Host cell interactions of novel antigenic membrane proteins of Mycoplasma agalactiae.

BACKGROUND: Mycoplasma agalactiae is the main etiological agent of Contagious Agalactia syndrome of small ruminants notifiable to the World Organization for Animal Health. Despite serious economic losses, successful vaccines are unavailable, largely because its colonization and invasion factors are not well understood. This study evaluates the role of two recently identified antigenic proteins (MAG_1560, MAG_6130) and the cytadhesin P40 in pathogenicity related phenotypes. RESULTS: Adhesion to HeLa and sheep primary mammary stromal cells (MSC) was evaluated using ELISA, as well as in vitro adhesion assays on monolayer cell cultures. The results demonstrated MAG_6130 as a novel adhesin of M. agalactiae whose capacity to adhere to eukaryotic cells was significantly reduced by specific antiserum. Additionally, these proteins exhibited significant binding to plasminogen and extracellular matrix (ECM) proteins like lactoferrin, fibrinogen and fibronectin, a feature that could potentially support the pathogen in host colonization, tissue migration and immune evasion. Furthermore, these proteins played a detrimental role on the host cell proliferation and viability and were observed to activate pro-apoptotic genes indicating their involvement in cell death when eukaryotic cells were infected with M. agalactiae. CONCLUSIONS: To summarize, the hypothetical protein corresponding to MAG_6130 has not only been assigned novel adhesion functions but together with P40 it is demonstrated for the first time to bind to lactoferrin and ECM proteins thereby playing important roles in host colonization and pathogenicity.

Vpma phase variation is important for survival and persistence of Mycoplasma agalactiae in the immunocompetent host.

Despite very small genomes, mycoplasmas retain large multigene families encoding variable antigens whose exact role in pathogenesis needs to be proven. To understand their in vivo significance, we used Mycoplasma agalactiae as a model exhibiting high-frequency variations of a family of immunodominant Vpma lipoproteins via Xer1-mediated site-specific recombinations. Phase-Locked Mutants (PLMs) expressing single stable Vpma products served as first breakthrough tools in mycoplasmology to study the role of such sophisticated antigenic variation systems. Comparing the general clinical features of sheep infected with a mixture of phase-invariable PLMs (PLMU and PLMY) and the wild type strain, it was earlier concluded that Vpma phase variation is not necessary for infection. Conversely, the current study demonstrates the in vivo indispensability of Vpma switching as inferred from the Vpma phenotypic and genotypic analyses of reisolates obtained during sheep infection and necropsy. PLMY and PLMU stably expressing VpmaY and VpmaU, respectively, for numerous in vitro generations, switched to new Vpma phenotypes inside the sheep. Molecular genetic analysis of selected 'switchover' clones confirmed xer1 disruption and revealed complex new rearrangements like chimeras, deletions and duplications in the vpma loci that were previously unknown in type strain PG2. Another novel finding is the differential infection potential of Vpma variants, as local infection sites demonstrated an almost complete dominance of PLMY over PLMU especially during early stages of both conjunctival and intramammary co-challenge infections, indicating a comparatively better in vivo fitness of VpmaY expressors. The data suggest that Vpma antigenic variation is imperative for survival and persistence inside the immunocompetent host, and although Xer1 is necessary for causing Vpma variation in vitro, it is not a virulence factor because alternative Xer1-independent mechanisms operate in vivo, likely under the selection pressure of the host-induced immune response. This singular study highlights exciting new aspects of mycoplasma antigenic variation systems, including the regulation of expression by host factors.

Novel role of Vpmas as major adhesins of Mycoplasma agalactiae mediating differential cell adhesion and invasion of Vpma expression variants.

