Validation of the suppressive subtractive hybridization method in Mycoplasma agalactiae species by the comparison of a field strain with the type strain PG2.
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The subtractive suppressive hybridization (SSH), a method that allows the identification of sequences that are present in one genome (tester) but not in the other (driver), is a promising technique for the comparison of Mycoplasma agalactiae pathogenic strains. The optimal conditions for SSH were established by subtracting the M. agalactiae type strain PG2 DNA from the M. agalactiae strain 5632 DNA. Because these two strains possess different vpma gene repertoires, 5632-specific vpma sequences (and possibly other 5632-specific sequences) were predicted to be retrieved by SSH. The subtracted tester DNA was PCR-amplified and cloned into the pGEM-T easy E. coli vector. Two independent libraries were generated and used to prepare individual probes that were tested by Southern blot with genomic DNA from various field isolates and mycoplasma reference strains. Sequence analysis of two overlapping clones showed that they potentially code for a large carboxyterminal portion of a new vpma ORF. Several DNA fragments homologous to insertion sequences were also found in 5632 and related strains. These preliminary data suggest that SSH is a powerful method to investigate differences between mycoplasma strains, and may be applied to molecular epidemiology, diagnostic, and host specificity or pathogenicity determinant discovery. (+info)
Mycotoxin fumonisin B1 alters the cytokine profile and decreases the vaccinal antibody titer in pigs.
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Fumonisin B1 (FB1), a mycotoxin produced by Fusarium verticillioides, may contaminate feed and food. In the present study, we investigated the effect of FB1 on the modulation of the cytokine profile and on the establishment of a vaccinal antibody response. In vitro investigations on pig peripheral blood mononuclear cells (PBMC) indicate that FB1 decreased interleukin-4 (IL-4) and increased interferon-gamma (IFN-gamma) synthesis at both the protein and mRNA levels. A short in vivo exposure (7 days) of weanling piglets to 1.5 mg/kg body weight of purified FB1 altered the cytokine balance in mesenteric lymph nodes and spleen similarly to the in vitro PBMC results. We also investigated the effect of FB1 on the antibody response during a vaccination process. A prolonged in vivo exposure (28 days) of weanling piglets to feed contaminated with 8 mg FB1/kg significantly decreased the expression of IL-4 mRNA by porcine whole blood cells and diminished the specific antibody titer after vaccination against Mycoplasma agalactiae. By contrast, ingestion of the contaminated feed had no effect on the serum concentration of the immunoglobulin subset (IgG, IgA, and IgM). Taken together, our data suggest that FB1 alters the cytokine profile and decreases the specific antibody response built during a vaccination protocol. These results may have implications for humans or animals eating contaminated food or feed. (+info)
Suppression subtractive hybridization as a basis to assess Mycoplasma agalactiae and Mycoplasma bovis genomic diversity and species-specific sequences.
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The phylogenically related Mycoplasma agalactiae and Mycoplasma bovis species are two ruminant pathogens difficult to differentiate and for which a limited amount of sequence data are available. To assess the degree of genomic diversity existing between and within these mycoplasma species, sets of DNA fragments specific for M. bovis type-strain PG45 or for M. agalactiae type-strain PG2 were isolated by suppression subtractive hybridization and used as probes on a panel of M. agalactiae and M. bovis field isolates. Results indicated that approximately 70 % of the DNA fragments specific to one or the other type strain are represented in all field isolates of the corresponding species. Only one M. bovis isolate, which was first classified as M. agalactiae, reacted with 15 % of the PG2-specific probes, while several M. agalactiae isolates reacted with 15 % of the PG45-specific probes. Sequence analyses indicated that most of the genomic diversity observed within one species is related to ORFs with (i) no homologies to proteins recorded in the databases or (ii) homologies to proteins encoded by restriction modification systems. Reminiscent of gene transfer as a means for genomic diversity, a PG45-specific DNA fragment with significant homologies to a central protein of an integrative conjugative element of Mycoplasma fermentans (ICEF) was found in most M. bovis field isolates and in a few M. agalactiae isolates. Finally, sequences encoding part of DNA polymerase III were found in both sets of M. agalactiae- and M. bovis-specific DNA fragments and were used to design a species-specific PCR assay for the identification and differentiation of M. agalactiae and M. bovis. (+info)
A new integrative conjugative element occurs in Mycoplasma agalactiae as chromosomal and free circular forms.
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An integrative conjugative element, ICEA, was characterized in Mycoplasma agalactiae strain 5632, in which it occurs as multiple chromosomal copies and as a free circular form. The distribution of ICEA sequences in M. agalactiae strains and their occurrence in Mycoplasma bovis suggest the spreading of the element within or between species. (+info)
Flow cytometric determination of the effects of antibacterial agents on Mycoplasma agalactiae, Mycoplasma putrefaciens, Mycoplasma capricolum subsp. capricolum, and Mycoplasma mycoides subsp. mycoides large colony type.
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Flow cytometry together with SYBR green I and propidium iodide was used to study the effects of enrofloxacin, ciprofloxacin, gentamicin, chloramphenicol, oxytetracycline, and tylosin on four mycoplasma species. Inhibition of mycoplasma growth could be detected by as early as 3 h after the start of treatment. The strongest effect was observed with enrofloxacin- and ciprofloxacin-treated cells. (+info)
Flow cytometric method for the assessment of the minimal inhibitory concentrations of antibacterial agents to Mycoplasma agalactiae.
