Use of Amplified Fragment Length Polymorphism To I
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微生物临床杂志 2005年第2期
Department of Statutory and Exotic Bacterial Diseases, Veterinary Laboratories Agency, Addlestone, Surrey, United Kingdom
ABSTRACT
Amplified fragment length polymorphism (AFLP) is a whole-genome fingerprinting method that relies on the selective PCR amplification of restriction fragments. The potential of this approach for the discrimination of Brucella isolates at the species and intraspecies level was assessed. A number of different combinations of restriction enzymes and selective primers were examined, and one, using EcoRI and MseI with additional selective TC bases on the MseI primer, was selected for full assessment against a panel of Brucella isolates. The technique could readily differentiate Brucella spp. from all Ochrobactrum spp. representing the group of organisms most closely related to Brucella spp. Application of AFLP highlighted the genetic homogeneity of Brucella. In spite of this determination of AFLP profiles of large numbers of isolates of human and animal origin, including Brucella abortus, B. melitensis, B. ovis, B. neotomae, marine mammal isolates (no species name), B. canis, and B. suis, confirmed that all but the latter two species could be separated into distinct clusters based on characteristic and conserved differences in profile. Only B. suis and B. canis isolates clustered together and could not be distinguished by this approach, adding to questions regarding the validity of species assignments in this group. Under the conditions examined in the present study only limited intraspecies genomic differences were detected, and thus this AFLP approach is likely to prove most useful for identification to the species level. However, combination of several of the useful restriction enzyme-primer combinations identified in the present study could substantially add to the discriminatory power of AFLP when applied to Brucella and enhance the value of this approach.
INTRODUCTION
Brucella spp. comprise a closely related group of organisms that classical taxonomists divided into six species based on subtle phenotypic and antigenic differences and host specificity: Brucella abortus (bovine), B. melitensis (caprine and ovine), B. ovis (ovine), B. canis (canine), B. suis (porcine), and B. neotomae (only seen in the desert wood rat). The situation has recently been complicated by the identification of Brucella isolates in marine mammals that do not fit into any of the recognized species and themselves show intragroup diversity (4, 14). Some of the species are classically divided into biovars such that several B. abortus, B. melitensis, and B. suis biovars are recognized. In addition, host specificity is not absolute; thus, B. melitensis and B. suis are important causes of bovine disease in some countries, and B. suis biovars 2, 4, and 5 have been associated with hares, reindeer, and rodents, respectively. The traditional view on Brucella taxonomy was challenged some time ago on the basis of the high degree of homology indicated by DNA hybridization experiments (26) and the inability to differentiate Brucella spp. by 16S rRNA sequencing. Although these findings conform better with the view of a single species within which the six classical species would be considered biovars, this scheme has not achieved widespread acceptance largely on practical grounds.
Brucellosis remains an important disease in both humans, in whom it leads to a chronic debilitating infection, and in domesticated animals, in which the main symptom is reproductive failure. A number of characteristics make Brucella spp. attractive targets for weaponization, and the organism remains on the list of CDC category B potential biological warfare agents (22). In humans, brucellosis caused by B. melitensis is by far the most important clinically apparent disease and is usually associated with occupational exposure or the consumption of unpasteurized dairy products. However, B. abortus, B. suis and, more rarely, B. canis and the marine mammal Brucella can also cause human infection. Brucellosis remains a major problem in many parts of the world, notably in the Mediterranean region, western Asia, and parts of Africa and Latin America (8). Brucellosis has been eradicated or severely curtailed in some Western countries by a combination of strict veterinary hygiene measures, monitoring programs, and improved food safety measures. However, regions recognized as "officially brucellosis free" are under constant threat of reintroduction via livestock trading, reinforcing the requirement for reliable molecular tools for the identification and typing of Brucella. The development of such discriminatory molecular tools has long been problematic, reflecting the lack of genetic polymorphism in Brucella spp., and many laboratories rely on conventional biotyping to identify and speciate Brucella. Furthermore, whereas the development of genus-specific and, in some cases, species-specific PCR assays for identification has been possible (2), typing tools of sufficient resolution to permit epidemiological tracing of outbreaks are still lacking. One of the most promising molecular approaches to date utilizes DNA polymorphism, reflecting the variable distribution of an insertion sequence, IS711, in the chromosome. The use of an IS711 based probe reveals that between 5 and 30 copies of this element can be present. In most cases species can be differentiated by their distinct patterns, although this is not absolute and further discrimination between biovars is limited (3). An alternative approach is the PCR-restriction fragment length polymorphism analysis of outer membrane protein encoding genes that can differentiate the six species of Brucella (23) and furthermore can differentiate between some biovars and field isolates of individual species (5, 6). However, findings to date indicate that neither of these approaches offers sufficient power of resolution to be confidently used in epidemiological tracing.
Here we investigate the application of an alternative DNA fingerprinting approach, AFLP, to Brucella. AFLP is based on the amplification of subsets of genomic restriction fragments by using PCR (15). DNA is cut with restriction enzymes, and double-stranded adaptors are ligated to the ends of DNA fragments to generate template DNA for PCR amplification. The sequence of the adaptors and adjacent restriction site serve as primer binding sites for subsequent amplification of the restriction fragments. Selective nucleotides can be included at the 3' ends of the PCR primers, which therefore prime DNA synthesis from only a subset of the restriction sites. Labeling of one of the primers (usually the one that corresponds to the less frequently cutting restriction enzyme) with a fluorescent dye permits visualization of a banding pattern after electrophoresis. Although amplified fragment length polymorphism (AFLP) requires no prior sequence knowledge, the restriction enzyme and primer combinations selected will substantially affect the discriminatory power of the method and thus suitable combinations must be selected for each organism being examined. AFLP has now been applied to a substantial number of microorganisms and has been shown to be highly discriminatory, rapid, and reproducible (20) and has proven to be a useful tool in bacterial taxonomy (10, 12, 13). We describe here the development of an AFLP approach to identify and speciate Brucella isolates and assess whether this approach may have value as an epidemiological tool.