Mycoplasma agalactiae exhibits antigenic variation by switching the expression of multiple surface lipoproteins called Vpmas. Although implicated to have a significant influence on the pathogenicity, their exact role in pathogen-host interactions has not been investigated so far. Initial attachment to host cells is regarded as one of the most important steps for colonization but this pathogen lacks the typical mycoplasma attachment organelle. The aim of this study was to determine the role of Vpmas in adhesion of M. agalactiae to host cells. 'Phase-Locked' Mutants (PLMs) steadily expressing single well-characterized Vpma lipoproteins served as ideal tools to evaluate the role of each of the six Vpmas in cytadhesion, which was otherwise not possible due to the high-frequency switching of Vpmas in the wildtype strain PG2. Using in vitro adhesion assays with HeLa and sheep mammary epithelial (MECs) and stromal (MSCs) cells, we could demonstrate differences in the adhesion capabilities of each of the six PLMs compared to the wildtype strain. The PLMV mutant expressing VpmaV exhibited the highest adhesion rate, whereas PLMU, which expresses VpmaU showed the lowest adhesion values explaining the reduced in vivo fitness of PLMU in sheep during experimental intramammary and conjunctival infections. Furthermore, adhesion inhibition assays using Vpma-specific polyclonal antisera were performed to confirm the role of Vpmas in M. agalactiae cytadhesion. This led to a significant decrease (p<0.05) in the adhesion percentage of each PLM. Immunofluorescence staining of TX-114 phase proteins extracted from each PLM showed binding of the respective Vpma to HeLa cells and MECs proving the direct role of Vpmas in cytadhesion. Furthermore, as adhesion is a prerequisite for cell invasion, the ability of the six PLMs to invade HeLa cells was also evaluated using the gentamicin protection assay. The results showed a strong correlation between the adhesion rates and invasion frequencies of the individual PLMs. This is the first report that describes a novel function of Vpma proteins in cell adhesion and invasion. Besides the variability of these proteins causing surface antigenic variation, the newly identified phenotypes are likely to play critical roles in the pathogenicity potential of this ruminant pathogen.

The oppD Gene and Putative Peptidase Genes May Be Required for Virulence in Mycoplasma gallisepticum.

Relatively few virulence genes have been identified in pathogenic mycoplasmas, so we used signature-tagged mutagenesis to identify mutants of the avian pathogen Mycoplasma gallisepticum with a reduced capacity to persist in vivo and compared the levels of virulence of selected mutants in experimentally infected chickens. Four mutants had insertions in one of the two incomplete oppABCDF operons, and a further three had insertions in distinct hypothetical genes, two containing peptidase motifs and one containing a member of a gene family. The three hypothetical gene mutants and the two with insertions in oppD1 were used to infect chickens, and all five were shown to have a reduced capacity to induce respiratory tract lesions. One oppD1 mutant and the MGA_1102 and MGA_1079 mutants had a greatly reduced capacity to persist in the respiratory tract and to induce systemic antibody responses against M. gallisepticum The other oppD1 mutant and the MGA_0588 mutant had less capacity than the wild type to persist in the respiratory tract but did elicit systemic antibody responses. Although M. gallisepticum carries two incomplete opp operons, one of which has been acquired by horizontal gene transfer, our results suggest that one of the copies of oppD may be required for full expression of virulence. We have also shown that three hypothetical genes, two of which encode putative peptidases, may be required for full expression of virulence in M. gallisepticum. None of these genes has previously been shown to influence virulence in pathogenic mycoplasmas.

Xer1-independent mechanisms of Vpma phase variation in Mycoplasma agalactiae are triggered by Vpma-specific antibodies.

Despite their small genomes mycoplasmas maintain large multigene families devoted to surface antigenic variation. Although implicated as important factors for mycoplasma pathogenicity and persistence, the role of these antigenic switches in host immune evasion has never been unequivocally proven in these minimalist microbes. Mycoplasma agalactiae exhibits antigenic variation due to Xer1-mediated site-specific DNA inversions of vpma genes encoding abundant multiple surface lipoproteins. To evaluate the biological significance of Vpma oscillations the xer1 recombinase gene has been disrupted in earlier studies to abolish Vpma switching and to generate stable phase-locked mutants (PLMs) steadily expressing a single Vpma product. However, in previous animal infection studies, surprisingly these PLMs switched to new different Vpma phenotypes. The aim of the current study was to demonstrate the influence of anti-Vpma antibodies on change of Vpma expression in PLMs as well as on the wildtype strain. In in vitro assays it is shown that wild type M. agalactiae escapes the negative effects of Vpma-specific antibodies by high-frequency Xer1-mediated switching to alternative Vpma phenoytpes. Even for Xer1-disrupted PLMs that stably expressed the same Vpma for several in vitro generations, the presence of the corresponding Vpma-specific antibody caused repression of the target Vpma and induction of new Vpma phenotypes by novel complex vpma rearrangements like intragenic deletions and gene chimeras. These Xer1-independent vpma recombinations correlated very well with similar PLM switches observed in vivo in an earlier independent study, clearly demonstrating that Vpma phase variation is necessary to express 'Vpma immune evasion proteins' in order to escape the immune response and to survive in the immunocompetent host. The data clearly demonstrate that although the Xer1 recombinase is the sole factor responsible for Vpma switching of wild type M. agalactiae in vitro, other alternative molecular switches operate in its absence under the selective pressure of the immune response. Furthermore, this evasion from the immune attack of the host involves complex vpma rearrangements, a causal relationship that was so far never demonstrated for M. agalactiae, thereby illustrating novel features of its regulation under immune pressure. The results are anticipated to have a direct impact on understanding the in vivo role of surface antigenic variation systems and the immune evasion tactics of other pathogenic mycoplasma species.

