and the intergenic spacer region between the 16S and the 23S rRNA genes). GTS revealed 12 sequence types (ST) that were found to spread over two clonal complexes (CC) and five singletons. Emergence of enrofloxacin and spiramycin resistance among M. gallisepticum local isolates has been revealed using the broth microdilution method. Causal mutations have been identified by sequencing the quinolone-resistance determining region and domain II and V of 23S rRNA as well as the rplD and rplV genes for enrofloxacin- and macrolide-resistant isolates, respectively. The emergence of antibiotic resistance to enrofloxacin and spiramycin has been identified as being related to a distinctive clonal complex formed by four different STs (ST2, ST3, ST4, and ST5), which would suggest that this phenotype was clonally disseminated.

More than one million new cancer cases occur worldwide every year. Although many clinical trials are applied and recent diagnostic tools are employed, curing cancer disease is still a great challenge for mankind. Heredity and epigenetics are the main risk factors often related to cancer. Although, the infectious etiological role in carcinogenesis was also theorized. By establishing chronic infection and inflammation in their hosts, several microorganisms were suggested to cause cell transformation. Of these suspicious microorganisms, mycoplasmas were well regarded because of their intimate parasitism with host cells, as well as their silent and insidious role during infections. This assumption has opened many questions about the real role played by mycoplasmas in oncogenesis. Herein, we presented a sum up of many studies among the hundreds which had addressed the Mycoplasma-cancer topic over the past 50 years. Research studies in this field have first started by approving the mycoplasmas malignancy potential. Indeed, using animal models and in vitro experiments in various cell lines from human and other mammalians, many mycoplasmas were proven to cause varied modifications leading to cell transformation. Moreover, many studies have looked upon the Mycoplasma-cancer subject from an epidemiological point of view. Diverse techniques were used to assess the mycoplasmas prevalence in patients with cancer from different countries. Not less than 10 Mycoplasma species were detected in the context of at least 15 cancer types affecting the brain, the breast, the lymphatic system, and different organs in the genitourinary, respiratory, gastrointestinal, and urinary tracts. Based on these revelations, one should concede that detection of mycoplasmas often linked to ‘‘wolf in sheep's clothing” is not a coincidence and might have a role in cancer. Thorough investigations are needed to better elucidate this role. This would have a substantial impact on the improvement of cancer diagnosis and its prevention.

Antimicrobial resistance in a number of bacterial pathogens has been shown to spread clonally. To our knowledge, data about the phylodistribution of drug resistance in Mycoplasma hominis are very scarce. The aims of this study were to assess the antimicrobial susceptibility of Mycoplasma hominis clinical strains in Tunisia, to identify the molecular basis of antibiotic resistance, and to investigate the phylogenetic relationships of resistant strains. This study included 65 molecularly typed Mycoplasma hominis clinical strains recovered from Tunisian patients over 18 years (2000–2018). The antimicrobial susceptibility was tested against nine antibacterial agents using the broth microdilution method. Minimum spanning tree was constructed to establish the phylogenetic relationships among resistant isolates. Fluoroquinolones, doxycycline, and josamycine were found to be the most effective antibacterial agents. However, 22 strains belonging to 11 expanded multilocus sequence types (eSTs) proved resistant to tetracycline. The majority of these eSTs were genetically related, indicative of clonal expansion of tetracycline resistance. The present study provides relevant information on the antibiotic susceptibility of Tunisian M. hominis clinical strains, lending support to a clonal transmission of tetracycline resistance. This is likely to have an important implication in monitoring the spread of drug resistance among M. hominis.

