Phase-locked mutants of Mycoplasma agalactiae: defining the molecular switch of high-frequency Vpma antigenic variation

Mycoplasma agalactiae, an important pathogen of small ruminants, exhibits antigenic diversity by switching the expression of multiple surface lipoproteins called Vpmas (Variable proteins of M. agalactiae). Although phase variation has been shown to play important roles in many host–pathogen interactions, the biological significance and the mechanism of Vpma oscillations remain largely unclear. Here, we demonstrate that all six Vpma proteins are expressed in the type strain PG2 and all undergo phase variation at an unusually high frequency. Furthermore, targeted gene disruption of the xer1 gene encoding a putative site-specific recombinase adjacent to the vpma locus was accomplished via homologous recombination using a replicon-based vector. Inactivation of xer1 abolished further Vpma switching and the ‘phase-locked’ mutants (PLMs) continued to steadily express only a single Vpma product. Complementation of the wild-type xer1 gene in PLMs restored Vpma phase variation thereby proving that Xer1 is essential for vpma inversions. The study is not only instrumental in enhancing our ability to understand the role of Vpmas in M. agalactiae infections but also provides useful molecular approaches to study potential disease factors in other ‘difficult-to-manipulate’ mycoplasmas

Chronic pneumonia in calves after experimental infection with Mycoplasma bovis strain 1067: Characterization of lung pathology, persistence of variable surface protein antigens and local immune response

Abstract Background Mycoplasma bovis is associated with pneumonia in calves characterized by the development of chronic caseonecrotic lesions with the agent persisting within the lesion. The purposes of this study were to characterize the morphology of lung lesions, examine the presence of M. bovis variable surface protein (Vsp) antigens and study the local immune responses in calves after infection with M. bovis strain 1067. Methods Lung tissue samples from eight calves euthanased three weeks after experimental infection with M. bovis were examined by bacteriology and pathology. Lung lesions were evaluated by immunohistochemical (IHC) staining for wide spectrum cytokeratin and for M. bovis Vsp antigens and pMB67 antigen. IHC identification and quantitative evaluation of CD4+ and CD8+ T lymphocytes and immunoglobulin (IgG1, IgG2, IgM, IgA)-containing plasma cells was performed. Additionally, expression of major histocompatibility complex class II (MHC class II) was studied by IHC. Results Suppurative pneumonic lesions were found in all calves. In two calves with caseonecrotic pneumonia, necrotic foci were surrounded by epithelial cells resembling bronchial or bronchiolar epithelium. In all calves, M. bovis Vsp antigens were constantly present in the cytoplasm of macrophages and were also present extracellularly at the periphery of necrotic foci. There was a considerable increase in numbers of IgG1- and IgG2-positive plasma cells among which IgG1-containing plasma cells clearly predominated. Statistical evaluation of the numbers of CD4+ and CD8+ T cells, however, did not reveal statistically significant differences between inoculated and control calves. In M. bovis infected calves, hyperplasia of bronchus-associated lymphoid tissue (BALT) was characterized by strong MHC class II expression of lymphoid cells, but only few of the macrophages demarcating the caseonecrotic foci were positive for MHC class II. Conclusions The results from this study show that infection of calves with M. bovis results in various lung lesions including caseonecrotic pneumonia originating from bronchioli and bronchi. There is long-term persistence of M. bovis as demonstrated by bacteriology and immunohistochemistry for M. bovis antigens, i.e. Vsp antigens and pMB67. The persistence of the pathogen and its ability to evade the specific immune response may in part result from local downregulation of antigen presenting mechanisms and an ineffective humoral immune response with prevalence of IgG1 antibodies that, compared to IgG2 antibodies, are poor opsonins. </jats:sec

Characterization of selected Gram-negative non-fermenting bacteria isolated from honey bees (Apis mellifera carnica)

International audienceThis study was conducted to improve the knowledge about bacteria associated with honey bees, Apis mellifera carnica. In this survey, the diversity of Gram-negative non-fermenting bacteria isolated and cultivated from pollen loads, honey sac, freshly stored nectar, and honey was investigated. Bacteria were characterized by a polyphasic approach. Based on morphological and physiological characteristics and comparison of isolates protein patterns after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 11 protein similarity groups were established and confirmed by enterobacterial repetitive intergenic consensus PCR. One isolate, representing a protein similarity group (representative strain), was characterized in more detail by analysis of respiratory quinones, amplified ribosomal DNA restriction analysis, and 16S rDNA sequence analysis. Based on the results of these examinations, seven representative strains were identified as members of the genus Pseudomonas. The remaining representative strains were allocated to the genera Acinetobacter, Chryseobacterium, Stenotrophomonas, and Commamonas

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

In Vitro Cell Invasion of Mycoplasma gallisepticum

The ability of the widespread avian pathogen Mycoplasma gallisepticum to invade cultured human epithelial cells (HeLa-229) and chicken embryo fibroblasts (CEF) was investigated by using the gentamicin invasion assay and a double immunofluorescence microscopic technique for accurate localization of cell-associated mycoplasmas. The presence of intracellular mycoplasmas in both cell lines was clearly demonstrated, with organisms entering the eukaryotic cells within 20 min. Internalized mycoplasmas have the ability to leave the cell, but also to survive within the intracellular space over a 48-h period. Frequencies of invasion were shown to differ between the two cell lines, but were also considerably dependent on the mycoplasma input population. Of the prototype strain R, a low-passage population in artificial medium, R(low), was capable of active cell invasion, while a high-passage population, R(high), showed adherence to but nearly no uptake into HeLa-229 and CEF. By passaging R(low) and R(high) multiple times through HeLa-229 cells, the invasion frequency was significantly increased. Taken together, these findings demonstrate that M. gallisepticum has the capability of entering nonphagocytic host cells that may provide this pathogen with the opportunity for resisting host defenses and selective antibiotic therapy, establishing chronic infections, and passing through the respiratory mucosal barrier to cause systemic infections

Rapid detection of Mycobacterium avium subsp. paratuberculosis from cattle and zoo animals by Nested PCR

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis, a suspect causative agent of Crohn's disease in man, is an emerging disease of international proportions affecting all ruminants. Early stage detection of Mycobacterium avium subsp. paratuberculosis infection would accelerate progress in control programmes. Despite new molecular approaches the standard diagnostic test for this disease is at present still the time consuming classic isolation procedure. Therefore, alternative diagnostic tests such as PCR, are needed for quick detection of infected animals. In this study, the conventional enrichment and isolation procedure and two IS900-based PCR methods for detection of Mycobactrium avium subsp. paratuberculosis in clinical samples from zoo animals and cattle were compared. A total number of 48 different clinical specimens obtained from animals suspected of having paratuberculosis were examined. The samples included faeces (n = 15) and organ tissues (n = 33). Of the faecal specimens two were identified as positive by nested PCR, whereas none was positive by single PCR or by culture. 28 organ specimens were found positive by culture. Mycobactrium avium subsp. paratuberculosis DNA was detected by nested PCR in 82% of the organ specimens identified positive by culture (23 samples) as opposed to 57% by single PCR (16 samples). Nested PCR also identified two positive samples that were not detected by either culture or single PCR. These findings show the great potential of nested PCR as a useful tool for the rapid diagnosis of paratuberculosis in animals