1 UMR INRA-ENVT 1225, Ecole Nationale Vétérinaire de Toulouse, 23 Ch des Capelles, 31076 Toulouse, France
2 AFSSA Lyon, 31 Av Tony Garnier BP 7033, 69342 Lyon, France
Correspondence
Marc S. Marenda
m.marenda{at}envt.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are: AGA-02 to -90, CL844514 to CL844536; AGA-4 to -89, CL844537 to CL844551; BOV-01 to -93, CL844552 to CL844574; BOV-7 to -94, CL844575 to CL844585.
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INTRODUCTION |
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The comparison of M. agalactiae and M. bovis whole genome sequences could offer a broader approach to identify species-specific target DNA sequences, and could result in the development of new diagnostic assays and molecular epidemiological tools. In addition, this comparison could contribute to the identification of host-specificity factors, if present, and to a better understanding of the evolution of these two ruminant pathogens. Up to now, the amount of sequence information available in the database required to perform such analysis has been too limited. An alternative approach is the use of the suppression subtractive hybridization (SSH) method, which allows the isolation of DNA fragments that are present in one set of DNA sequences (tester) but not in another (driver). SSH is well suited for biodiversity studies, molecular epidemiology, identification of species-specific markers and identification of virulence factors of bacteria (Winstanley, 2002). In the present study, two sets of DNA fragments were identified by SSH that were specific to the M. bovis and M. agalactiae type strains. The distribution of these sequences within field isolates was further tested using a large panel representative of each mycoplasma species, which revealed the genetic diversity that exists among strains of the same species. Using this approach, M. agalactiae and M. bovis species-specific sequences were identified which were represented in all the field isolates tested in this study. Among these, a region of the polC gene present in both species was shown in a PCR assay to provide a valuable target for the discrimination of M. agalactiae from M. bovis, even when using field isolates with ambiguous status. Overall, these sets of specific DNA fragments offer a large choice of candidates for the development of new molecular diagnostic and epidemiological tools.
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METHODS |
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After complete digestion of the tester DNA (2 µg) by Sau3AI, a 100 ng aliquot of the digested products was ligated to 20 pmol of adapter A1 while a second 100 ng aliquot was ligated to A2. For the first round of hybridization, 10 ng of each adapter-ligated DNA subset was mixed with an excess of Sau3AI-digested driver DNA (600 ng), denatured for 1 min at 98 °C and incubated for 1·5 h at 55 °C. In the second round of hybridization, the two subsets were then quickly mixed in the presence of additional denatured driver DNA (300 ng) and incubated for 18 h at 55 °C. The resulting subtracted products were subjected to a first PCR using the primer R24 (5'-AGCACTCTCCAGCCTCTCACCGAG-3') with the following conditions: fill-in step for 2 min at 72 °C, followed by 26 cycles of 30 s at 94 °C, 30 s at 52 °C, and 2 min at 68 °C. The resulting products were then subjected to a nested PCR using the primers J24 (5'-ACCGACGTCGACTATCCATGAACG-3') and N24 (5'-AGGCAACTGTGCTATCCGAGGGAG-3') with the following conditions: 23 cycles of 30 s at 94 °C, 30 s at 49 °C, and 2 min at 68 °C. PCR assays were performed on an MJresearch PT100 thermocycler with recombinant Taq polymerase and reaction buffer from Invitrogen.
Establishment of a subtracted PG2 and PG45 DNA library.
Approximately 5 ng of PCR products obtained after SSH using PG2 or PG45 DNA as tester were ligated into 5 ng pGEM-T-easy plasmid (Promega). Ligation reactions were performed with the LigaFast ligation system (Promega). Each ligation mixture was used to transform E. coli DH5, and the recombinant clones were selected on LB agar plates supplemented with ampicillin (50 µg ml1) in the presence of IPTG and X-Gal. Ninety five clones of each library were randomly picked for further studies. Purification of plasmid DNA was performed with the WizardSV miniprep system (Promega), and the sequencing of cloned inserts was performed by PCR amplification, according to Applied Biosystems (Big Dye Terminator) or Amersham Pharmacia Biotech (ET terminator) protocols; the samples were processed onto capillary or slab gel sequencers by Genome-Express.
Southern blots.
