Herd-level risk factors for the seropositivity to Mycoplasma hyopneumoniae and the occurrence of enzootic pneumonia among fattening pigs in areas of endemic infection and high pig density.

The aim of the present study was to identify potential risk factors for the occurrence of enzootic pneumonia (EP) in herds situated in a region of high pig density, where a majority of herds is endemically infected with Mycoplasma hyopneumoniae. Between 2006 and 2010, overall 100 herds were enrolled in a case-control study. Data were collected through personal interview with the farmers, clinical examination of pigs and their environments, and serological testing for M. hyopneumoniae, swine influenza virus and porcine reproductive and respiratory syndrome virus. There were 40 case herds (coughing index high, seroprevalence high) with a mean coughing index of 4.3 and a seroprevalence of 86.6%. There were two control groups. Control group I consisted of 25 herds (coughing index low, seroprevalence low) with mean values of 0.7 and 11.2%, and 35 herds were allocated to control group II (coughing index low, seroprevalence high) where the mean coughing index was 0.9 and seroprevalence 86.3%. Case herds and control II herds had an increased age of piglets at weaning compared to control I herds. Any contact between fattening pigs of different age during restocking of compartments increased the risk for the occurrence of EP in a herd. Finally, farms that use living animals for the exposure to gilts during the acclimatization and farms that had increased number of weaned piglets per sow and year were less likely to test positive for M. hyopneumoniae and less likely to develop clinical symptoms of EP in fattening pigs.

Multilocus sequence typing of Mycoplasma agalactiae.

Mycoplasma agalactiae is the main cause of contagious agalactia, a serious disease of sheep and goats, which has major clinical and economic impacts. We have developed a multilocus sequence typing (MLST) scheme using the sequenced genomes of the M. agalactiae strains PG2 and 5632. An MLST scheme based on the genes gltX, metS, gyrB, tufA and dnaA was designed and in total 3468 bp of sequence were analysed for each strain. MLST offers a highly discriminatory typing method for M. agalactiae and was capable of subdividing 53 strains into 17 distinct sequence types, largely according to geographical origin. MLST detected unexpected diversity in recent isolates from Spain, identifying two novel outliers, and enabled typing of novel Mongolian isolates for the first time. Genetic diversity in the sequenced regions was largely due to mutation, with recombination playing a much smaller role. A web-accessible database has been set up for this MLST scheme for M. agalactiae: http://pubmlst.org/magalactiae/. MLST offers a robust, objective molecular epidemiological tool for M. agalactiae that that enables interlaboratory comparison of data.

Detection of Mycoplasma bovis by in-situ hybridization and expression of inducible nitric oxide synthase, nitrotyrosine and manganese superoxide dismutase in the lungs of experimentally-infected calves.

Pneumonic lesions occurring in calves after respiratory infection with Mycoplasma bovis are characterized by subacute or chronic suppurative bronchopneumonia with multiple foci of necrosis and by persistence of M. bovis antigen, which is frequently associated with phagocytes at the periphery of the necrotic foci. The aims of this study were: (1) to investigate the expression of inducible nitric oxide synthase (iNOS), nitrotyrosine (NT) and manganese superoxide dismutase (Mn-SOD) in the lung lesions of calves infected experimentally with M. bovis, and (2) to analyse the distribution and localization of M. bovis DNA by in-situ hybridization and correlate these findings with the immunohistochemical detection of M. bovis antigen. Phagocytic cells infiltrating the lung tissue were characterized using the markers CD68, S100A8 and S100A9. Lung tissue from 18 infected calves and three non-infected controls were examined. All infected calves had an increased number of cells expressing iNOS, NT and Mn-SOD in the inflamed lung tissue. These molecules were most strongly expressed by macrophages demarcating necrotic areas, by altered bronchiolar epithelial cells and by macrophages within obliterated bronchioles. Co-localization of M. bovis DNA, M. bovis antigen and macrophages expressing iNOS, NT and Mn-SOD was observed. These findings suggest that the generation of reactive oxygen and nitrogen species is involved in the development of severe chronic lung damage in M. bovis infection.

Expression of Mycoplasma bovis variable surface membrane proteins in the respiratory tract of calves after experimental infection with a clonal variant of Mycoplasma bovis type strain PG45.