(Preliminary portions of this study were presented 28 to 31 May 2003 at the IMAB-NATO Conference on Risk Infections and Possibilities for Medical BioTerrorism in Varna, Bulgaria.)
MATERIALS AND METHODS
Strains. Details of all strains described in the present study are given in Table 1. Brucella strains were routinely cultured on serum dextrose agar plates at 37°C in the presence of 10% CO2 for 3 days.
DNA preparation. DNA was extracted by standard procedures. Briefly, growth from two to four spread plates was harvested into 2 ml of sterile water per plate, and the final volume was made up to 20 ml. The resulting cell suspension was harvested by centrifugation, the supernatant was removed, and the cells were resuspended in 5 ml of 1x TNE buffer (0.1 M Tris-HCl [pH 8], 0.1 M EDTA [pH 8], 0.1 M NaCl), 1% (wt/vol) sodium dodecyl sulfate, and 1% (wt/vol) Sarcosine. After a vortexing step, a 150-μl aliquot of proteinase K (20 mg ml–1) was added to the cell suspensions, which were then incubated for 1 h at 65°C, followed by the addition of further proteinase K aliquots if necessary, until complete cell lysis had occurred. Phenol-chloroform extractions were performed by adding 5 ml of Ultrapure buffer saturated phenol (Invitrogen), mixing the suspension thoroughly, centrifuging the mixture at 4,000 rpm for 5 min, and removing the aqueous phase. The extraction procedure was repeated twice more by using Ultrapure phenol-chloroform-isoamyl alcohol (25:24:1; Invitrogen), and DNA was precipitated by adding the final aqueous phase to ca. 15 ml of chilled (–20°C) ethanol. After precipitation, the pellet was washed in 70% ethanol, dried, and resuspended in Tris-EDTA buffer.
AFLP procedure. In initial testing, some 43 different restriction enzyme-primer combinations were screened for suitability by using DNA extracted from four Brucella type strains (B. abortus biovars 1 and 2, B. melitensis biovar 1, and B. ovis). The sequences of all adapters used and the core AFLP primers are given in Table 2. DNA was digested with appropriate restriction enzymes according to the manufacturer's instructions and subjected to standard AFLP procedures (15, 16). Full details of the final protocol adopted for the "EcoRI+0-MseI+TC AFLP" approach selected for large-scale study are as follows. Chromosomal DNA concentrations were adjusted to 0.25 μg/μl, and 3 μl of each DNA to be tested was digested with MseI in React 1 buffer (Invitrogen) in a 10-μl final volume for 1 h at 37°C. After this, 1 μl of React 1, 2 μl of 1 M NaCl, 1 μl of EcoRI (Invitrogen), and 6 μl of water were added, followed by incubation for a further 1 h at 37°C. Double-stranded adapters were prepared by mixing equal volumes of the two relevant adapter primers to give final concentrations of 25 μM for the MseI adapter and 2.5 μM for the EcoRI adapter. The mixes were heated to 95°C for 10 min and then allowed to anneal by cooling to room temperature for 15 min. Adapters were ligated to genomic DNA digests in a mix containing 2 μl of 5x ligase buffer, 1 μl of T4 DNA ligase, 2 μl of EcoRI adaptors (5 pmol), 2 μl of MseI adaptors (50pmol), 1 μl of digest from above, and 2 μl of water, followed by incubation at 12°C for a minimum of 4 h. A first-round PCR was then performed with nonselective primers specific for EcoRI and MseI adaptors. Each PCR mix consisted of 1 μl of Promega PCR buffer, 1 μl of 2 mM concentrations of deoxynucleoside triphosphates, 0.2 μl of 50 μM EcoRI+0 primer (FAM labeled), 0.2 μl of 50 μM MseI+0 primer, 0.6 μl of magnesium chloride (25 mM), 4.3 μl of water, 0.2 μl of Promega Taq polymerase, and 2.5 μl of the appropriate ligation mix. Cycling conditions were as follows: 94°C for 5 min; followed by 13 cycles of 94°C for 1 min, 65°C for 1 min, and 72°C for 1 min, reducing the annealing temperature by 1°C each cycle; followed in turn by 17 cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 1 min; with a final finishing step of 72°C for 30 min. Freshly prepared 1:10 dilutions of the first-round PCRs were used as templates for a second-round PCR containing 2 μl of Promega PCR buffer, 2 μl of 2 mM concentrations of deoxynucleoside triphosphates, 0.4 μl of 50 μM EcoRI+0 primer (FAM labeled), 0.4 μl of 50 μM MseI+TC primer, 1.2 μl of 25 mM magnesium chloride, 8.8 μl of water, 0.2 μl of Promega Taq polymerase, and 5 μl of the appropriate diluted first-round PCR. Cycling conditions were as for the first-round amplification, and reactions were stored at –20°C until ready to precipitate.
Preparation of samples for fragment analysis. Since unpurified AFLP reactions were found to result in unacceptable levels of background when subjected to electrophoresis, all samples were purified prior to this step. Samples were prepared for fragment analysis by removing 4 μl of PCR product and adding 16 μl of water, 50 μl of 95% ethanol, and 1 μl of 3 M sodium acetate (pH 5.2) to each sample. Samples were precipitated by centrifugation, washed in 70% (vol/vol) ethanol, dried, and resuspended in 4 μl of water. This procedure yielded significantly improved profile quality compared to equivalent reactions run without purification. For fragment analysis, 1 μl of each purified sample was mixed with 0.7 μl of formamide-blue dextran loading buffer (300 μl of blue dextran [50 mg ml–1] in 1 ml of deionized formamide) and 0.3 μl of Genescan-500ROX size standard (Applied Biosystems). Samples were heated at 95°C for 2 min immediately prior to being loaded onto 36-cm ABI377 sequencing gels.