DIGlabelling and detection systems (Roche) were used according to the manufacturer's instructions. Plasmid DNA inserts were labelled by Dig-11-dUTP PCR using the primers J24 and N24, recombinant Taq polymerase and reaction buffer from Invitrogen on an MJresearch PT100 thermocycler, with a Dig-11-dUTP : TTP ratio of 1 : 19. Approximately 1 µg genomic DNA was digested with EcoRI, submitted to 1 % agarose gel electrophoresis and transferred onto nylon membranes (Roche), which were incubated in Church buffer (Church & Gilbert, 1984) overnight at 60 °C with approximately 1 pmol of heat-denatured probe. The membranes were briefly washed with 0·2x SSC/0·1 % SDS at room temperature, then in 0·2x SSC/0·1 % SDS for 1 h at 60 °C. Detection of hybridized probes was performed with alkaline phosphatase-conjugated anti-Dig Fab antibodies and CDPstar reagents (Roche).
Sequencing analyses.
The BLAST suite of programs (http://www.ncbi.nlm.nih.gov/blast/blast.cgi) was used for sequence homology searches on non-redundant databases and on the specialized database Molligen (http://cbi.labri.fr/outils/molligen/). Alignments were performed with the Lfasta program (Chao et al., 1992) at the infobiogen web site (http://www.infobiogen.fr/services/analyseq/cgi-bin/lfastan_in.pl). The AGA and BOV DNA sequences listed in Tables 5 and 6
were deposited in the Genome Survey Sequences database (dbGSS) division of GenBank as follows: AGA-02 to -90 (Table 5
, first section), CL844514 to CL844536; AGA-4 to -89 (Table 5
, second section), CL844537 to CL844551; BOV-01 to -93 (Table 6
, first section), CL844552 to CL844574; BOV-7 to -94 (Table 6
, second section), CL844575 to CL844585.
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PCR conditions for species identification.
The species-specific uvrC and polC-based PCR was performed on an Eppendorf Mastercycler Gradient thermocycler with Taq polymerase and reaction buffer from New England Biolabs, using 10 ng M. agalactiae or M. bovis genomic DNA as template. The uvrC-based PCR was performed as previously described (Subramaniam et al., 1998) at the following annealing temperatures: 44 °C for the M. agalactiae uvrC PCR and 52 °C for the M. bovis uvrC PCR. The M. agalactiae-specific polC-based PCR was performed with the primer pair MAPol-1F (5'-CATTGAACCTCTTATGTCATTTACTTTG-3') and MAPol-5R (5'-CTATGTCATCAGCTTTTGGGTGA-3'); the M. bovis-specific polC-based PCR was performed with the primer pairs MBPol-6F (5'-TATCGAGCCTTTGATGTCGTTTACACTA-3') and MBPol-8R (5'-TAATATCATCTGCTTTTGAATGG-3'), or MBPol-7F (5'-CTTATCAAATGATGAAGTTGAACTAC-3') and MBPol-8R at a unique annealing temperature of 49 °C. The M. agalactiae and M. bovis polC-based PCR was performed with the primer pairs MABPol1-F (5'-TTTGTTAGACAAATGCTTAATGA-3') and MABPolR (5'-GGCTTATCAATCATATTTT-3'), or MABPol2-F (5'-CTTAGTCACGGAACTAATGTTTG-3') and MABPolR at a unique annealing temperature of 42 °C. Both uvrC and polC PCR were performed in 25 µl reaction mixtures containing 0·4 µM of each primer, 2·5 µl 10x reaction buffer, 400 µM dNTPs, 2 mM Mg2+ and 2·5 U Taq polymerase, for 30 cycles of 30 s at 94 °C, 30 s at the annealing temperature, and 30s at 72 °C.
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RESULTS |
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Identification of M. agalactiae and M. bovis species-specific DNA sequences
To define the prevalence of the AGA and BOV DNA fragments in each mycoplasma species, the reactivity of each probe was individually assessed by Southern blot using EcoRI-digested genomic DNA from a panel of M. agalactiae and M. bovis field isolates collected (i) from different hosts, (ii) from various geographical origins, (iii) from different outbreaks and (iv) from animals presenting different clinical manifestations (Tables 1 and 2). Since the phenotypic and genotypic diversity of M. agalactiae has been previously documented using several molecular markers, only 18 isolates were chosen to represent this species. In contrast, little was known regarding the biodiversity of M. bovis, prompting us to include 56 isolates in the M. bovis collection to make it more representative.