Additional file 2: File S1. Multiple sequence alignment of the 5′-end of mba gene of the reference strain UPA1 and 19 Ureaplasma serovar 1 clinical strains. File S2. Multiple sequence alignment of the 5′-end of mba gene of the reference strain UPA3 and 35 Ureaplasma serovar 3 clinical strains. File S3. Multiple sequence alignment of the 5′-end of mba gene of the reference strain UPA6 and 19 Ureaplasma serovar 6 clinical strains. File S4. Multiple sequence alignment of the 5′-end of mba gene of the reference strains UUR4, UUR10, UUR12, UUR13 and 19 Ureaplasma serovars 4, 10, 12, 13 clinical strains. File S5. Multiple sequence alignment of the 5′-end of mba gene of the reference strains UUR2, UUR5, UUR8, UUR9 and 19 Ureaplasma serovars 2, 5, 8, 9 clinical strains. File S6. Partial sequence alignment of ParC protein of 26 Ureaplasma isolates resistant to fluoroquinolones (ParC_consensus correspond to ParC protein of UPA3 and UUR8 reference strains). File S7. Partial sequence alignment of ParE protein of 26 Ureaplasma isolates resistant to fluoroquinolones (ParE_consensus correspond to ParE protein of UPA3 and UUR8 reference strains). File S8. Partial sequence alignment of L22 protein of 26 Ureaplasma isolates resistant to macrolides (L22_consensus correspond to L22 protein of UPA3 and UUR8 reference strains). (538.5 KB).

Background Ureaplasma spp. have been implicated in a variety of clinical conditions and certain serovars are likely to be disease-associated. Hence, the ascending trend of Ureaplasma spp. resistance to antimicrobials should deserve more attention. Here we assessed the extent of antimicrobial resistance of Ureaplasma serovars in Tunisia, and investigated the underlying molecular basis. Methods This study included 101 molecularly typed Ureaplasma spp. clinical strains isolated over a 12-year time period (2005–2017). The antimicrobial susceptibility was tested against nine antibacterial agents using the broth microdilution method. Neighbor-joining tree was constructed to establish the phylogenetic relationships among isolates. Results We found that all ureaplasma isolates were resistant to ciprofloxacin and erythromycin, intermediately resistant to azithromycin, and susceptible to doxycycline, moxifloxacin and josamycin. Ofloxacin and levofloxacin resistance was found in 73.27 and 17.82%, respectively, while 37.62% of isolates proved resistant to tetracycline. Consequently, we detected an elevated multidrug resistance rate among ureaplasma isolates (37.62%), particularly among serovars 2, 5, 8, and 9 (77.77% overall), as well as serovars 4, 10, 12, and 13 (52.63% overall). In most cases, drug resistance was found to be associated with known molecular mechanisms, yet we have identified two novel mutations in the L22 protein, which might be associated with macrolide-resistance. Conclusion To our knowledge, this is the first study that reports the widespread expansion of multidrug resistance among Ureaplasma serovars, a finding of importance in terms of both surveillance and antimicrobial usage.

Natural interfaces have revolutionized the way we interact with computers. They have provided in many fields a comfortable and efficient mechanism that requires no computer knowledge, nor artificial controlling devices, but allow as to interoperate via natural gestures. Diverse fields such as entertainment, remote control, medicine, fitness exercise are finding improvements with the introduction of this technology. However, most of these sensorial interfaces only provide support for basic gestures. In this work we show how it is possible to construct your own complex gestures using the underlying capabilities of these sensor devices. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Mycoplasma meleagridis and Mycoplasma gallinarum are bacteria that affect birds, but little is known about the genetic basis of their interaction with chickens and other poultry. Here, we sequenced the genomes of M. meleagridis strain MM_26B8_IPT and M. gallinarum strain Mgn_IPT, both isolated from chickens showing respiratory symptoms, poor growth, reduction in hatchability, and loss of production.

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.

Mycoplasma meleagridis is a prominent turkey bacterial pathogen associated with airsacculitis and reproductive disorders. Notwithstanding the economic losses caused by M. meleagridis , its genome has still not been sequenced. For a better understanding of its genetic background and pathogenicity mechanisms, we sequenced the genome of M. meleagridis type strain ATCC 25294.

