Large-scale analysis of the mycoplasma bovis genome identified non-essential, adhesion- and virulence-related genes

Mycoplasma bovis is an important pathogen of cattle causing bovine mycoplasmosis. Clinical manifestations are numerous, but pneumonia, mastitis, and arthritis cases are mainly reported. Currently, no efficient vaccine is available and antibiotic treatments are not always satisfactory. The design of new, efficient prophylactic and therapeutic approaches requires a better understanding of the molecular mechanisms responsible for M. bovis pathogenicity. Random transposon mutagenesis has been widely used in Mycoplasma species to identify potential gene functions. Such an approach can also be used to screen genomes and search for essential and non-essential genes for growth. Here, we generated a random transposon mutant library of M. bovis strain JF4278 containing approximately 4000 independent insertion sites. We then coupled high-throughput screening of this mutant library to transposon sequencing and bioinformatic analysis to identify M. bovis non-essential, adhesion- and virulence- related genes. Three hundred and fifty-two genes of M. bovis were assigned as essential for growth in rich medium. Among the remaining non-essential genes, putative virulence-related factors were subsequently identified. The complete mutant library was screened for adhesion using primary bovine mammary gland epithelial cells. Data from this assay resulted in a list of conditional-essential genes with putative adhesion-related functions by identifying non-essential genes for growth that are essential for host cell-adhesion. By individually assessing the adhesion capacity of six selected mutants, two previously unknown factors and the adhesin TrmFO were associated with a reduced adhesion phenotype. Overall, our study (i) uncovers new, putative virulence-related genes; (ii) offers a list of putative adhesion-related factors; and (iii) provides valuable information for vaccine design and for exploring M. bovis biology, pathogenesis, and host-interaction

Bovine Epithelial in vitro Infection Models for Mycoplasma bovis

Mycoplasma bovis causes bovine mycoplasmosis. The major clinical manifestations are pneumonia and mastitis. Recently an increase in the severity of mastitis cases was reported in Switzerland. At the molecular level, there is limited understanding of the mechanisms of pathogenicity of M. bovis. Host–pathogen interactions were primarily studied using primary bovine blood cells. Therefore, little is known about the impact of M. bovis on other cell types present in infected tissues. Clear in vitro phenotypes linked to the virulence of M. bovis strains or tissue predilection of specific M. bovis strains have not yet been described. We adapted bovine in vitro systems to investigate infection of epithelial cells with M. bovis using a cell line (MDBK: Madin-Darby bovine kidney cells) and two primary cells (PECT: bovine embryonic turbinate cells and bMec: bovine mammary gland epithelial cells). Two strains isolated before and after the emergence of severe mastitis cases were selected. Strain JF4278 isolated from a cow with mastitis and pneumonia in 2008 and strain L22/93 isolated in 1993 were used to assess the virulence of M. bovis genotypes toward epithelial cells with particular emphasis on mammary gland cells. Our findings indicate that M. bovis is able to adhere to and invade different epithelial cell types. Higher titers of JF4278 than L22/93 were observed in co-cultures with cells. The differences in titers reached between the two strains was more prominent for bMec cells than for MDBK and PECT cells. Moreover, M. bovis strain L22/93 induced apoptosis in MDBK cells and cytotoxicity in PECT cells but not in bMec cells. Dose-dependent variations in proliferation of primary epithelial cells were observed after M. bovis infection. Nevertheless, an indisputable phenotype that could be related to the increased virulence toward mammary gland cells is not obvious

Virulence, persistence and dissemination of Mycoplasma bovis

Bovine mycoplasmosis due to Mycoplasma bovis causes several important bovine diseases such as pneumonia, mastitis, arthritis, otitis, genital disorders or keratoconjunctivitis. Variable surface lipoproteins, adhesion, invasion of host cells, modulation of the host immune system, biofilm formation and the release of secondary metabolites like hydrogen peroxide, as well as synergistic infections with other bacterial or viral pathogens are among the more significantly studied characteristics of the bacterium. The aim of this review is to summarize the current knowledge regarding the virulence of M. bovis and additionally, factors contributing to the dissemination and persistence of this pathogen in the bovine host will be discussed