The majority of the AGA probes (41 out of 59, or 69·5 %) recognized all the 18 isolates previously identified as M. agalactiae (Table 3), with the exception of 8062, as illustrated in Fig. 1
(a). Strain 8062 failed to react with 50 AGA probes (Table 1
) and its status was subsequently re-evaluated based on its particular features, as discussed below. With the exclusion of the data obtained with isolate 8062, none of the 59 AGA probes reacted with any of the M. bovis isolates. However, only 12 M. agalactiae isolates (not considering 8062) were recognized by all 59 AGA probes (reactivity pattern 1, MA-RP1; see Table 3
), since five isolates (4025, 4054, 4055, 5632 and 8064) failed to react with between eight and 14 AGA probes out of a group of 18, resulting in five individual reactivity patterns (Table 3
). Such lack of reactivity is illustrated in Fig. 1(b)
. A more detailed analysis revealed that isolates displaying the same reactivity pattern may differ in their hybridization profile. For instance, probe AGA-45, which reacted with the entire M. agalactiae collection (with the exception of isolate 8062), revealed an RFLP among some isolates (see Fig. 1a
, isolates 4025, 4054, 4055 and 8064).
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Characterization of M. bovis and M. agalactiae species-specific DNA sequences
To define new potential DNA targets which can be used in PCR assays to discriminate between the two closely related mycoplasma species, M. bovis and M. agalactiae, the DNA inserts of 58 and 57 clones, corresponding to the AGA and BOV sets of probes, respectively, were sequenced. Sequencing analyses revealed the presence of a Sau3A restriction site in 18 AGA and 23 BOV fragments. Since Sau3A was used to digest the tester DNA prior to ligation with the adapters, such sequences may represent chimerical DNA resulting from the concatenation of non-adjacent Sau3A fragments. Because these might represent non-adjacent sequences within the PG2 and PG45 genomes, they were excluded from further studies. The remaining DNA sequences were compared to those contained in non-redundant databases (Tables 5 and 6
) using the BLASTX algorithm (Altschul et al., 1990
). Since the availability of M. agalactiae and M. bovis genomic sequences in the database is limited, most similarities and/or homologies were found with putative ORFs or proteins encoded by the Mycoplasma pulmonis genome, which represents the most closely related fully sequenced mycoplasma species. Overall, significant alignments were obtained with: (i) sequences related to DNA modification, recombination and repair, or to insertion sequences; (ii) sequences involved in metabolic pathways or in transporters; (iii) sequences encoding proteins putatively surface exposed, but also with sequences encoding conserved hypothetical proteins or involved in DNA replication or translation. Interestingly, no significant alignment was obtained with 12 PG2 and 10 PG45 DNA fragments (Tables 5 and 6
).
Housekeeping or constitutive genes that provide basic functions, such as replication, transcription and translation, are good candidates for genetic differentiation of microbial species. Five DNA inserts that were specific for M. agalactiae produced an alignment with sequences of M. pulmonis encoding (i) two distinct portions of the subunit A of DNA gyrase (AGA-49 and AGA-45), (ii) a portion of the chromosomal replication initiator protein DnaA (AGA-33), (iii) a portion of the helicase PcrA/UvrD (AGA-14), and (iv) a portion of DNA polymerase III or PolC (AGA-34). The BOV-25 DNA fragment identified above as M. bovis species-specific aligned with the M. pulmonis DNA polymerase, but with a portion different from that contained in AGA-34. In Southern analyses, AGA-34 and BOV-25 did not hybridize with genomic DNA from various mycoplasma species type strains that are commonly found in ruminants and from more distantly related species (see Methods; data not shown).