Genetic loci of Mycoplasma agalactiae involved in systemic spreading during experimental intramammary infection of sheep.

Mycoplasmas are amongst the most successful pathogens of both humans and animals yet the molecular basis of mycoplasma pathogenesis is poorly understood. This is partly due to the lack of classical virulence factors and little similarity to common bacterial pathogenic determinants. Using Mycoplasma agalactiae as a model we initiated research in this direction by screening a transposon mutant library in the natural sheep host using a negative selection method. Having successfully identified putative factors involved in the colonization of local infection and lymphogenic sites, the current study assessed mutants unable to spread systemically in sheep after experimental intramammary infection. Analysis of distant body sites for complete absence of mutants via SSM PCR revealed that additional set of genes, such as pdhB, oppC, oppB, gtsB, MAG1890, MAG5520 and MAG3650 are required for systemic spreading apart from those that were necessary for initial colonization. Additional in vitro studies with the mutants absent at these systemic sites confirmed the potential role of some of the respective gene products concerning their interaction with host cells. Mutants of pdhB, oppC and MAG4460 exhibited significantly slower growth in the presence of HeLa cells in MEM medium. This first attempt to identify genes exclusively required for systemic spreading provides a basis for further in-depth research to understand the exact mechanism of chronicity and persistence of M. agalactiae.

Carriage of meticillin-resistant staphylococci between humans and animals on a small farm.

BACKGROUND: Meticillin-resistant staphylococci (MRS) are pathogens of increasing importance to human and animal health worldwide. Transmission of meticillin-resistant Staphylococcus aureus (MRSA) between animals and humans has been well documented. By contrast, information about transmission of other Staphylococcus spp. is limited. HYPOTHESIS/OBJECTIVES: The aim of this study was to screen animals and humans on a small farm for nasal carriage of MRS and to assess interspecies exchange. METHODS: After detection of MRSA in a lung sample of a deceased cat, which lived on a small mixed farm, nasal swabs were taken within two weeks, four and 16 months from other animals of various species and humans living on the farm. Swabs were cultured for MRS which were then characterized molecularly. RESULTS: MRSA and meticillin-resistant coagulase negative staphylococci (MRCoNS), including Staphylococcus haemolyticus, S. epidermidis and S. fleurettii, were isolated from humans and different animal species. Typing of the MRS revealed isolates with the same characteristics in different human and animal hosts. CONCLUSIONS AND CLINICAL IMPORTANCE: To the best of the authors' knowledge, this is the first report of carriage of both MRSA and MRCoNS among humans and various animals within a shared environment. The detection of strains with indistinguishable molecular characteristics strongly suggested transmission of these MRS between the various animal species and humans.

Simultaneous Identification of Potential Pathogenicity Factors of Mycoplasma agalactiae in the Natural Ovine Host by Negative Selection.