A dominant lineage of Mycoplasma bovis is associated with an increased number of severe mastitis cases in cattle

Mycoplasma bovis is the most frequent etiologic agent of bovine mycoplasmosis. It causes various diseases in bovines and considerable economic loss due to the lack of effective treatment or preventive measures such as vaccination. In contrast to the US, where M. bovis-mastitis has been reported for a long time, M. bovis infections in Switzerland and Austria were predominantly associated with pneumonia and subclinical mastitis. However, since 2007 the situation has changed with the emergence of severe M. bovis-associated mastitis cases in both countries. In order to evaluate the molecular epidemiology of the bacteria isolated from these infections, recent and old Swiss, along with recent Austrian M. bovis isolates were analyzed by a typing method displaying intermediate resolution of evolutionary relationships among isolates called Multi-Locus Sequence Typing (MLST). The analysis of Swiss and Austrian M. bovis isolates revealed two major lineages. Isolates collected since 2007 in both countries cluster in the lineage I including ST5, ST33, ST34, 36, and ST38-40 (clonal complex 1), while all Swiss isolates recovered before 2007 cluster in the lineage II comprising ST17 and ST35 (clonal complex 5). Further investigations are necessary to understand if lineage I has a higher predilection or virulence toward mammary gland cells than the old lineage or if other factors are involved in the increased number of severe mastitis cases

Cytotoxin CctA, a major virulence factor of Clostridium chauvoei conferring protective immunity against myonecrosis.

OBJECTIVE The purpose of this study was to determine the identity of the major toxin of Clostridium chauvoei, an important pathogen of cattle causing black leg and to determine its value as a protective antigen in vaccines against myonecrosis. METHODS Genomic sequence analysis was used to determine potential virulence genes of C. chauvoei. Subsequently, the putative toxin candidate gene was cloned and expressed to obtain recombinant toxin. This toxin was investigated for its cytotoxic activity, hemolysis and its potential as a protective antigen in the guinea pig potency assay. RESULTS A novel protein toxin, named Clostridium chauvoei toxin A (CctA) that belongs to the family of β-barrel pore forming toxins of the leucocidin superfamily of bacterial toxins was discovered by whole genome sequence analysis. The corresponding gene cctA was found in all strains of C. chauvoei analyzed, isolated from various geographical areas over the globe during the last 50 years, but not in other pathogenic Clostridium species. Native CctA and recombinant rCctA produced in Escherichia coli in the form of a rCctA::NusA fusion protein or thrombin processed rCctA were highly cytotoxic for Embryonic Calf Nasal Epithelial (ECaNEp) cells and had high haemolytic activity against sheep erythrocytes in standard haemolysis assays. Polyclonal anti-rCctA rabbit antibodies fully neutralized the cytotoxic and haemolytic activity, not only of rCctA but also of supernatants from cultures of the various C. chauvoei strains, indicating that CctA is the main cytotoxic and haemolytic substance secreted by C. chauvoei. Using a standard vaccine release procedure, we demonstrated that vaccination of guinea pigs with CctA in the form of a fusion protein with the E. coli heat labile toxin B subunit (rCctA::LTB) as a peptide adjuvant protected the animals against challenge with spores of virulent C. chauvoei. CONCLUSIONS CctA is the major virulence factor of C. chauvoei and the main protective antigen in vaccines against blackleg