Data analysis. After electrophoresis initial data collection was performed by using the ABI Genescan software (Applied Biosystems). Each gel track was then imported into the Bionumerics package (Applied Maths) by using the program ABICON (Applied Maths) and normalized with reference to the ROX-labeled size standard included in each sample. After normalization, the levels of genetic diversity between the AFLP patterns were calculated by using the Pearson product-moment correlation coefficient. Cluster analysis was performed by using the unweighted pair group method with arithmetic averages. The reliability of clustering was determined by calculation of cophenetic correlation values (Bionumerics). This method calculates the correlation between dendrogram derived similarities and matrix similarities, providing a measure of statistical reliability of each of the clusters. In addition, where linkage levels are discussed the standard deviation for the relevant branch is given, providing an indication of the stability and significance of clustering.
RESULTS
Preliminary assessment of discriminatory enzyme-primer combinations. Initial stages of the present study involved selection of appropriate restriction enzyme combinations to digest genomic DNA. A large number of different enzyme combinations have been applied to other organisms; however, the success of particular enzyme combinations and reaction conditions in producing a good banding pattern (i.e., a manageable number of bands, a good spread of bands, and low background) cannot be predicted in advance for a new organism. Thus, a small panel of type strains of Brucella was used to assess the usefulness of a large selection of enzyme-primer combinations. Forty-three different restriction enzyme-primer combinations (illustrated in Table 3) were assessed for suitability in AFLP analysis with four Brucella reference strains (B. abortus biovars 1 and 2, B. melitensis biovar 1, and B. ovis). Most enzymes were selected on the basis of having been used successfully in other AFLP typing schemes; however, some attempt was made to focus on potential areas of diversity by selecting enzymes such as HindIII and HinfI that cut within the Brucella-specific insertion sequence IS711. The rationale behind this was that current knowledge of Brucella suggests that they are genetically highly homogeneous, and thus such an approach might enhance the probability of good strain resolution.
First-round PCR was performed with nonselective primer combinations, and products were subjected to electrophoresis to examine the resultant banding pattern. A number of enzyme combinations were found to give good banding patterns, particularly EcoRI-MseI, EcoRI-TaqI, and ApaI-MseI. Other combinations appeared to be far less useful since there were either few bands or the bands were of a poor size range. No combination with HindIII appeared to be useful, and HinfI cut Brucella DNA very poorly. Although some of these combinations were subjected to further trials with the addition of a single selective base, all were ultimately abandoned so that we could examine more useful combinations. Since the patterns obtained with the more useful combinations were too complex for analysis, it was necessary to subject the product of the first-round PCR to a further PCR by using primers with additional selective bases. In the case of the EcoRI-MseI and EcoRI-TaqI combinations, the addition of a single selective base to the MseI primer did not reduce complexity sufficiently, and it proved necessary to examine the use of combinations with the addition of two selective bases to the MseI primer. In the case of the ApaI-MseI combination, the addition of a single selective base to the MseI primer adequately reduced complexity, presumably reflecting less-frequent cutting of Brucella DNA by ApaI relative to EcoRI. The final choice of the enzyme combination to pursue for larger scale studies was based on the "quality" of the banding pattern, with combinations that gave clear banding patterns, a manageable number of bands, and a good spread of bands across a usable size range (50 to 450 bp) being selected. In addition, because of the need to balance the reduction in pattern complexity needed to permit analysis with the concomitant reduction in discriminatory ability that inevitably resulted, combinations in which clear differences between reference strains were apparent were selected. As a result, the primer combination EcoRI+0-MseI+TC was selected for further study. A number of other combinations also gave promising results in this preliminary assessment and, although they were not pursued in depth in the present study, may merit further investigation elsewhere. These combinations are indicated in Table 3.
Assessment of AFLP for species identification of Brucella. The ability of the EcoRI+0-MseI+TC AFLP reaction to assign isolates of Brucella to the currently recognized species was assessed by using the panel of isolates described in Table 1. These isolates were selected to include as much breadth of diversity as possible by including isolates of different biovars representing diverse geographical and temporal sources. A dendrogram based on the AFLP profiles of the corresponding isolates is shown in Fig. 1. Despite the fact that AFLP profiles were found to be very highly conserved isolates representing B. ovis, B. melitensis, B. abortus, B. neotomae, and the marine mammal Brucella all fall into separate clusters. The remaining two species—B. suis and B. canis—form a further cluster, but there does not appear to be any differentiation between isolates of these two species. The cophenetic correlation values determined for each of the clusters were as follows: B. ovis, 81; B. melitensis, 78; B. abortus, 71; B. neotomae, 100; marine mammal Brucella, 79; and B. suis-B. canis, 78. The value for the whole dendrogram was 93.
A number of major bands that consistently discriminate between Brucella species were identified and are highlighted in the electropherogram traces shown in Fig. 2. Thus, for example, B. melitensis can be discriminated by the absence of a peak of 129 bp (B. ovis) or 130 bp (all other species), whereas B. ovis, in addition to the unique peak at 129 bp, lacks the peak at 107 bp seen in all other species. B. abortus isolates possess a peak at 235 bp not seen in any other species and are further differentiated from B. suis-B. canis by the absence of a 252-bp product and an additional peak at 196 bp in the latter. Although the number of B. neotomae isolates examined was small, they can be differentiated from all other species by an additional peak at 135 bp.
Assessment of AFLP for identifying Brucella. Reflecting the relative genetic homogeneity of the genus, unidentified isolates can confidently be identified as Brucella by using this AFLP approach. To illustrate this, we applied the same AFLP approach to various Ochrobactrum species. Members of this genus represent the closest known relatives of Brucella spp. and display >98% sequence identity in 16S rRNA analysis (25). A dendrogram comparing the profiles of the type strains of the five known Ochrobactrum spp. with representatives of each of the Brucella spp. is shown in Fig. 3. The AFLP profile of all of these isolates is very distinct from that of the Brucella spp. that separate into a cluster with a linkage level of 70.75% ± 5.77%. The cophenetic correlation value of the Brucella cluster was 93 with a value of 99 for the whole dendrogram. This analysis once again highlights the striking genetic homogeneity within the Brucella in contrast to the very diverse patterns observed with different species of Ochrobactrum. The practical application of this was demonstrated during the course of the present study when this AFLP approach was used to examine several isolates phenotypically resembling Brucella spp. Examination of AFLP profiles categorically demonstrated that these isolates were not Brucella, and sequence analysis of the 16S rRNA confirmed that these isolates showed highest similarity with Ochrobactrum sequences (data not shown).