Mycoplasmas possess complex pathogenicity determinants that are largely unknown at the molecular level. Mycoplasma agalactiae serves as a useful model to study the molecular basis of mycoplasma pathogenicity. The generation and in vivo screening of a transposon mutant library of M. agalactiae were employed to unravel its host colonization factors. Tn4001mod mutants were sequenced using a novel sequencing method, and functionally heterogeneous pools containing 15 to 19 selected mutants were screened simultaneously through two successive cycles of sheep intramammary infections. A PCR-based negative selection method was employed to identify mutants that failed to colonize the udders and draining lymph nodes in the animals. A total of 14 different mutants found to be absent from ≥ 95% of samples were identified and subsequently verified via a second round of stringent confirmatory screening where 100% absence was considered attenuation. Using this criterion, seven mutants with insertions in genes MAG1050, MAG2540, MAG3390, uhpT, eutD, adhT, and MAG4460 were not recovered from any of the infected animals. Among the attenuated mutants, many contain disruptions in hypothetical genes, implying their previously unknown role in M. agalactiae pathogenicity. These data indicate the putative role of functionally different genes, including hypothetical ones, in the pathogenesis of M. agalactiae. Defining the precise functions of the identified genes is anticipated to increase our understanding of M. agalactiae infections and to develop successful intervention strategies against it.

World Health Organization International Standard To Harmonize Assays for Detection of Mycoplasma DNA.

Nucleic acid amplification technique (NAT)-based assays (referred to here as NAT assays) are increasingly used as an alternative to culture-based approaches for the detection of mycoplasma contamination of cell cultures. Assay features, like the limit of detection or quantification, vary widely between different mycoplasma NAT assays. Biological reference materials may be useful for harmonization of mycoplasma NAT assays. An international feasibility study included lyophilized preparations of four distantly related mycoplasma species (Acholeplasma laidlawii, Mycoplasma fermentans, M. orale, M. pneumoniae) at different concentrations which were analyzed by 21 laboratories using 26 NAT assays with a qualitative, semiquantitative, or quantitative design. An M. fermentans preparation was shown to decrease the interassay variation when used as a common reference material. The preparation was remanufactured and characterized in a comparability study, and its potency (in NAT-detectable units) across different NATs was determined. The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) established this preparation to be the "1st World Health Organization international standard for mycoplasma DNA for nucleic acid amplification technique-based assays designed for generic mycoplasma detection" (WHO Tech Rep Ser 987:42, 2014) with a potency of 200,000 IU/ml. This WHO international standard is now available as a reference preparation for characterization of NAT assays, e.g., for determination of analytic sensitivity, for calibration of quantitative assays in a common unitage, and for defining regulatory requirements in the field of mycoplasma testing.

In vitro and in vivo cell invasion and systemic spreading of Mycoplasma agalactiae in the sheep infection model.

Generally regarded as extracellular pathogens, molecular mechanisms of mycoplasma persistence, chronicity and disease spread are largely unknown. Mycoplasma agalactiae, an economically important pathogen of small ruminants, causes chronic infections that are difficult to eradicate. Animals continue to shed the agent for several months and even years after the initial infection, in spite of long antibiotic treatment. However, little is known about the strategies that M. agalactiae employs to survive and spread within an immunocompetent host to cause chronic disease. Here, we demonstrate for the first time its ability to invade cultured human (HeLa) and ruminant (BEND and BLF) host cells. Presence of intracellular mycoplasmas is clearly substantiated using differential immunofluorescence technique and quantitative gentamicin invasion assays. Internalized M. agalactiae could survive and exit the cells in a viable state to repopulate the extracellular environment after complete removal of extracellular bacteria with gentamicin. Furthermore, an experimental sheep intramammary infection was carried out to evaluate its systemic spread to organs and host niches distant from the site of initial infection. Positive results obtained via PCR, culture and immunohistochemistry, especially the latter depicting the presence of M. agalactiae in the cytoplasm of mammary duct epithelium and macrophages, clearly provide the first formal proof of M. agalactiae's capability to translocate across the mammary epithelium and systemically disseminate to distant inner organs. Altogether, the findings of these in vitro and in vivo studies indicate that M. agalactiae is capable of entering host cells and this might be the strategy that it employs at a population level to ward off the host immune response and antibiotic action, and to disseminate to new and safer niches to later egress and once again proliferate upon the return of favorable conditions to cause persistent chronic infections.

Mycoplasma agalactiae Vaccines: Current Status, Hurdles, and Opportunities Due to Advances in Pathogenicity Studies.