The pathomorphological findings and the expression and distribution of variable surface protein antigens (Vsp) of Mycoplasma (M.) bovis were characterised immunohistochemically in lungs of eight calves following inoculation with a Vsp A-expressing clonal variant of M. bovis type strain PG45. Within 48 h post inoculation (p.i.) an innate immune response dominated by macrophages and neutrophils develops. The monoclonal antibodies (mAbs) 1A1 and 1E5 detected M. bovis Vsp antigens in paraffin tissue sections of seven calves. Vsp antigens were widely distributed and were already present at day two p.i. within macrophages and other lung compartments. Taken together, the results demonstrate that the bovine is unable to eliminate M. bovis during the time period examined. Based on the different immunohistochemical labelling patterns obtained with the mAbs, the results also support the speculation that the in vivo variability of Vsps together with immunological factors may contribute to the chronicity of pulmonary disease.

Prevalence of potentially pathogenic bacteria as genital pathogens in dairy cattle.

Bacteria on the genital mucosa have been studied less in healthy, non-puerperal cows than in cows with puerperal endometritis. We have thus analysed bacteria in swabs from the vagina and cervix of post-puerperal cattle (n = 644). Out of the animals, 6.8% had aborted within the last 12 months, 2.6% and 11.6% showed signs of vaginitis and endometritis, respectively. In 17.2% of cervical swabs pathogenic gram-positive and in 11.5% pathogenic gram-negative bacteria were found. Arcanobacterium pyogenes was isolated from 41.3% of cows with endometritis and from 3.5% without endometritis (p < 0.05). From 12.5% of cows with abortion but from no cow without abortion, Staphylococcus aureus was recovered (p < 0.05). Out of 383 vaginal swabs, 88.3% were positive. In 3.4% of swabs pathogenic gram-positive and in 16.7% pathogenic gram-negative microorganisms were found. The percentage of positive vaginal swabs did not differ between pregnant and non-pregnant animals. In the genital tract, the percentage of swabs positive for normal mucosal bacteria decreased from caudally to cranially (p < 0.05). Pathogenic bacteria were found more often in cervical than in vaginal swabs (p < 0.05). In conclusion, bacteria on the vaginal and cervical mucosa in cattle involve a wide range of species. In animals without endometritis or vaginitis, colonization of the mucosa rather than infection has to be assumed.

Identification and differentiation of canine Mycoplasma isolates by 16S-23S rDNA PCR-RFLP.

Conventional serological methods for the identification of canine mycoplasma isolates depend on the availability of a panel of species-specific diagnostic antisera and are not always reliable in terms of specificity. To enable simultaneous identification of field isolates, PCR-RFLP analysis of the 16S-23S rRNA intergenic spacer region was used to characterize the type strains of the 12 currently described canine mycoplasmas of the Genus Mycoplasma which represent the "classic" non-hemotropic species. The use of 16S-23S rDNA PCR in the first step of this analysis revealed specific size differences of amplicons which allowed to classify these 12 canine Mycoplasma species into three groups. Depending on the length of the amplicon, subsequent RFLP analysis of PCR products using two restriction endonucleases in a single digest (ApoI/DdeI or TaqI/VspI) generated unique banding patterns. For further evaluation of the 16S-23S rDNA PCR-RFLP assay system as identification and differentiation tool, a total of 262 field isolates collected from the canine genital tract were tested. PCR-RFLP results for 251 field isolates correlated with traditional serological test results. The remaining 11 isolates had an RFLP pattern distinct from the type strains included in this study and were identified by 16S rDNA sequencing as closely related to M. sp. HRC689. The PCR-RFLP assay established in this study enabled a rapid, accurate and easily performed identification and differentiation of all 12 currently described non-hemotropic canine Mycoplasma species.

Outbreak of pneumonia and arthritis in beef calves associated with Mycoplasma bovis and Mycoplasma californicum.

During an outbreak of pneumonia and arthritis in beef calves and young cattle on a large farm in north-west Germany, Mycoplasma bovis and Mycoplasma californicum were isolated from tracheobronchial lavage fluids and synovial fluids. The microbiological findings in dead and living animals and the immunohistochemical demonstration of M californicum antigen in lung and arthritic joint tissue, indicated that under poor housing conditions and possibly other predisposing factors, this mycoplasma, like M bovis, can colonise the respiratory tract and may be able to cross the respiratory mucosal barrier to spread through an infected animal and cause systemic infections that may contribute to severe arthritis.