Intraspecies variability in field isolates assessed by AFLP. As illustrated in Fig. 1, there is limited diversity within the individual Brucella spp. and little obvious genetic relationship between members of distinct biovars. All of the species clusters are defined by linkage levels of >93% (B. ovis, 93.78% ± 2.07%; B. melitensis, 93.17% ± 1.97%; B. suis-B. canis, 93.90% ± 1.99%; B. abortus, 95.37% ± 2.01%; B. neotomae, 94.73%; and marine mammal Brucella, 95.59% ± 1.35%). In addition to the isolates described here we assessed a large number of additional field isolates so that, in all, 40 B. abortus, 52 B. melitensis, 69 marine mammal Brucella, 43 B. ovis, 14 B. suis, 6 B. canis, and 3 B. neotomae isolates were subject to the EcoRI+0-MseI+TC AFLP. These studies again confirmed the extreme homogeneity within the classical Brucella species. Although minor intraspecies diversity was apparent, such as a major band at 162 bp that differentiates the B. suis biovar 2 strains F12/02, F13/02, F5-03-2, and 74/12 or a slight size shift of a 74-bp band in B. melitensis Ether, 80/95, and UK5/02, differences are so rare that this AFLP approach, although useful for identification and speciation, seems unlikely to provide adequate resolution for epidemiological traceback purposes.
DISCUSSION
This study is the first published application of the widely used DNA fingerprinting technique AFLP to the differentiation of Brucella isolates. A number of promising enzyme-primer combinations were identified by initial screening and one, EcoRI+0-MseI+TC, was chosen for use in a full-scale assessment of the applicability of the approach in the identification and typing of Brucella. The well-known lack of genetic heterogeneity of Brucella spp. (26) has frustrated attempts to develop molecular tools to fully differentiate Brucella isolates in the past. The present study further highlights the high degree of DNA homology in Brucella with a technique that has been applied to many organisms on the basis of its powerful discriminatory capacity, revealing only very limited diversity between the classical Brucella species. This contrasts with the extensive diversity seen both within species and between species of a genus when AFLP has been applied to many other bacteria (see, for example, references 1, 9, 10, 17, and 24) or, for example, between the Ochrobactrum strains examined in the present study. Despite this apparent genetic homogeneity, we have developed a reproducible AFLP method that will readily permit both the identification of an isolate as Brucella and, with one notable exception (B. suis-B. canis), the identification of field isolates as members of one of the six classical species.
A number of characteristic differences in profile between members of the classical Brucella species that reflect the clustering pattern shown in Fig. 1 were identified, as highlighted in Fig. 2, and these were found to be consistent across large numbers of field isolates examined. Field and reference isolates of B. abortus, B. ovis, B. melitensis, and B. neotomae all fall into distinct clusters consistent with the long-standing separation of these groups based on phenotypic observations. Only B. canis and B. suis isolates could not be separated into distinct clusters by this AFLP approach. This finding mirrors a number of previous observations, including chromosomal maps (18), omp2 profiling (5, 6), multilocus enzyme electrophoresis (11), and insertion sequence typing (21), that have demonstrated little or no difference between isolates of B. suis and B. canis. Although all field strains clustered into their appropriate species groups, we noted that one reference strain, not included in the dendrogram, failed to cluster as expected. The B. suis biovar 5 reference strain 513 lacked the 196-bp band and possessed the 252-bp band and thus appears to be most closely related to B. abortus. However, it is notable the status of the B. suis group as a biologically meaningful species has been questioned (19). For example, the B. suis biovar 2 type strain Thomsen is reported to give very different IRS-PCR profiles than other B. suis biovars, although this profile was shared with other B. suis biovar 2 field isolates (7). Furthermore, this isolate is reported to have a completely different IS6501 (IS711) profile from the highly conserved pattern seen in B. canis and B suis biovars 1, 3, and 4 (21). Less work has been carried done with B. suis biovar 5, but it has been reported to share multilocus enzyme electrophoresis profiles with B. melitensis and not with other B. suis isolates (11).
One positive aspect of the extreme homogeneity of the Brucella spp. is that the conserved AFLP profile permits confident genus identification of Brucella. Thus, it was immediately apparent that a number of isolates investigated as suspect Brucella spp. during the course of the present study were not Brucella. Although the approach used here is clearly useful for identification and speciation of Brucella isolates, there was only very limited genetic diversity detected within any of the classical Brucella species. Levels of similarity were generally determined to be 93%, with only a few band differences noted. With the possible exception of B. suis biovar 2, there was no obvious relationship between any of the classical biovars and AFLP profile, although the numbers of many of the individual biovars examined were very small, and a much more detailed study is needed to investigate this further. This lack of diversity and the fact that similarity levels within the species clusters fall within the 90 to 95% value equivalent to that considered normal between AFLP replicates (10, 15) makes application of the AFLP approach described here to epidemiological traceback unlikely. The potential to increase AFLP discriminatory capacity for an organism as genetically conserved as Brucella with a single set of conditions is limited by the inherent need to balance discriminatory power with the need to maintain a manageable number of bands for analysis. Thus, for example, it was apparent during the course of the present study that EcoRI+0-MseI combinations with only a single selective base reveal more diversity but increase the number of bands above levels that the analysis programs can handle. One approach that might substantially increase resolution and thus enhance the potential of the technique as an epidemiological tool would be to obtain AFLP profiles from isolates by using a number of different enzyme and/or selective primer combinations and then amalgamate the data. If this approach were to be pursued, the combinations identified in Table 3 as potentially useful for Brucella AFLP provide a starting point for further work in this direction.
In summary, we describe an AFLP approach developed for Brucella that, reflecting the substantial database of profiles built up in the course of the present study, allows confident identification of an isolate as Brucella and, with the exception of B. suis and B. canis, placement within one of the classically recognized species. The present study provides an additional molecular tool for use in confirmation and characterization of this important human and veterinary pathogen and outlines potentially useful avenues for further development and refinement of the technique. AFLP is relatively straightforward to perform, and reproducible, with high throughput, and the storage of data in electronic forms should facilitate the development of a database that permits comparisons of isolates over long time periods.