Contagious agalactia (CA) is a serious multietiological disease whose classic etiological agent is Mycoplasma agalactiae and which causes high morbidity and mortality rates in infected herds. CA is classified as a notifiable disease by the World Organization for Animal Health due to its significant worldwide economic impact on livestock, primarily involving goat and sheep farms. The emergence of atypical symptoms and strains of M. agalactiae in wildlife ungulates reestablishes its highly plastic genome and is also of great epidemiological significance. Antimicrobial therapy is the main form of control, although several factors, such as intrinsic antibiotic resistance and the selection of resistant strains, must be considered. Available vaccines are few and mostly inefficient. The virulence and pathogenicity mechanisms of M. agalactiae mainly rely on surface molecules that have direct contact with the host. Because of this, they are essential for the development of vaccines. This review highlights the currently available vaccines and their limitations and the development of new vaccine possibilities, especially considering the challenge of antigenic variation and dynamic genome in this microorganism.

Role of the GapA and CrmA cytadhesins of Mycoplasma gallisepticum in promoting virulence and host colonization.

Mycoplasma gallisepticum is an important avian pathogen that commonly induces chronic respiratory disease in chicken. To better understand the mycoplasma factors involved in host colonization, chickens were infected via aerosol with two hemadsorption-negative (HA(-)) mutants, mHAD3 and RCL2, that were derived from a low passage of the pathogenic strain R (Rlow) and are both deficient in the two major cytadhesins GapA and CrmA. After 9 days of infection, chickens were monitored for air sac lesions and for the presence of mycoplasmas in various organs. The data showed that mHAD3, in which the crmA gene has been disrupted, did not promote efficient colonization or significant air sac lesions. In contrast, the spontaneous HA(-) RCL2 mutant, which contains a point mutation in the gapA structural gene, successfully colonized the respiratory tract and displayed an attenuated virulence compared to that of Rlow. It has previously been shown in vitro that the point mutation of RCL2 spontaneously reverts with a high frequency, resulting in on-and-off switching of the HA phenotype. Detailed analyses further revealed that such an event is not responsible for the observed in vivo outcome, since 98.4% of the mycoplasma populations recovered from RCL2-infected chickens still display the mutation and the associated phenotype. Unlike Rlow, however, RCL2 was unable to colonize inner organs. These findings demonstrate the major role played by the GapA and CrmA proteins in M. gallisepticum host colonization and virulence.

MalF is essential for persistence of Mycoplasma gallisepticum in vivo.

There is limited understanding of the molecular basis of virulence in the important avian pathogen Mycoplasma gallisepticum. To define genes that may be involved in colonization of chickens, a collection of mutants of the virulent Ap3AS strain of M. gallisepticum were generated by signature-tagged transposon mutagenesis. The collection included mutants with single insertions in the genes encoding the adhesin GapA and the cytadherence-related protein CrmA, and Western blotting confirmed that these mutants did not express these proteins. In two separate in vivo screenings, two GapA-deficient mutants (ST mutants 02-1 and 06-1) were occasionally recovered from birds, suggesting that GapA expression may not always be essential for persistence of strain Ap3AS. CrmA-deficient ST mutant 33-1 colonized birds poorly and had reduced virulence, indicating that CrmA was a significant virulence factor, but was not absolutely essential for colonization. ST mutant 04-1 contained a single transposon insertion in malF, a predicted ABC sugar transport permease, and could not be reisolated even when inoculated by itself into a group of birds, suggesting that expression of MalF was essential for persistence of M. galliseptium strain Ap3AS in infected birds.

Characterization of methicillin-resistant Staphylococcus spp. carrying the mecC gene, isolated from wildlife.

OBJECTIVES: A recently identified mecA homologue, mecC, in methicillin-resistant Staphylococcus aureus (MRSA) has been isolated from humans and different animal hosts. The aim of this study was to determine antimicrobial resistance and provide molecular characterization of MRSA and methicillin-resistant non-Staphylococcus aureus staphylococci (MRnSA) isolated from wildlife that carried the gene mecC. METHODS: Five S. aureus and one coagulase-negative Staphylococcus isolate displaying phenotypic oxacillin resistance, but not recognized with conventional PCR for mecA, were further characterized by a polyphasic approach. The presence of mecC in all isolates was determined using specific PCR. PCR targeting Panton-Valentine leucocidin (PVL) genes of MRSA was performed. MRSA isolates were genotyped by spa typing and multilocus sequence typing. All isolates were genotyped by staphylococcal cassette chromosome mec (SCCmec) typing. 16S rDNA sequence analysis for MRnSA identification was performed. Antimicrobial susceptibility testing was performed for all isolates. RESULTS: All five MRSA isolates contained the mecC gene, were PVL negative, carried SCCmec type XI and belonged to ST130 (where ST stands for sequence type), with spa types t843, t10513 or t3256, or to ST2620, with spa type t4335. The MRnSA isolate, most closely related to Staphylococcus stepanovicii, carried mecA and blaZ genes related to SCCmec XI. MRSA isolates exhibited resistance to the ß-lactams only. CONCLUSIONS: The MRSA isolates described in this study represent the first detection of mecC-positive MRSA in a European otter (Lutra lutra) and a European brown hare (Lepus europaeus). The MRnSA isolate represents the first isolation of MRnSA from a Eurasian lynx (Lynx lynx).