Serological prevalence of Mycoplasma bovis infection in suckling beef cattle in France.

The prevalence of Mycoplasma bovis infection in France was assessed by means of a serological survey of suckling beef cattle, using an ELISA. The survey included 824 randomly selected herds in eight French counties and a total of 32,197 animals more than one year old. In each county, the number of herds tested was determined statistically on the basis of the hypothesis that about 40 per cent of herds are infected. The proportion of herds containing at least one infected animal ranged from 28 to 90 per cent depending on the county. Among the 32,197 sera tested, the animal infection rate ranged between 2 per cent and 13 per cent. In infected herds, the average number of positive animals per herd was between 10 and 20 per cent, and the infection was unevenly distributed among the areas tested.

Phase variation among major surface antigens of Mycoplasma penetrans.

The pathogenicity and prevalence of Mycoplasma penetrans, a Mycoplasma species recently isolated from humans, are still debated. A major P35 antigen, which is used as target epitope in serological assays, was shown to be a phase-variable lipid-associated membrane protein (LAMP). In this study, we performed a comparative analysis of the LAMP patterns from five M. penetrans clinical isolates and from the type strain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and immunoblots with sera serially collected from an M. penetrans-infected patient indicated that these strains expressed different LAMP repertoires. Furthermore, the intraclonal variation in the expression of LAMPs (P34A, P34B, P35, and P38) was monitored by immunoblot analysis with three specific monoclonal antibodies (MAbs) developed in this study and MAb 7 to P35. The phase variation of these LAMPs occurs in an independent manner, with frequencies of variation ranging from 10(-2) to 10(-4) per cell per generation. Consistent with their amphipathic nature, the P34B and P38 antigens were found exposed at the cell surface. The DNA sequence encoding the P38 antigen was defined and found to be related to those of the P35 gene and other putative LAMP-encoding genes, suggesting that these variable antigens are encoded by a family of related genes. Finally, the serum samples from an M. penetrans-infected patient contained antibodies that reacted with a P36 antigen expressed in different M. penetrans strains but not in the isolate recovered from this patient. This result suggested that in vivo phase variation of P36 occurred, which would support a role for these LAMP variations in avoiding the host's immune vigilance.

Classification of a Brevundimonas strain detectable after PCR with a Helicobacter-specific primer pair.

In a study for the isolation of new Helicobacter strains, biopsy samples were taken from the gastric mucosa of dogs and subjected to PCR amplification using a Helicobacter-specific primer pair (H276f and H676r, directed to the 16S rDNA) to identify members of this genus in the specimens. From one Helicobacter positive sample, a bacterial strain was isolated which displayed a characteristic band after PCR amplification with the Helicobacter-specific primer pair. The isolate designated H2/98-FUNDUS was motile, oxidase-, catalase- and aminopeptidase-positive and grew only under microaerophilic conditions at 37 degrees C. The bacterium was classified by a polyphasic approach, including analysis of the isoprenoid quinones, fatty acids, polar lipids and partial 16S rDNA sequence. Analysis of the 16S rDNA sequence (1003 bases) indicated that the strain H2/98-FUNDUS is a member of the genus Brevundimonas and most closely related to Brevundimonas aurantiaca DSM 4731T (99.5% sequence similarity). Isolate H2/98-FUNDUS contained a predominant ubiquinone Q-10 and a fatty acid profile with the major compounds C18:1 and C16:0. In the polar lipid extract, phosphatidylglycerol, six unknown phospholipids, one unknown phosphoglycolipid, two unknown glycolipids and two unknown aminolipids were detected. All these results indicate that H2/98-FUNDUS represents a new member of the genus Brevundimonas which gives a positive signal upon PCR employing the Helicobacter-specific primer pair.

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 Mycobacterium 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. Mycobacterium 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.

Keratinopathogenic mould fungi and dermatophytes in healthy and diseased hooves of horses.