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ABSTRACT
Amplified fragment length polymorphism (AFLP) is a whole-genome fingerprinting method that relies on the selective PCR amplification of restriction fragments. The potential of this approach for the discrimination of Brucella isolates at the species and intraspecies level was assessed. A number of different combinations of restriction enzymes and selective primers were examined, and one, using EcoRI and MseI with additional selective TC bases on the MseI primer, was selected for full assessment against a panel of Brucella isolates. The technique could readily differentiate Brucella spp. from all Ochrobactrum spp. representing the group of organisms most closely related to Brucella spp. Application of AFLP highlighted the genetic homogeneity of Brucella. In spite of this determination of AFLP profiles of large numbers of isolates of human and animal origin, including Brucella abortus, B. melitensis, B. ovis, B. neotomae, marine mammal isolates (no species name), B. canis, and B. suis, confirmed that all but the latter two species could be separated into distinct clusters based on characteristic and conserved differences in profile. Only B. suis and B. canis isolates clustered together and could not be distinguished by this approach, adding to questions regarding the validity of species assignments in this group. Under the conditions examined in the present study only limited intraspecies genomic differences were detected, and thus this AFLP approach is likely to prove most useful for identification to the species level. However, combination of several of the useful restriction enzyme-primer combinations identified in the present study could substantially add to the discriminatory power of AFLP when applied to Brucella and enhance the value of this approach.
INTRODUCTION
Brucella spp. comprise a closely related group of organisms that classical taxonomists divided into six species based on subtle phenotypic and antigenic differences and host specificity: Brucella abortus (bovine), B. melitensis (caprine and ovine), B. ovis (ovine), B. canis (canine), B. suis (porcine), and B. neotomae (only seen in the desert wood rat). The situation has recently been complicated by the identification of Brucella isolates in marine mammals that do not fit into any of the recognized species and themselves show intragroup diversity (4, 14). Some of the species are classically divided into biovars such that several B. abortus, B. melitensis, and B. suis biovars are recognized. In addition, host specificity is not absolute; thus, B. melitensis and B. suis are important causes of bovine disease in some countries, and B. suis biovars 2, 4, and 5 have been associated with hares, reindeer, and rodents, respectively. The traditional view on Brucella taxonomy was challenged some time ago on the basis of the high degree of homology indicated by DNA hybridization experiments (26) and the inability to differentiate Brucella spp. by 16S rRNA sequencing. Although these findings conform better with the view of a single species within which the six classical species would be considered biovars, this scheme has not achieved widespread acceptance largely on practical grounds.
Brucellosis remains an important disease in both humans, in whom it leads to a chronic debilitating infection, and in domesticated animals, in which the main symptom is reproductive failure. A number of characteristics make Brucella spp. attractive targets for weaponization, and the organism remains on the list of CDC category B potential biological warfare agents (22). In humans, brucellosis caused by B. melitensis is by far the most important clinically apparent disease and is usually associated with occupational exposure or the consumption of unpasteurized dairy products. However, B. abortus, B. suis and, more rarely, B. canis and the marine mammal Brucella can also cause human infection. Brucellosis remains a major problem in many parts of the world, notably in the Mediterranean region, western Asia, and parts of Africa and Latin America (8). Brucellosis has been eradicated or severely curtailed in some Western countries by a combination of strict veterinary hygiene measures, monitoring programs, and improved food safety measures. However, regions recognized as "officially brucellosis free" are under constant threat of reintroduction via livestock trading, reinforcing the requirement for reliable molecular tools for the identification and typing of Brucella. The development of such discriminatory molecular tools has long been problematic, reflecting the lack of genetic polymorphism in Brucella spp., and many laboratories rely on conventional biotyping to identify and speciate Brucella. Furthermore, whereas the development of genus-specific and, in some cases, species-specific PCR assays for identification has been possible (2), typing tools of sufficient resolution to permit epidemiological tracing of outbreaks are still lacking. One of the most promising molecular approaches to date utilizes DNA polymorphism, reflecting the variable distribution of an insertion sequence, IS711, in the chromosome. The use of an IS711 based probe reveals that between 5 and 30 copies of this element can be present. In most cases species can be differentiated by their distinct patterns, although this is not absolute and further discrimination between biovars is limited (3). An alternative approach is the PCR-restriction fragment length polymorphism analysis of outer membrane protein encoding genes that can differentiate the six species of Brucella (23) and furthermore can differentiate between some biovars and field isolates of individual species (5, 6). However, findings to date indicate that neither of these approaches offers sufficient power of resolution to be confidently used in epidemiological tracing.
Here we investigate the application of an alternative DNA fingerprinting approach, AFLP, to Brucella. AFLP is based on the amplification of subsets of genomic restriction fragments by using PCR (15). DNA is cut with restriction enzymes, and double-stranded adaptors are ligated to the ends of DNA fragments to generate template DNA for PCR amplification. The sequence of the adaptors and adjacent restriction site serve as primer binding sites for subsequent amplification of the restriction fragments. Selective nucleotides can be included at the 3' ends of the PCR primers, which therefore prime DNA synthesis from only a subset of the restriction sites. Labeling of one of the primers (usually the one that corresponds to the less frequently cutting restriction enzyme) with a fluorescent dye permits visualization of a banding pattern after electrophoresis. Although amplified fragment length polymorphism (AFLP) requires no prior sequence knowledge, the restriction enzyme and primer combinations selected will substantially affect the discriminatory power of the method and thus suitable combinations must be selected for each organism being examined. AFLP has now been applied to a substantial number of microorganisms and has been shown to be highly discriminatory, rapid, and reproducible (20) and has proven to be a useful tool in bacterial taxonomy (10, 12, 13). We describe here the development of an AFLP approach to identify and speciate Brucella isolates and assess whether this approach may have value as an epidemiological tool.
(Preliminary portions of this study were presented 28 to 31 May 2003 at the IMAB-NATO Conference on Risk Infections and Possibilities for Medical BioTerrorism in Varna, Bulgaria.)