In this work, we studied the effect of deposition temperature on the characteristics of silicon thin films deposited by pulsed DC magnetron sputtering. This pulse consists of a successive fine layers (about 25 to 30 nm) deposition separated by a relaxation time. This last is ensured by the injection of a high quantity of hydrogen without stopping plasma. When we increase the temperature of deposition (from 200 °C to 550 °C); some characteristics of the material show variations similar to the results met in the literature. We thus obtain a diminution in the optical gap (from 2.00 eV to 1.66 eV) and a reduction in the hydrogen relative concentration (from 30% to 5%). Otherwise, in the range of deposition temperature between 300 °C and 400 °C, the microstructural parameter “R2090”, the dark conductivity “σobs” and its activation energy “Ea” present a particular evolution. In addition, the samples Raman shift spectra shows that the material deposited at 350 °C presents, in its structure, a microcrystalline phase in addition to the amorphous one.

A μ(2)-process in the Ullmann-type C-O coupling of potassium phenolate and 4-chloropyridine was successfully performed in a combined microwave (MW) and microflow process. Selective MW absorption in a micro-fixed-bed reactor (μ-FBR) by using a supported Cu nanocatalyst resulted in an increased activity compared to an oil-bath heated process. Yields of up to 80 % were attained by using a multisegmented μ-FBR without significant catalyst deactivation. The μ-FBR was packed with beads coated with Cu/TiO(2) and CuZn/TiO(2) catalysts. Temperature measurements along axial positions of the reactor were performed by using a fiber-optic probe in the catalyst bed. The simultaneous application of MW power and temperature sensors resulted in an isothermal reactor at 20 W. Initially, only solvent was used to adjust the MW field density in the cavity and optimize the power utility. Subsequently, the reaction mixture was added to ensure the maximum MW power transfer by adjusting the waveguide stub tuners to steady-state operations as a result of the changed reaction mixture composition and, therefore, the dielectric properties. Finally, the beneficial influence of the Cu/TiO(2)- and CuZn/TiO(2)-coated glass beads (200 μm) on the MW absorption as a result of the additional absorbing effect of the metallic Cu nanoparticles was optimized in a fine-tuning step. For the catalyst synthesis, various sol-gel, deposition, and impregnation methods provided Cu catalyst loadings of around 1 wt %. The addition of Zn to the Cu nanocatalyst revealed an increased catalyst activity owing to the presence of stable Cu(0). Multilaminar mixing was necessary because of the large difference in fluid viscosities. To the best of our knowledge, this work is the first extended experimental survey of the decisive parameters to combine microprocess and single-mode MW technology following the concepts of "novel process windows" for organic syntheses.

Resistance to tetracyclines in genital mycoplasmas is due mainly to acquisition of the tet(M) determinant, which is frequently associated with conjugative transposon elements of the Tn916/Tn1545 family. The aim of the present work was to evaluate the prevalence of tet(M) in Tunisian isolates and to gain an insight into its origin and evolution. Twenty Ureaplasma parvum, two Ureaplasma urealyticum and 48 Mycoplasma hominis isolates, recovered from Tunisian patients with urogenital and infertility disorders, were evaluated for their resistance to tetracyclines and interrogated by PCR amplification for the presence of tet(M) and int-Tn, the gene encoding the integrase of Tn916/Tn1545-like transposons. The resistance rates to tetracyclines were 22.72 and 25.0 % among U. parvum and M. hominis isolates, respectively, with high-level resistance observed in 11 of the 12 resistant M. hominis isolates. All resistant isolates harboured both tet(M) and int-Tn sequences. Nucleotide sequence analysis of the tet(M) amplicon revealed a unique sequence shared by all tetracycline-resistant clinical isolates of both species. Molecular typing indicated that the tetracycline-resistant U. parvum and M. hominis isolates were not clonal. Taken together, these data indicate that a single tet(M) gene sequence type, most probably transmitted via a Tn916/Tn1545-like transposon, contributes to most of the tetracycline resistance in U. parvum and M. hominis isolates in Tunisia. Because this tet(M) gene sequence type was harboured by different Mycoplasma spp. and by phylogenetically distinct isolates within these species, one could reasonably argue that it may have benefited from an efficient horizontal transfer context, making it highly competent to spread.