Species-specific amplification to discriminate between M. agalactiae and M. bovis, and re-evaluation of the status of strain 8062
To better evaluate the differences and homologies existing between the polC sequences of M. bovis and M. agalactiae, BOV-25 was used as a probe to screen an E. coli library containing EcoRV-digested PG45 genomic DNA fragments ligated into the pBluescript plasmid at the unique EcoRV site of the polylinker. A recombinant plasmid that carried a 1091 bp insert and hybridized with the probe was selected and designated pMM31-1. Sequence analysis of its DNA insert revealed that nucleotides 1 to 88 are identical to the 3' end of BOV-25, while nucleotides 139 to 739 display 85 % identity with the first 610 nucleotides of AGA34 (Fig. 2). Two primer pairs, MAPol (MAPol-1F/MAPol-5R) and MBPol (MBPol-6F/MBPol-8R), were then designed to specifically amplify a 265 bp DNA fragment of the PG2 and PG45 genomes, respectively (Fig. 2
). To assess the specificity of these primers, the genomic DNA from the M. agalactiae and M. bovis panels (Tables 1 and 2
) and from an additional 14 M. agalactiae field isolates (included in Table 1
, see Methods) was subjected to two identical PCR assays using either the MAPol or the MBPol primer pair. As expected, all M. agalactiae isolates yield an amplicon with the MAPol but not with the MBPol primer pair and all the M. bovis isolates yeld an amplicon with the MBPol primer pair but not the MAPol primer pair, with the exception of three isolates: 8062 and 8063, which were previously identified as M. agalactiae, but were amplified by the MBPol primers and not the MAPol primers, and 2094, which was previously identified as M. bovis, but was never amplified. To further confirm our results, the same M. bovis and M. agalactiae DNA templates were subjected to a PCR assay based on the uvrC gene sequences previously used to discriminate the two mycoplasma species (Subramaniam et al., 1998
). In our hands, at the recommended annealing temperature (50 °C), the M. agalactiae-specific uvrC primers amplified all the M. agalactiae isolates except 4054, 4212, 5632, 8062, 8063 and 8064; additionally, none of the M. bovis isolates were amplified. Lowering the annealing temperature of the M. agalactiae-uvrC PCR to 44 °C yielded amplicons with 4054, 4212, 5632 and 8064, along with the rest of the M. agalactiae isolates, but not with 8062 and 8063, nor with the M. bovis isolates. Similarly, at the recommended annealing temperature (52 °C), the M. bovis-specific uvrC primers amplified all the M. bovis isolates, except 2094, and none of the M. agalactiae isolates, except 8062 and 8063. Increasing the annealing temperature to 58 °C did not allow a differential amplification between 8062 or 8063 and the rest of the M. bovis isolates. We therefore propose that the 8062 and 8063 isolates should be considered as M. bovis and not M. agalactiae strains. Nevertheless, when an alternative primer, MBPol-7F, which was designed from the pMM31-1 plasmid sequence downstream from the MBPol-6F primer (Fig. 2
), was used in a PCR assay with the MBPol-8R primer, no amplification was observed with 8062 and 8063 DNA, while PG45 and all the M. bovis isolates (except 2094) yielded the expected 211 bp fragment. This indicated that unlike the rest of the M. bovis isolates, some sequence differences exist between PG45 and the 8062 and 8063 isolates, at least in the locus corresponding to the MBPol-7F primer. In order to analyse such sequence divergence in 8062 and 8063, two additional forward primers, MABPol-1F and MABPol-2F, and one reverse primer, MABPol-R, were designed from polC sequences which surround the region amplified by MBPol-6F/MBPol-8R and which are conserved between PG2 and PG45 (Fig. 2
), with the aim of cloning and sequencing the corresponding region of interest from 8062 and 8063. Surprisingly, these primers did not allow the PCR amplification of 8062 or 8063 DNA, whereas PG2 and PG45 DNA were amplified as expected. These results indicated that the 8062 and 8063 isolates occupy a distinct position in our collections that deserves further investigation.
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DISCUSSION |
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More than 70 % of the DNA fragments obtained from the PG2 versus PG45 and PG45 versus PG2 subtractive hybridizations were tester specific (that is, present in one type strain and absent or substantially different in the other). This ratio is slightly higher than those reported in similar SSH studies, which range between 50 % to 60 % (Akopyants et al., 1998; Bogush et al., 1999
), and include the comparison of related but distinct bacterial species, such as E. coli and S. typhimurium. The effectiveness of SSH in our hands is illustrated (i) by the presence in the M. agalactiae PG2 library of independent clones containing subtracted fragments corresponding to different portions of the same gene, i.e. AGA-45 and -49, which encode products with a high identity level to the M. pulmonis DNA gyrase subunit A (67 % for AGA-45 and 72 % for AGA-49 at the amino-acid level); and (ii) by the absence of redundant tester specific sequences in randomly selected clones derived from the PG2- or PG45-subtracted library. This is further supported by the identification of DNA sequences encoding part of the same gene in both subtracted genomes (i.e. AGA-34 of the PG2 library and BOV-25 of the PG-45 library, which encode different regions of the polC gene), even though the number of clones tested for each library was relatively low.