Bovine Epithelial in vitro Infection Models for Mycoplasma bovis

Mycoplasma bovis causes bovine mycoplasmosis. The major clinical manifestations are pneumonia and mastitis. Recently an increase in the severity of mastitis cases was reported in Switzerland. At the molecular level, there is limited understanding of the mechanisms of pathogenicity of M. bovis. Host–pathogen interactions were primarily studied using primary bovine blood cells. Therefore, little is known about the impact of M. bovis on other cell types present in infected tissues. Clear in vitro phenotypes linked to the virulence of M. bovis strains or tissue predilection of specific M. bovis strains have not yet been described. We adapted bovine in vitro systems to investigate infection of epithelial cells with M. bovis using a cell line (MDBK: Madin-Darby bovine kidney cells) and two primary cells (PECT: bovine embryonic turbinate cells and bMec: bovine mammary gland epithelial cells). Two strains isolated before and after the emergence of severe mastitis cases were selected. Strain JF4278 isolated from a cow with mastitis and pneumonia in 2008 and strain L22/93 isolated in 1993 were used to assess the virulence of M. bovis genotypes toward epithelial cells with particular emphasis on mammary gland cells. Our findings indicate that M. bovis is able to adhere to and invade different epithelial cell types. Higher titers of JF4278 than L22/93 were observed in co-cultures with cells. The differences in titers reached between the two strains was more prominent for bMec cells than for MDBK and PECT cells. Moreover, M. bovis strain L22/93 induced apoptosis in MDBK cells and cytotoxicity in PECT cells but not in bMec cells. Dose-dependent variations in proliferation of primary epithelial cells were observed after M. bovis infection. Nevertheless, an indisputable phenotype that could be related to the increased virulence toward mammary gland cells is not obvious

Sites et monuments : bulletin de la Société pour la protection des paysages et de l'esthétique générale de la France

janvier 19681968/01 (N41)-1968/03

Invasion and persistence of Mycoplasma bovis in embryonic calf turbinate cells.

Mycoplasma bovis is a wall-less bacterium causing bovine mycoplasmosis, a disease showing a broad range of clinical manifestations in cattle. It leads to enormous economic losses to the beef and dairy industries. Antibiotic treatments are not efficacious and currently no efficient vaccine is available. Moreover, mechanisms of pathogenicity of this bacterium are not clear, as few virulence attributes are known. Microscopic observations of necropsy material suggest the possibility of an intracellular stage of M. bovis. We used a combination of a gentamicin protection assay, a variety of chemical treatments to block mycoplasmas entry in eukaryotic cells, and fluorescence and transmission electron microscopy to investigate the intracellular life of M. bovis in calf turbinate cells. Our findings indicate that M. bovis invades and persists in primary embryonic calf turbinate cells. Moreover, M. bovis can multiply within these cells. The intracellular phase of M. bovis may represent a protective niche for this pathogen and contribute to its escape from the host's immune defense as well as avoidance of antimicrobial agents

Occurrence of Brachyspira hyodysenteriae in multiplier pig herds in Switzerland

OBJECTIVE: This research was aimed to determine the occurrence of Brachyspira (B.) hyodysenteriae in Swiss multiplier pig herds. MATERIALS AND METHODS: In a pilot study a direct real-time polymerase chain reaction (PCR) method for B. hyodysenteriae was compared to culture followed by PCR on 106 samples from three herds. Subsequently 40 multiplier herds were epidemiologically characterized and analysed for the presence of B. hyodysenteriae using direct PCR on 1412 rectal swabs. For external validation 20 swabs obtained from two positive conventional herds were analysed. RESULTS: The comparison of direct PCR with culture followed by PCR resulted in a moderate agreement (kappa index: 0.58). In the two conventional herds, 35% of the samples (7/20) tested positive. Samples from 39 multipliers tested negative. In one multiplier herd, 25% (9/36) of the samples tested PCR positive. Risk factors in the multiplier herd may have been rodents or birds, but not pig purchase. CONCLUSION AND CLINICAL RELEVANCE: B. hyodysenteriae have been detected in a Swiss multiplier herd, which underlines the threat of potential spread by replacement pigs. Consequently, a Brachyspira monitoring programme was established for Swiss multiplier herds