The Mycoplasma synoviae haemagglutinin gene, vlhA, encodes two major immunodominant and surface-exposed membrane proteins, MSPB and MSPA. Both products are antigenically variable but only MSPA mediates binding to erythrocytes. Previously we have shown that M. synoviae type strain WVU 1853 could express a variant vlhA gene, referred to as MS2/28.1, with a considerably shorter and divergent MSPA region. A finding that prompted detailed characterization of its antigenic and functional properties. Here we mutagenized each of the six opal codons of the variant MS2/28.1 vlhA member into tryptophan, thus allowing its expression in Escherichia coli as well as its cleavage products, MSPB and MSPA. In addition, we expressed 5 contiguous regions of MS2/28.1 extending from the last part of MSPB to the C-terminus of MSPA. Colony immunostaining with region-specific antisera mapped antigenic variation to the N-terminal half of MS2/28.1 MSPA. No haemagglutinating activity was observed for MSPB, but consistent haemadsorption inhibition was mapped to the region extending from amino acid 325 to 344. Inhibition of both haemagglutination and haemadsorption activities were obtained with sera directed against the C-terminal region of MSPA, with the highest titers (1/320 and 1/160, respectively) being recorded for its last 60 residues. Importantly, antibodies to this region also yielded the highest metabolic inhibition titer of 1/1280. Overall, aside from mapping the functional domains of a M. synoviae highly divergent haemagglutinin gene, this study shows that the C-terminal half of its MSPA region induced the highest titers of antibodies inhibiting haemagglutination, haemadsorption, and metabolism.

Background In Mycoplasma synoviae, type strain WVU 1853, a single member of the haemaglutinin vlhA gene family has been previously shown to be expressed. Variants of vlhA are expressed from the same unique vlhA promoter by recruiting pseudogene sequences via site-specific recombination events, thus generating antigenic variability. Using a bacterial stock of M. synoviae WVU 1853 that had been colony purified thrice and maintained in our laboratory at low passage level, we previously identified a vlhA gene-related partial coding sequence, referred to as MS2/28.1. The E. coli-expressed product of this partial coding sequence was found to be immunodominant, suggesting that it might be expressed. Results Reverse transcription-PCR amplification (RT-PCR), using a sense primer located at the 5'-end region of the expected vlhA transcript and a reverse primer located at the 3' end of MS2/28.1 coding sequence, yielded a consistent amplification product showing that MS2/28.1 was indeed transcribed. Nucleotide sequence analysis of the RT-PCR product identified an 1815-nucleotide full-length open reading frame (ORF), immediately preceded by a nucleotide sequence identical to that previously reported for expressed vlhA genes. PCR amplifications using genomic DNA isolated from single colonies further confirmed that the full-length ORF of MS2/28.1 was located downstream of the unique vlhA promoter sequence. The deduced 604-amino acid (aa) sequence showed a perfect sequence identity to the previously reported vlhA expressed genes along the first 224 residues, then highly diverged with only 37.6% aa identity. Despite the fact that this M. synoviae clone expressed a highly divergent and considerably shorter C-terminal haemagglutinin product, it was found to be expressed at the surface of the bacterium and was able to haemagglutinate chicken erythrocytes. Importantly, the E. coli-expressed C-terminal highly divergent 60 residues of MS2/28.1 proved haemagglutination competent. Conclusions In contrast to the previously characterized vlhA expressed variants, MS2/28.1 displayed a highly divergent sequence, while still able to haemagglutinate erythrocytes. Overall, the data provide an indication as to which extent the M. synoviae vlhA gene could vary its antigenic repertoire.

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.