Southern blot analyses revealed that the vast majority of the PG2- or PG45-specific DNA sequences (69 % and 85 %, respectively) specifically recognized a panel of M. agalactiae or M. bovis strains collected in the field, with the exception of 2094 and 8062 (see below). The paired subtractions of the two ruminant mycoplasma type-strains provided large sets of species-specific DNA fragments, which for the most part confirm the previous classification of the field isolates by classical bacteriology methods. This further indicates that the routine mycoplasma identification method, which is based on reactivity to sera and which was used to assemble our species-representative strain collections, correlates well with the degree of genomic relatedness existing between the type strain and the field isolates of the same mycoplasma species. This observation prompted us to re-examine the status of (i) strain 2094, which failed to react with any of the PG2 or PG45 probes, although it was first identified by serological assays as M. bovis, and (ii) strain 8062, first classified as M. agalactiae, but more frequently recognized by probes specific to PG45. Re-examination of strain 2094 by dot blot and PCR revealed that it belongs neither to M. bovis nor M. agalactiae, and that it remains to be identified. It is possible that strain 2094 does not belong to M. bovis but shares with PG45 antigens that induce a cross-reaction in MF dot. It should be noted that this strain was isolated from a bovine uterine infection, a rather unusual isolation site for M. bovis. Whether the 2094 strain belongs to a particular subset of M. bovis that is substantially different from the rest of the isolates (antigenically and clinically), or to another mycoplasma species, remains to be determined. The status of strain 8062 was more ambiguous as: (i) it exhibited cross-reactivity in serological assays with the rabbit antisera used for the identification of M. bovis and M. agalactiae; (ii) it was shown to be positive for M. agalactiae and negative for M. bovis by PCR using the uvrC gene as target by Subramaniam et al. (1998); (iii) it was found nevertheless in our hands to be positive for M. bovis and negative for M. agalactiae using the same PCR assay. A recent collaborative trial for the evaluation of the current PCR systems used for the identification and differentiation for M. agalactiae and M. bovis indicated that strain 8062 belongs to M. bovis (Bashiruddin et al., 2004
). However, it is noteworthy that out of five laboratories involved in this trial, only two could identify strain 8062 without ambiguity. In this regard, the polC-based PCR proposed in this paper appears to be an interesting complement to the uvrC-based PCR for species-diagnostic purposes. Both PCR tests offer similar sensitivity (data not shown), and correlate well with the antisera-based MF dot assay, but the polC-based PCR offers several interesting advantages: (i) a unique annealing temperature that allows specific amplification of the polC target sequence, while two different annealing temperatures are required for the uvrC-based PCR; (ii) some M. agalactiae strains require annealing temperature adjustments with the uvrC-based PCR, a problem that was not encountered in the polC-based PCR; (iii) the isolate 8062, which gave inconsistent results with the uvrC-based PCR in other studies, was unambiguously amplified by the polC-based PCR under the standard conditions using MBPol-6F and MBPol-8R primers. At this point, it should be noted that one of the M. bovis-specific polC primers, MBPol-7F, which allows amplification of all the isolates from the M. bovis collection, failed to amplify 8062 and 8063, while the M. bovis-specific uvrC-based PCR, although it may give inconsistent results in certain laboratories, properly amplified these isolates in our hands. In conclusion, the existence of isolates such as 8062 or 8063 in ruminants demonstrates that a PCR identification assay should rely on more than one target sequence in order to improve its sensitivity and specificity; the combined use of PCR based on uvrC, polC and possibly other housekeeping genes should therefore be considered for developing future diagnostic assays.