MATERIALS AND METHODS
Strains. Details of all strains described in the present study are given in Table 1. Brucella strains were routinely cultured on serum dextrose agar plates at 37°C in the presence of 10% CO2 for 3 days.
DNA preparation. DNA was extracted by standard procedures. Briefly, growth from two to four spread plates was harvested into 2 ml of sterile water per plate, and the final volume was made up to 20 ml. The resulting cell suspension was harvested by centrifugation, the supernatant was removed, and the cells were resuspended in 5 ml of 1x TNE buffer (0.1 M Tris-HCl [pH 8], 0.1 M EDTA [pH 8], 0.1 M NaCl), 1% (wt/vol) sodium dodecyl sulfate, and 1% (wt/vol) Sarcosine. After a vortexing step, a 150-μl aliquot of proteinase K (20 mg ml–1) was added to the cell suspensions, which were then incubated for 1 h at 65°C, followed by the addition of further proteinase K aliquots if necessary, until complete cell lysis had occurred. Phenol-chloroform extractions were performed by adding 5 ml of Ultrapure buffer saturated phenol (Invitrogen), mixing the suspension thoroughly, centrifuging the mixture at 4,000 rpm for 5 min, and removing the aqueous phase. The extraction procedure was repeated twice more by using Ultrapure phenol-chloroform-isoamyl alcohol (25:24:1; Invitrogen), and DNA was precipitated by adding the final aqueous phase to ca. 15 ml of chilled (–20°C) ethanol. After precipitation, the pellet was washed in 70% ethanol, dried, and resuspended in Tris-EDTA buffer.
AFLP procedure. In initial testing, some 43 different restriction enzyme-primer combinations were screened for suitability by using DNA extracted from four Brucella type strains (B. abortus biovars 1 and 2, B. melitensis biovar 1, and B. ovis). The sequences of all adapters used and the core AFLP primers are given in Table 2. DNA was digested with appropriate restriction enzymes according to the manufacturer's instructions and subjected to standard AFLP procedures (15, 16). Full details of the final protocol adopted for the "EcoRI+0-MseI+TC AFLP" approach selected for large-scale study are as follows. Chromosomal DNA concentrations were adjusted to 0.25 μg/μl, and 3 μl of each DNA to be tested was digested with MseI in React 1 buffer (Invitrogen) in a 10-μl final volume for 1 h at 37°C. After this, 1 μl of React 1, 2 μl of 1 M NaCl, 1 μl of EcoRI (Invitrogen), and 6 μl of water were added, followed by incubation for a further 1 h at 37°C. Double-stranded adapters were prepared by mixing equal volumes of the two relevant adapter primers to give final concentrations of 25 μM for the MseI adapter and 2.5 μM for the EcoRI adapter. The mixes were heated to 95°C for 10 min and then allowed to anneal by cooling to room temperature for 15 min. Adapters were ligated to genomic DNA digests in a mix containing 2 μl of 5x ligase buffer, 1 μl of T4 DNA ligase, 2 μl of EcoRI adaptors (5 pmol), 2 μl of MseI adaptors (50pmol), 1 μl of digest from above, and 2 μl of water, followed by incubation at 12°C for a minimum of 4 h. A first-round PCR was then performed with nonselective primers specific for EcoRI and MseI adaptors. Each PCR mix consisted of 1 μl of Promega PCR buffer, 1 μl of 2 mM concentrations of deoxynucleoside triphosphates, 0.2 μl of 50 μM EcoRI+0 primer (FAM labeled), 0.2 μl of 50 μM MseI+0 primer, 0.6 μl of magnesium chloride (25 mM), 4.3 μl of water, 0.2 μl of Promega Taq polymerase, and 2.5 μl of the appropriate ligation mix. Cycling conditions were as follows: 94°C for 5 min; followed by 13 cycles of 94°C for 1 min, 65°C for 1 min, and 72°C for 1 min, reducing the annealing temperature by 1°C each cycle; followed in turn by 17 cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 1 min; with a final finishing step of 72°C for 30 min. Freshly prepared 1:10 dilutions of the first-round PCRs were used as templates for a second-round PCR containing 2 μl of Promega PCR buffer, 2 μl of 2 mM concentrations of deoxynucleoside triphosphates, 0.4 μl of 50 μM EcoRI+0 primer (FAM labeled), 0.4 μl of 50 μM MseI+TC primer, 1.2 μl of 25 mM magnesium chloride, 8.8 μl of water, 0.2 μl of Promega Taq polymerase, and 5 μl of the appropriate diluted first-round PCR. Cycling conditions were as for the first-round amplification, and reactions were stored at –20°C until ready to precipitate.
Preparation of samples for fragment analysis. Since unpurified AFLP reactions were found to result in unacceptable levels of background when subjected to electrophoresis, all samples were purified prior to this step. Samples were prepared for fragment analysis by removing 4 μl of PCR product and adding 16 μl of water, 50 μl of 95% ethanol, and 1 μl of 3 M sodium acetate (pH 5.2) to each sample. Samples were precipitated by centrifugation, washed in 70% (vol/vol) ethanol, dried, and resuspended in 4 μl of water. This procedure yielded significantly improved profile quality compared to equivalent reactions run without purification. For fragment analysis, 1 μl of each purified sample was mixed with 0.7 μl of formamide-blue dextran loading buffer (300 μl of blue dextran [50 mg ml–1] in 1 ml of deionized formamide) and 0.3 μl of Genescan-500ROX size standard (Applied Biosystems). Samples were heated at 95°C for 2 min immediately prior to being loaded onto 36-cm ABI377 sequencing gels.
Data analysis. After electrophoresis initial data collection was performed by using the ABI Genescan software (Applied Biosystems). Each gel track was then imported into the Bionumerics package (Applied Maths) by using the program ABICON (Applied Maths) and normalized with reference to the ROX-labeled size standard included in each sample. After normalization, the levels of genetic diversity between the AFLP patterns were calculated by using the Pearson product-moment correlation coefficient. Cluster analysis was performed by using the unweighted pair group method with arithmetic averages. The reliability of clustering was determined by calculation of cophenetic correlation values (Bionumerics). This method calculates the correlation between dendrogram derived similarities and matrix similarities, providing a measure of statistical reliability of each of the clusters. In addition, where linkage levels are discussed the standard deviation for the relevant branch is given, providing an indication of the stability and significance of clustering.