Background Among the surface antigens of Mycoplasma hominis, the P120' protein was previously shown to elicit a subtle antibody response and appears to be relatively conserved. To get better insight into the evolution of this protein, we analysed the genetic variability of its surface exposed region in 27 M. hominis isolates recovered from the genital tract of Tunisian patients with infertility disorders. Methods All specimens were processed for culture and PCR amplification of the N-terminal surface exposed region of p120' gene. PCR products were sequenced to evaluate the genetic variability, to test for adaptive selection, and to infer the phylogenetic relationship of the M. hominis isolates. Results Sequence analysis showed a total of 25 single nucleotide polymorphisms distributed through 23 polymorphic sites, yielding 13 haplotypes. All but one mutation were confined within three distinct regions. Analysis of the amino acid-based phylogenetic tree showed a predominant group of 17 closely related isolates while the remaining appear to have significantly diverged. Conclusion By analysing a larger sample of M. hominis recovered from patients with urogenital infections, we show here that the P120' protein undergoes substantial level of genetic variability at its surface exposed region.

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.

We developed a duplex PCR assay targeting the hemagglutinin multigene families, vlhA and pMGA, of Mycoplasma synoviae and Mycoplasma gallisepticum , respectively. The assay proved to be specific and sensitive enough to justify its use for the simultaneous detection of the two major avian mycoplasma species from field isolates.

A genomic library of Mycoplasma synoviae (MS) was generated by using bacteriophage lambda gt11 as a cloning and expression vector. Identification of recombinant clones highly specific to MS was achieved by screening the library for expression of MS proteins with polyclonal antiserum that had been preadsorbed with 6 heterologous avian mycoplasma species antigens. Expression of the recombinant clones in Escherichia coli followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the total cell lysates and immunoblot yielded a predominant reactive fusion protein of 165 kD. Two clones (MS2/28 and MS2/12) that yielded inserts of different size were selected. The 2 MS DNA inserts were subcloned in a plasmid vector, labeled with digoxigenin, and used as probes for the specific recognition of several MS strains. A high degree of conservation was demonstrated for the MS2/12 and MS2/28 genes in tested MS strains. In addition, neither DNA fragment recognized any other avian mycoplasma species ( M. gallisepticum, M. meleagridis, M. gallinarum, M. iners. M. anatis, and M. iowae), thus indicating their high specificity to MS. The sensitivity of the slot blot hybridization method using digoxigenin-labeled MS2/12 and MS2/28 probes for direct detection of MS from broth cultures of field isolates was 105 colony-forming units/ml. These results demonstrate the effectiveness of adsorbed antisera for the isolation of species-specific mycoplasma DNA and the potential for its use as probes for the specific and direct detection of MS from broth cultures of field isolates.

Cross-reactivities among seven avian mycoplasma species (Mycoplasma gallisepticum, M. synoviae, M. meleagridis, M. iowae, M. anatis, M. gallinarum, and M. iners) were examined using rabbit polyclonal antisera and two monoclonal antibodies (MAbs) against M. pneumoniae: Myc-9 and Myc-4. The major antigens and cross-reacting antigens of the seven mycoplasma species were demonstrated by immunoblotting assay using homologous and heterologous antisera. Two-way cross-reacting antigens of MS and MG, with apparent molecular weights of 44,000 and 36,000, were identified. Additionally, the 36,000 protein of MG was recognized by all of the heterologous antisera. A large number of cross-reactivities, which have been never investigated before, were shown between M. meleagridis and M. synoviae, between M. meleagridis and M. gallinarum, between M. meleagridis and M. iners, and between M. gallinarum and M. iners. All of the cross-reactions existing between the majority of the above species were considerably reduced or eliminated by adsorbing the polyclonal antisera with a pool of heterologous antigens. Two MAbs against a M. pneumoniae protein of a molecular weight of 150,000 were also able to recognize several avian mycoplasma epitopes, of which two common epitopes with molecular weights of 44,000 and 42,000 were present in most of the seven mycoplasma species.