Ureaplasma antigenic variation beyond MBA phase variation: DNA inversions generating chimeric structures and switching in expression of the MBA N-terminal paralogue UU172.

Phase variation of the major ureaplasma surface membrane protein, the multiple-banded antigen (MBA), with its counterpart, the UU376 protein, was recently discussed as a result of DNA inversion occurring at specific inverted repeats. Two similar inverted repeats to the ones within the mba locus were found in the genome of Ureaplasma parvum serovar 3; one within the MBA N-terminal paralogue UU172 and another in the adjacent intergenic spacer region. In this report, we demonstrate on both genomic and protein level that DNA inversion at these inverted repeats leads to alternating expression between UU172 and the neighbouring conserved hypothetical ORF UU171. Sequence analysis of this phase-variable 'UU172 element' from both U. parvum and U. urealyticum strains revealed that it is highly conserved among both species and that it also includes the orthologue of UU144. A third inverted repeat region in UU144 is proposed to serve as an additional potential inversion site from which chimeric genes can evolve. Our results indicate that site-specific recombination events in the genome of U. parvum serovar 3 are dynamic and frequent, leading to a broad spectrum of antigenic variation by which the organism may evade host immune responses.

Predominant Single Stable VpmaV Expression in Strain GM139 and Major Differences with Mycoplasma agalactiae Type Strain PG2.

Although mycoplasmas have a reduced genome and no cell wall, they have important mechanisms for the antigenic variation in surface lipoproteins that modulate their interactions with the host. Mycoplasma agalactiae, the main etiological agent of contagious agalactia, has a multigene family involved in the high-frequency phase variation in surface lipoproteins called variable proteins of M. agalactiae (Vpmas). The Vpma lipoproteins are involved in the immune evasion, colonization, dissemination, and persistence of M. agalactiae in the host. In this paper, we evaluate the Vpma phenotypic profiles of two different strains of M. agalactiae, namely, GM139 and the type strain PG2, to assess possible correlations between Vpma phase variability and the geographic localization, animal origin, and pathogenicity of these two strains. Using monospecific Vpma antibodies against individual Vpmas in immunoblots, we demonstrate that, unlike PG2, which expresses six Vpma proteins with high-frequency phase variation, colonies of GM139 predominantly express VpmaV and do not exhibit any sectoring phenotype for any Vpma. Since VpmaV is one of the most important Vpmas for cell adhesion and invasion, its predominant sole expression in GM139 without high-frequency variation may be the basis of the differential pathogenicity of GM139 and PG2. Additionally, MALDI-ToF MS analysis also demonstrates significant differences between these two strains and their relatedness with other M. agalactiae strains.

Sheep Infection Trials with 'Phase-Locked' Vpma Expression Variants of Mycoplasma agalactiae-Towards Elucidating the Role of a Multigene Family Encoding Variable Surface Lipoproteins in Infection and Disease.

The significance of large multigene families causing high-frequency surface variations in mycoplasmas is not well-understood. Previously, VpmaY and VpmaU clonal variants of the Vpma family of lipoproteins of M. agalactiae were compared via experimental sheep infections using the two corresponding 'Phase-Locked Mutants'. However, nothing is known about the infectivity of the remaining four Vpma expression variants VpmaX, VpmaW, VpmaZ and VpmaV as they were never evaluated in vivo. Here, in vivo infection and disease progression of all six Vpma expressers constituting the Vpma family of type strain PG2 were compared using the corresponding xer1-disrupted PLMs expressing single well-characterized Vpmas. Each of the six PLMs were separately evaluated using the intramammary sheep infection model along with the control phase-variable wildtype strain PG2. Thorough bacteriological, pathological and clinical examinations were performed, including assessment of milk quality, quantity and somatic cell counts. Altogether, the results indicated that the inability to vary the Vpma expression phase does not hamper the initiation of infection leading to mastitis for all six PLMs, except for PLMU, which showed a defect in host colonization and multiplication for the first 24 h p.i. and pathological/bacteriological analysis indicated a higher potential for systemic spread for PLMV and PLMX. This is the first study in which all isogenic expression variants of a large mycoplasma multigene family are tested in the natural host.