Specimens of hoof horn from 187 horses were examined for a possible relationship between clinically affected hooves and the occurrence of pathogenic fungi. Specimens were taken from the coronary band and from the stratum externum and medium of the coronary horn and transferred on to Sabouraud dextrose agar, with and without cycloheximide, and incubated at 28 degrees C. Dermatophytes and mould fungi were identified by their macroscopic and microscopic characteristics. The 732 isolates could be assigned to 26 species of moulds, two different species of the dermatophyte Microsporum and three different species of the dermatophyte Trichophyton. Depending on their pathogenic potential they were assigned to three groups: (i) fungi known to be keratinopathogenic (Acremonium blochii, Alternaria alternata, Alternaria chlamydospora, Geotrichum candidum, Microsporum ferrugineum, Microsporum gypseum, Scopulariopsis brevicaulis, Trichophyton species, Trichophyton mentagrophytes, Trichophyton schönleinii, 57 isolates), (ii) a group of uncertain pathogenicity (223 isolates), and (iii) a group of non-pathogenic species (452 isolates). Eighty per cent of the samples from horses with hoof horn lesions and 66.7 per cent of the samples from horses with slightly affected hoof horn contained fungi of the keratinopathogenic group, whereas only 8.9 per cent of the samples from horses with healthy hoof horn contained fungi of this group. There were no significant correlations between the clinical data and the age, sex or breed of the horses or their bedding and hygiene. Twelve species of fungi were isolated from the air in the horses' stables, but none of them belonged to the keratinopathogenic group.

Host-pathogen interactions in mycoplasma pathogenesis: virulence and survival strategies of minimalist prokaryotes.

Despite their very small genomes mycoplasmas are successful pathogens of man and a wide range of animal hosts. Because of the lack of effective therapeutics and vaccines, mycoplasma diseases continue to be a significant problem for public health as well as livestock production with major socio-economic consequences worldwide. Recent outbreaks and epidemiological studies predict that the incidence of human and animal mycoplasma diseases might increase which indicates the urgent need to develop new approaches for prevention and therapy. Development of such reagents, however, requires a solid understanding of the molecular biology of mycoplasma infections. Knowledge in this field has considerably increased during the past decade since new techniques have been developed and adapted to mycoplasmas that allow these organisms to be studied at the molecular level. Research on the two human pathogens Mycoplasma pneumoniae and Mycoplasma genitalium of which the genome sequences have recently been completed as well as the substantial number of studies carried out on the AIDS-associated mycoplasmas, Mycoplasma penetrans and Mycoplasma fermentans, has led the way, but a number of animal mycoplasmas are becoming increasingly appreciated as models for the study of the molecular basis of mycoplasma diseases. This review summarizes and highlights some of the recent findings concerning the molecular interactions that occur between pathogenic mycoplasmas and their hosts, both the common strategies as well as some unique approaches evolved by particular mycoplasma pathogens, including adherence to and uptake into non-phagocytic host cells, as well as mechanisms of escaping the host immune system.

Characterization of a multigene family undergoing high-frequency DNA rearrangements and coding for abundant variable surface proteins in Mycoplasma agalactiae.

A family of abundant surface proteins (Vpmas [variable proteins of Mycoplasma agalactiae]) undergoing phase variation in M. agalactiae has been characterized using monoclonal antibodies and specific polyclonal sera. Two expressed members of 39 kDa (Vpma39) and 34 kDa (Vpma34), which varied in expression between clones of a lineage, shared a common amino-terminal sequence but were immunologically distinct. An amino-terminal oligonucleotide probe identified multiple vpma genes which were clustered within a 14-kb ClaI genomic fragment. Rearrangements were found to have occurred within the vpma locus between clones which correlated with changes in their Vpma phenotype. Two neighboring vpma genes were cloned and sequenced from one M. agalactiae clonal variant expressing Vpma39. The two genes, vpmaX and vpmaY, were orientated divergently and shared highly homologous 5' untranslated regions, 25-amino-acid (aa) lipoprotein leader sequences, and amino-terminal sequences. The vpmaY gene coded for 346 aa and 84% of the open reading frame, comprised of 1.5 units of a large repeat of 186 aa. Although the sequence for an entire second vpmaY repeat was present, it was prematurely terminated by insertion of two nucleotides. The vpmaX gene encoded 221 aa and possessed 102 aa of the 186-aa repeat of vpmaY. Many of the features in common between the vpma genes were also found to be shared by the vsp genes of M. bovis, which also undergo DNA rearrangements concomitant with phenotypic changes. Since M. bovis is the closest phylogenetic relative to M. agalactiae, the vpma and vsp gene families most probably represent homologous systems.