RESULTS
Preliminary assessment of discriminatory enzyme-primer combinations. Initial stages of the present study involved selection of appropriate restriction enzyme combinations to digest genomic DNA. A large number of different enzyme combinations have been applied to other organisms; however, the success of particular enzyme combinations and reaction conditions in producing a good banding pattern (i.e., a manageable number of bands, a good spread of bands, and low background) cannot be predicted in advance for a new organism. Thus, a small panel of type strains of Brucella was used to assess the usefulness of a large selection of enzyme-primer combinations. Forty-three different restriction enzyme-primer combinations (illustrated in Table 3) were assessed for suitability in AFLP analysis with four Brucella reference strains (B. abortus biovars 1 and 2, B. melitensis biovar 1, and B. ovis). Most enzymes were selected on the basis of having been used successfully in other AFLP typing schemes; however, some attempt was made to focus on potential areas of diversity by selecting enzymes such as HindIII and HinfI that cut within the Brucella-specific insertion sequence IS711. The rationale behind this was that current knowledge of Brucella suggests that they are genetically highly homogeneous, and thus such an approach might enhance the probability of good strain resolution.
First-round PCR was performed with nonselective primer combinations, and products were subjected to electrophoresis to examine the resultant banding pattern. A number of enzyme combinations were found to give good banding patterns, particularly EcoRI-MseI, EcoRI-TaqI, and ApaI-MseI. Other combinations appeared to be far less useful since there were either few bands or the bands were of a poor size range. No combination with HindIII appeared to be useful, and HinfI cut Brucella DNA very poorly. Although some of these combinations were subjected to further trials with the addition of a single selective base, all were ultimately abandoned so that we could examine more useful combinations. Since the patterns obtained with the more useful combinations were too complex for analysis, it was necessary to subject the product of the first-round PCR to a further PCR by using primers with additional selective bases. In the case of the EcoRI-MseI and EcoRI-TaqI combinations, the addition of a single selective base to the MseI primer did not reduce complexity sufficiently, and it proved necessary to examine the use of combinations with the addition of two selective bases to the MseI primer. In the case of the ApaI-MseI combination, the addition of a single selective base to the MseI primer adequately reduced complexity, presumably reflecting less-frequent cutting of Brucella DNA by ApaI relative to EcoRI. The final choice of the enzyme combination to pursue for larger scale studies was based on the "quality" of the banding pattern, with combinations that gave clear banding patterns, a manageable number of bands, and a good spread of bands across a usable size range (50 to 450 bp) being selected. In addition, because of the need to balance the reduction in pattern complexity needed to permit analysis with the concomitant reduction in discriminatory ability that inevitably resulted, combinations in which clear differences between reference strains were apparent were selected. As a result, the primer combination EcoRI+0-MseI+TC was selected for further study. A number of other combinations also gave promising results in this preliminary assessment and, although they were not pursued in depth in the present study, may merit further investigation elsewhere. These combinations are indicated in Table 3.
Assessment of AFLP for species identification of Brucella. The ability of the EcoRI+0-MseI+TC AFLP reaction to assign isolates of Brucella to the currently recognized species was assessed by using the panel of isolates described in Table 1. These isolates were selected to include as much breadth of diversity as possible by including isolates of different biovars representing diverse geographical and temporal sources. A dendrogram based on the AFLP profiles of the corresponding isolates is shown in Fig. 1. Despite the fact that AFLP profiles were found to be very highly conserved isolates representing B. ovis, B. melitensis, B. abortus, B. neotomae, and the marine mammal Brucella all fall into separate clusters. The remaining two species—B. suis and B. canis—form a further cluster, but there does not appear to be any differentiation between isolates of these two species. The cophenetic correlation values determined for each of the clusters were as follows: B. ovis, 81; B. melitensis, 78; B. abortus, 71; B. neotomae, 100; marine mammal Brucella, 79; and B. suis-B. canis, 78. The value for the whole dendrogram was 93.
A number of major bands that consistently discriminate between Brucella species were identified and are highlighted in the electropherogram traces shown in Fig. 2. Thus, for example, B. melitensis can be discriminated by the absence of a peak of 129 bp (B. ovis) or 130 bp (all other species), whereas B. ovis, in addition to the unique peak at 129 bp, lacks the peak at 107 bp seen in all other species. B. abortus isolates possess a peak at 235 bp not seen in any other species and are further differentiated from B. suis-B. canis by the absence of a 252-bp product and an additional peak at 196 bp in the latter. Although the number of B. neotomae isolates examined was small, they can be differentiated from all other species by an additional peak at 135 bp.
Assessment of AFLP for identifying Brucella. Reflecting the relative genetic homogeneity of the genus, unidentified isolates can confidently be identified as Brucella by using this AFLP approach. To illustrate this, we applied the same AFLP approach to various Ochrobactrum species. Members of this genus represent the closest known relatives of Brucella spp. and display >98% sequence identity in 16S rRNA analysis (25). A dendrogram comparing the profiles of the type strains of the five known Ochrobactrum spp. with representatives of each of the Brucella spp. is shown in Fig. 3. The AFLP profile of all of these isolates is very distinct from that of the Brucella spp. that separate into a cluster with a linkage level of 70.75% ± 5.77%. The cophenetic correlation value of the Brucella cluster was 93 with a value of 99 for the whole dendrogram. This analysis once again highlights the striking genetic homogeneity within the Brucella in contrast to the very diverse patterns observed with different species of Ochrobactrum. The practical application of this was demonstrated during the course of the present study when this AFLP approach was used to examine several isolates phenotypically resembling Brucella spp. Examination of AFLP profiles categorically demonstrated that these isolates were not Brucella, and sequence analysis of the 16S rRNA confirmed that these isolates showed highest similarity with Ochrobactrum sequences (data not shown).