Mycoplasma mucosicanis sp. nov., isolated from the mucosa of dogs.

Fourteen Mycoplasma strains were isolated from the oral cavity and genital tract of asymptomatic dogs. Isolates had been preliminarily identified by conventional serological testing as Mycoplasma bovigenitalium, but in 16S-23S rRNA intergenic spacer PCR-RFLP assays the isolates exhibited an RFLP pattern distinct from M. bovigenitalium PG11(T). Analysis of the 16S rRNA gene placed a representative of the isolates (strain 1642(T)) in the M. bovigenitalium subcluster of the Mycoplasma bovis cluster of mycoplasmas, with the highest sequence similarities to Mycoplasma californicum ST-6(T) (96.4 %), M. bovigenitalium PG11(T) (96.3 %) and Mycoplasma phocirhinis 852(T) (96.2 %). 16S rRNA gene sequence similarities almost equidistant from three recognized species and results obtained by sequence analysis of the 16S-23S rRNA intergenic spacer region, polar lipid profiles and serological reactions indicated that this organism represents a novel species of the genus Mycoplasma for which the name Mycoplasma mucosicanis sp. nov. is proposed, with strain 1642(T) ( = ATCC BAA-1895(T)  = DSM 22457(T)) as the type strain.

Xer1-mediated site-specific DNA inversions and excisions in Mycoplasma agalactiae.

Surface antigen variation in Mycoplasma agalactiae, the etiologic agent of contagious agalactia in sheep and goats, is governed by site-specific recombination within the vpma multigene locus encoding the Vpma family of variable surface lipoproteins. This high-frequency Vpma phase switching was previously shown to be mediated by a Xer1 recombinase encoded adjacent to the vpma locus. In this study, it was demonstrated in Escherichia coli that the Xer1 recombinase is responsible for catalyzing vpma gene inversions between recombination sites (RS) located in the 5'-untranslated region (UTR) in all six vpma genes, causing cleavage and strand exchange within a 21-bp conserved region that serves as a recognition sequence. It was further shown that the outcome of the site-specific recombination event depends on the orientation of the two vpma RS, as direct or inverted repeats. While recombination between inverted vpma RS led to inversions, recombination between direct repeat vpma RS led to excisions. Using a newly developed excision assay based on the lacZ reporter system, we were able to successfully demonstrate under native conditions that such Xer1-mediated excisions can indeed also occur in the M. agalactiae type strain PG2, whereas they were not observed in the control xer1-disrupted VpmaY phase-locked mutant (PLMY), which lacks Xer1 recombinase. Unless there are specific regulatory mechanisms preventing such excisions, this might be the cost that the pathogen has to render at the population level for maintaining this high-frequency phase variation machinery.

In situ hybridization for the detection of Mycoplasma bovis in paraffin-embedded lung tissue from experimentally infected calves.

Mycoplasma bovis DNA was detected in lung tissue of experimentally infected calves by in situ hybridization (ISH) with a nonradioactive, digoxigenin-labeled DNA probe. The 171-base pair DNA probe targeting part of the gene of the major immunodominant variable surface protein A, which is conserved among all vsp genes, was generated by polymerase chain reaction. Four calves between 57 and 63 days old were inoculated intratracheally with 30 ml of a suspension of M. bovis strain 1067 containing 7 x 10(4) colony forming units per milliliter. Two calves inoculated with 30 ml of sterile medium served as control animals. The calves were euthanized and then examined 21 days after inoculation. The ISH method developed in the current study was suitable for the detection of M. bovis DNA in formalin-fixed, paraffin-embedded lung tissue and may be a valuable tool for diagnostic purposes and for further investigating the pathogenesis of M. bovis infection.