Molecular characterization of the Mycoplasma gallisepticum pvpA gene which encodes a putative variable cytadhesin protein.

A putative cytadhesin-related protein (PvpA) undergoing variation in its expression was identified in the avian pathogen Mycoplasma gallisepticum. The pvpA gene was cloned, expressed in Escherichia coli, and sequenced. It exhibits 54 and 52% homology with the P30 and P32 cytadhesin proteins of the human pathogens Mycoplasma pneumoniae and Mycoplasma genitalium, respectively. In addition, 50% homology was found with the MGC2 cytadhesin of M. gallisepticum and 49% homology was found with a stretch of 205 amino acids of the cytadherence accessory protein HMW3 of M. pneumoniae. The PvpA molecule possesses a proline-rich carboxy-terminal region (28%) containing two identical directly repeated sequences of 52 amino acids and a tetrapeptide motif (Pro-Arg-Pro-X) which is repeated 14 times. Genetic analysis of several clonal isolates representing different expression states of the PvpA product ruled out chromosomal rearrangement as the mechanism for PvpA phase variation. The molecular basis of PvpA variation was revealed in a short tract of repeated GAA codons, encoding five successive glutamate resides, located in the N-terminal region and subject to frequent mutation generating an in-frame UAA stop codon. Size variation of the PvpA protein was observed among M. gallisepticum strains, ranging from 48 to 55 kDa and caused by several types of deletions occurring at the PvpA C-terminal end and within the two directly repeated sequences. By immunoelectron microscopy, the PvpA protein was localized on the mycoplasma cell surface, in particular on the terminal tip structure. Collectively, these findings suggest that PvpA is a newly identified variable surface cytadhesin protein of M. gallisepticum.

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.

Development of a recombinant antigen for antibody-based diagnosis of Mycoplasma bovis infection in cattle.

Mycoplasma bovis induces various clinical manifestations in cattle, such as mastitis, arthritis, and pneumonia. We have evaluated the immunoreactivity of three variable surface proteins (Vsps) of M. bovis, namely VspA, VspB, and VspC, with sera collected from herds with mycoplasmosis or from cattle experimentally infected with M. bovis. Western blot analysis revealed that the Vsps are the predominant antigens recognized by the host humoral response during M. bovis infection. The immunoreactivity of VspA, VspB, and VspC with host antibodies was independent of the clinical manifestations, the geographical origin of the M. bovis isolates, the mode of infection, and the animal's history. Moreover, the results showed that Vsp-specific host antibodies can be detected about 10 days after experimental infection and for up to several months. The full-length or truncated versions of the VspA product were overexpressed in Escherichia coli as fusion proteins (FP-VspA). Recombinant products showed strong immunoreactivity with the Vsp-specific monoclonal antibodies 1A1 and 1E5, with the corresponding epitopes localized at the VspA N-terminal and C-terminal ends, respectively. Anti-M. bovis sera of cattle naturally or experimentally infected also strongly recognized the full-length FP-VspA. The seroreactivity of sera collected from cattle between 6 and 10 days after experimental infection was weaker with truncated versions of VspA lacking the 1E5 epitope than with the full-length VspA or the truncated versions lacking the 1A1 epitope. Overall, the results indicate that the Vsps, despite their inter- and intraclonal variability, may be applied as target antigens in serodiagnostic assays for epidemiological studies.

Vsp antigens and vsp-related DNA sequences in field isolates of Mycoplasma bovis.

The expression of the 1E5 epitope which is common to the three characterized variable lipoproteins VspA, VspB and VspC of Mycoplasma bovis type strain PG45 and the presence of vsp gene DNA sequences were assessed in field isolates randomly collected from cattle showing clinical manifestations due to M. bovis infection. Among 250 isolates tested, only four failed to react with mAb 1E5. Southern blot analysis of these four isolates and of 20 isolates expressing the 1E5 epitope were performed using synthetic oligonucleotide probes corresponding to a sequence located in the Vsp signal peptide coding region common to all known Vsp products or to selected regions of previously characterized vsp genes, vspA, vspE and vspF. The results demonstrate the presence of multiple vsp-related DNA sequences in all M. bovis field isolates tested and indicate that the vsp repertoire varies in size and composition among isolates.