Interestingly, M. bovis strain 8062 has a geographical origin (Ethiopia) different from the rest of our mycoplasma collection, which mostly represents European isolates, and it was isolated from a goat, one of the usual hosts for M. agalactiae, and not from cattle, which are the usual hosts for M. bovis. The sequence differences observed between 8062 and the rest of the M. bovis isolates could be related to these features. In contrast to the 56 other M. bovis strains included in this study, 8062 is the only one that is recognized by PG2-specific DNA sequences (9/59 AGA probes) and fails to react with several PG45-specific DNA sequences (13/60 BOV probes). Apart from strain 8062, the M. bovis strains presenting the most genotypic divergence with the PG45 type-strain are 9583 and 9591, both of which only failed to react with 4/60 PG45 probes. Overall, the M. bovis collection is composed of three related clusters: the largest is composed of 26 strains (MB-RP1), which react with 60/60 PG45 probes; the two smaller groups comprise 16 (MB-RP2) and 9 (MB-RP3) strains, respectively, each lacking reactivity with only two PG45 probes. Most of the strains belonging to MP-RP3 (7/9) were isolated after 1998, whereas most of those belonging to MP-RP1 and MB-RP2 (39/42) were isolated before 1998. Whether this observation is due to a bias in our collection or whether it does correlate to a recent change in the epidemiology of M. bovis infections has to be further assessed. Finally, four out of 56 M. bovis isolates displayed unique individual reactivity profiles, based on the lack of a hybridization signal with between one and four PG45 probes. By contrast, the M. agalactiae collection contained a heterogeneous subset of five strains which hybridized with various PG45 probes (26/60 BOV probes). Compared to the majority of the M. agalactiae strains that display a PG2-like reactivity pattern, this subset failed to bind between 8 and 14 of the 59 AGA probes (Table 3). These strains harbour one or more unusual traits, including a particular serotype (Bergonier et al., 1996
), an original repertoire of vpma genes (M. Glew, personal communication), and the presence of insertion sequences also found in M. bovis PG45 (Marenda et al., 2004
; Pilo et al., 2003
). Taking into account the fact that none of these strains exhibits the same reactivity pattern, these findings might reflect the existence of a greater genotypic diversity among M. agalactiae field isolates than among those of M. bovis.
No M. bovis strains, other than those clustering in MB-RP2, lacked reactivity with BOV-60 and BOV-73, while strains reacting positively with BOV-20 but negatively with BOV-90, or vice versa, were found (Table 4). These data and the hybridization of both the BOV-60 and BOV-73 probes with M. agalactiae strain 5632 suggest that these two sequences might be genetically linked. While sequencing analyses of BOV-60 revealed no significant homology in the databases, BOV-73 was of particular interest, since it exhibited a high degree of homology (42 % identity and 61 % similarity at amino acid level) with an ORF recently identified as a central component of an integrative conjugal element (ICE) of M. fermentans (Calcutt et al., 2002
). The presence of this sequence in most of the M. bovis strains tested in this study (with the exception of those belonging to MB-RP3), and in M. agalactiae strains 5632, 8064 and 3990, is suggestive of the existence of conjugative systems in the two ruminant pathogens. If further confirmed, lateral gene transfer between mycoplasma strains or species might explain the presence of sequences common to both species that are restricted to particular isolates or groups of strains. As mentioned above, the M. agalactiae collection contains five strains that hybridized with a specific set of PG45-derived probes (Table 3
), whereas none of the M. bovis strains (except for 8062) were recognized by any of the PG2-derived probes (Table 4
). That this non-symmetrical observation could reflect the widespread occurrence of a putative conjugative system in M. bovis which is absent from most of the M. agalactiae strains, and which could account for the polar transfer of genetic information from M. bovis to M. agalactiae cells during transient co-infection of the same host, is an appealing hypothesis that has not yet been assessed.
The results of this study show the potential of SSH as a simple, easy and inexpensive method that can serve as a basis for mycoplasma species comparison, leading to the discovery of new molecular markers for diagnostics, molecular epidemiology and strain typing. Finally, approaches based on SSH appear to be the ideal complement to more complex studies, such as those combining whole genome sequencing and microarrays, and in particular may help to decipher the relationship existing between the genetic variability and plasticity of these pathogens and their virulence or host specificity.
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ACKNOWLEDGEMENTS |
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Received 26 August 2004;
revised 15 October 2004;
accepted 20 October 2004.
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