Intraspecies variability in field isolates assessed by AFLP. As illustrated in Fig. 1, there is limited diversity within the individual Brucella spp. and little obvious genetic relationship between members of distinct biovars. All of the species clusters are defined by linkage levels of >93% (B. ovis, 93.78% ± 2.07%; B. melitensis, 93.17% ± 1.97%; B. suis-B. canis, 93.90% ± 1.99%; B. abortus, 95.37% ± 2.01%; B. neotomae, 94.73%; and marine mammal Brucella, 95.59% ± 1.35%). In addition to the isolates described here we assessed a large number of additional field isolates so that, in all, 40 B. abortus, 52 B. melitensis, 69 marine mammal Brucella, 43 B. ovis, 14 B. suis, 6 B. canis, and 3 B. neotomae isolates were subject to the EcoRI+0-MseI+TC AFLP. These studies again confirmed the extreme homogeneity within the classical Brucella species. Although minor intraspecies diversity was apparent, such as a major band at 162 bp that differentiates the B. suis biovar 2 strains F12/02, F13/02, F5-03-2, and 74/12 or a slight size shift of a 74-bp band in B. melitensis Ether, 80/95, and UK5/02, differences are so rare that this AFLP approach, although useful for identification and speciation, seems unlikely to provide adequate resolution for epidemiological traceback purposes.
DISCUSSION
This study is the first published application of the widely used DNA fingerprinting technique AFLP to the differentiation of Brucella isolates. A number of promising enzyme-primer combinations were identified by initial screening and one, EcoRI+0-MseI+TC, was chosen for use in a full-scale assessment of the applicability of the approach in the identification and typing of Brucella. The well-known lack of genetic heterogeneity of Brucella spp. (26) has frustrated attempts to develop molecular tools to fully differentiate Brucella isolates in the past. The present study further highlights the high degree of DNA homology in Brucella with a technique that has been applied to many organisms on the basis of its powerful discriminatory capacity, revealing only very limited diversity between the classical Brucella species. This contrasts with the extensive diversity seen both within species and between species of a genus when AFLP has been applied to many other bacteria (see, for example, references 1, 9, 10, 17, and 24) or, for example, between the Ochrobactrum strains examined in the present study. Despite this apparent genetic homogeneity, we have developed a reproducible AFLP method that will readily permit both the identification of an isolate as Brucella and, with one notable exception (B. suis-B. canis), the identification of field isolates as members of one of the six classical species.
A number of characteristic differences in profile between members of the classical Brucella species that reflect the clustering pattern shown in Fig. 1 were identified, as highlighted in Fig. 2, and these were found to be consistent across large numbers of field isolates examined. Field and reference isolates of B. abortus, B. ovis, B. melitensis, and B. neotomae all fall into distinct clusters consistent with the long-standing separation of these groups based on phenotypic observations. Only B. canis and B. suis isolates could not be separated into distinct clusters by this AFLP approach. This finding mirrors a number of previous observations, including chromosomal maps (18), omp2 profiling (5, 6), multilocus enzyme electrophoresis (11), and insertion sequence typing (21), that have demonstrated little or no difference between isolates of B. suis and B. canis. Although all field strains clustered into their appropriate species groups, we noted that one reference strain, not included in the dendrogram, failed to cluster as expected. The B. suis biovar 5 reference strain 513 lacked the 196-bp band and possessed the 252-bp band and thus appears to be most closely related to B. abortus. However, it is notable the status of the B. suis group as a biologically meaningful species has been questioned (19). For example, the B. suis biovar 2 type strain Thomsen is reported to give very different IRS-PCR profiles than other B. suis biovars, although this profile was shared with other B. suis biovar 2 field isolates (7). Furthermore, this isolate is reported to have a completely different IS6501 (IS711) profile from the highly conserved pattern seen in B. canis and B suis biovars 1, 3, and 4 (21). Less work has been carried done with B. suis biovar 5, but it has been reported to share multilocus enzyme electrophoresis profiles with B. melitensis and not with other B. suis isolates (11).
One positive aspect of the extreme homogeneity of the Brucella spp. is that the conserved AFLP profile permits confident genus identification of Brucella. Thus, it was immediately apparent that a number of isolates investigated as suspect Brucella spp. during the course of the present study were not Brucella. Although the approach used here is clearly useful for identification and speciation of Brucella isolates, there was only very limited genetic diversity detected within any of the classical Brucella species. Levels of similarity were generally determined to be 93%, with only a few band differences noted. With the possible exception of B. suis biovar 2, there was no obvious relationship between any of the classical biovars and AFLP profile, although the numbers of many of the individual biovars examined were very small, and a much more detailed study is needed to investigate this further. This lack of diversity and the fact that similarity levels within the species clusters fall within the 90 to 95% value equivalent to that considered normal between AFLP replicates (10, 15) makes application of the AFLP approach described here to epidemiological traceback unlikely. The potential to increase AFLP discriminatory capacity for an organism as genetically conserved as Brucella with a single set of conditions is limited by the inherent need to balance discriminatory power with the need to maintain a manageable number of bands for analysis. Thus, for example, it was apparent during the course of the present study that EcoRI+0-MseI combinations with only a single selective base reveal more diversity but increase the number of bands above levels that the analysis programs can handle. One approach that might substantially increase resolution and thus enhance the potential of the technique as an epidemiological tool would be to obtain AFLP profiles from isolates by using a number of different enzyme and/or selective primer combinations and then amalgamate the data. If this approach were to be pursued, the combinations identified in Table 3 as potentially useful for Brucella AFLP provide a starting point for further work in this direction.
In summary, we describe an AFLP approach developed for Brucella that, reflecting the substantial database of profiles built up in the course of the present study, allows confident identification of an isolate as Brucella and, with the exception of B. suis and B. canis, placement within one of the classically recognized species. The present study provides an additional molecular tool for use in confirmation and characterization of this important human and veterinary pathogen and outlines potentially useful avenues for further development and refinement of the technique. AFLP is relatively straightforward to perform, and reproducible, with high throughput, and the storage of data in electronic forms should facilitate the development of a database that permits comparisons of isolates over long time periods.
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