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In this study, flow cytometry was evaluated for the determination of the minimal inhibitory concentrations (MIC) of seven antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, streptomycin, chloramphenicol, oxytetracycline, and tylosin) on Mycoplasma (M.) agalactiae. Flow cytometry was able to detect M. agalactiae inhibition from 6 h postincubation, although it seems that definitive MIC values determined by flow cytometry were only possible at 12-h postincubation. However, the results obtained by the traditional method were only obtained at 24 h, when a visible change in the medium had occurred. At 24 h, both methods gave the same result for six antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, streptomycin, chloramphenicol, and oxytetracycline); whereas flow cytometry gave slightly higher MIC for tylosin. This was attributed to the fact that the M. agalactiae growth that had occurred in the tubes containing tylosin was not enough to visibly change the color of the medium. Futhermore, flow cytometry detected that inhibitory concentrations of oxytetracycline, chloramphenicol, and tylosin as judged at 24 h were not able to inhibit the M. agalactiae growth after 48 h. MIC values of enrofloxacin and ciprofloxacin were sufficient only to maintain the total counts per milliliter throughout the time matched samples, whereas higher concentrations of theses antibacterial agents reduced the total counts per milliliter over the course of the experiment. The main advantage of the flow cytometric method is that MIC results for M. agalactiae can be obtained in a shorter time than is possible with the traditional method. The method presented makes identification of resistant populations of M. agalactiae possible and, unlike the traditional method, allows the effect of each antibacterial agent to be determined in real-time at the single-cell level. (+info)
Development of a sensitive and specific enzyme-linked immunosorbent assay based on recombinant antigens for rapid detection of antibodies against Mycoplasma agalactiae in sheep.
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We developed a new recombinant enzyme-linked immunosorbent assay (rELISA) for serodiagnosis of contagious agalactia (CA), a disease caused by Mycoplasma agalactiae in sheep and goats. The assay is based on two M. agalactiae surface proteins, namely, P80 and P55. Identification of these immunodominant and common antigens was accomplished by examining the antibody response elicited in sheep during experimental infection and comparing it to the protein expression profiles of 75 M. agalactiae field strains. Our rELISA was tested with 343 sera, collected from sheep with a laboratory-confirmed diagnosis of CA (n = 223) and from healthy animals (n = 120). All sera had previously been tested by Western blotting (WB) for reactivity against M. agalactiae. In addition, our rELISA was compared with a commercial routine ELISA based on inactivated antigens (CHEKiT). Among the 223 samples that were WB positive for M. agalactiae, 209 (93.7%) tested positive for rP80-P55 with our ELISA, whereas only 164 (73.8%) tested positive with the CHEKiT ELISA. Among the 120 samples tested that were WB negative for M. agalactiae, 96.7% were confirmed as negative with our rELISA, while only 75.8% were confirmed as negative with the CHEKiT ELISA. A comparison of the results with receiver operating characteristic curves indicated that the differences observed between our rELISA and the CHEKiT ELISA are statistically significant. The use of recombinant peptides instead of inactivated antigens could significantly improve the discrimination of positive and negative animals, bringing significant advantages in controlling the import/export of live animals and helping in eradication of this economically detrimental disease. (+info)
Being pathogenic, plastic, and sexual while living with a nearly minimal bacterial genome.
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Mycoplasmas are commonly described as the simplest self-replicating organisms, whose evolution was mainly characterized by genome downsizing with a proposed evolutionary scenario similar to that of obligate intracellular bacteria such as insect endosymbionts. Thus far, analysis of mycoplasma genomes indicates a low level of horizontal gene transfer (HGT) implying that DNA acquisition is strongly limited in these minimal bacteria. In this study, the genome of the ruminant pathogen Mycoplasma agalactiae was sequenced. Comparative genomic data and phylogenetic tree reconstruction revealed that approximately 18% of its small genome (877,438 bp) has undergone HGT with the phylogenetically distinct mycoides cluster, which is composed of significant ruminant pathogens. HGT involves genes often found as clusters, several of which encode lipoproteins that usually play an important role in mycoplasma-host interaction. A decayed form of a conjugative element also described in a member of the mycoides cluster was found in the M. agalactiae genome, suggesting that HGT may have occurred by mobilizing a related genetic element. The possibility of HGT events among other mycoplasmas was evaluated with the available sequenced genomes. Our data indicate marginal levels of HGT among Mycoplasma species except for those described above and, to a lesser extent, for those observed in between the two bird pathogens, M. gallisepticum and M. synoviae. This first description of large-scale HGT among mycoplasmas sharing the same ecological niche challenges the generally accepted evolutionary scenario in which gene loss is the main driving force of mycoplasma evolution. The latter clearly differs from that of other bacteria with small genomes, particularly obligate intracellular bacteria that are isolated within host cells. Consequently, mycoplasmas are not only able to subvert complex hosts but presumably have retained sexual competence, a trait that may prevent them from genome stasis and contribute to adaptation to new hosts. (+info)