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1Department of Sera and Vaccine Evaluation, National Institute of Hygiene, 24 Chocimska Str., 00-791 Warsaw, Poland 2Interfaculty Studies of Biotechnology, Warsaw Agricultural University, 159 Nowoursynowska Str., 00-776 Warsaw, Poland
Correspondence Anna Gzyl agzyl{at}pzh.gov.pl
Received April 5, 2004
Accepted November 19, 2004
Automated amplified fragment length polymorphism (AFLP) and randomly amplified polymorphic DNA (RAPD) techniques with fluorescently labelled primers were used to track differences among isolates of the eight known species of the Bordetella genus. Eighty-one representative strains of these species from international and Polish bacterial collections were genotyped according to RAPD protocols using primer 1254 or 1247, and AFLP involving EcoRI/MseI or newly designed SpeI/ApaI restriction/ligation/amplification procedures. By comparing AFLP and RAPD data, it was concluded that the discriminatory power of AFLP is higher in comparison with RAPD for both intra- and inter-species differentiation of isolates of the Bordetella genus. The most precise level of inter-species discrimination and the highest level of intra-species discrimination of the Bordetella isolates of the eight species were observed in the AFLP EcoRI/MseI and SpeI/ApaI sets, respectively. Both techniques might provide alternative tools for the identification of Bordetella at the genomic species and strain levels, and thus may be valuable in human and veterinary diagnostics as well as in epidemiology. By applying the AFLP technique presented in this article, more precise data on the emergence of newly acquired and/or on expanded clones and transmission routes of isolates of the Bordetella genus in the human and animal environments might be obtained.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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These three species can be differentiated by phenotype. However, the genetic diversity shown with many techniques [DNA–DNA hybridization, multilocus enzyme electrophoresis (MLEE), and 16S and 23S rRNA gene typing] has been found to be limited (Gerlach et al., 2001; Musser et al., 1986; van der Zee et al., 1997). Thus they are commonly considered as members of a single B. bronchiseptica cluster (Gerlach et al., 2001).
Bordetella avium colonizes mainly the respiratory tract of birds, inducing bird bordetellosis and turkey coryza (Gerlach et al., 2001). In poultry, B. avium infections result in coryza or rhinotracheitis, highly contagious diseases resulting in economic losses to the turkey/poultry industry (Register et al., 2003). Bordetella hinzii found in birds has recently been also associated with human infections seen in patients with AIDS or cystic fibrosis (Cookson et al., 1994; Dworkin et al., 1999; Funke et al., 1996; Kattar et al., 2000; Vandamme et al., 1995).
Bordetella holmesii has been recovered from patients with underlying disorders, including Hodgkin lymphoma, sickle-cell anaemia, pulmonary disease and asplenia, septicaemia and endocarditis (Russell et al., 2001; Tang et al., 1998; Yih et al., 1999). Surprisingly, in the last few years it has also been isolated from the nasopharynx of patients with pertussis-like symptoms (Mazengia et al., 2000).
Bordetella trematum, commonly isolated in humans from wounds or ears but not from the respiratory tract, is a species of unknown pathogenic potential (Gerlach et al., 2001).
Bordetella petrii, the first anaerobic environmental isolate of the Bordetella genus, was described in 2001 and still only a single strain is available (von Wintzingerode et al., 2001).
Tracking epidemiological routes is extremely important in the case of B. pertussis since it still affects large proportions of individuals and infection rates are rising unexpectedly in highly vaccinated populations (Mooi et al., 2000; van der Zee et al., 1996a). Similarly, studies of B. parapertussis transmission and co-infection with B. pertussis might elucidate many aspects of the epidemiology of whooping cough. Tracking differences among strains of B. bronchiseptica, B. avium, B. hinzii or B. holmesii as opportunistic infections of humans, together with differences found in strains isolated from natural animal hosts, might be important for human and animal microbiology.
Several studies have proved the usefulness of molecular tools in differentiating particular species of the Bordetella genus. Some of them were able to track genetic differences among/within two to three species simultaneously. For several years, amplified fragment length polymorphism (AFLP) and randomly amplified polymorphic DNA (RAPD) have been found to be very useful in studying the epidemiology of many pathogens, e.g. in order to follow transmission routes (van der Zwet et al., 1999) or to study phylogenetic relationships between organisms (Torriani et al., 2001). In this study we believe we present the first report on the application of AFLP for genotyping isolates within the Bordetella genus. The discriminatory power of AFLP fingerprinting has been compared with RAPD performed with protocols previously applied by Yuk et al. (1998) for B. parapertussis. In addition, we describe a newly designed SpeI/ApaI restriction enzymes/adapters/primers protocol not used previously for AFLP fingerprinting. Our preliminary study was aimed at investigating the possibility of Bordetella inter- and intra-species genotyping and evaluating the taxonomic potential of AFLP and RAPD in the delineation of species-specific identification.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Isolates of B. avium (nine), B. hinzii (four) and B. trematum (five) came from the BCCG/LMG Collection, Gent, Belgium. Among the 13 isolates of B. bronchiseptica, nine came from the BCCG/LMG Collection and one from the Institute of Immunology and Experimental Therapy in Wroclaw, Poland. Two B. bronchiseptica strains were obtained from Dr N. Guiso (Institut Pasteur, Paris). Regarding the B. holmesii strains analysed, three isolates came from the BCCG/LMG Collection and two were kindly obtained again from Dr N. Guiso (Institut Pasteur, Paris). Five and four isolates of B. parapertussis originated from the BCGG/LMG Collection and the parental Bacterial Institute Collection, NIH, Warsaw, Poland. B. petrii was kindly received from Professor R. Gross (Universität Wurzburg, Germany).
B. pertussis strains were cultured on Bordet–Gengou agar (Difco) supplemented with 15 % sheep blood and incubated at 35 °C for 2–3 days. The B. petrii strain was grown anaerobically on LB agar at 30 °C for 48 h (von Wintzingerode et al., 2001).
Isolation of total DNA.
DNA from 81 strains belonging to the Bordetella genus was isolated by means of a commercially available Qiagen DNA isolation kit. The concentration of the DNA was measured by electrophoresis of samples on 1 % agarose gel against diluted preparations of 
phage DNA of known concentrations.
Typing by the RAPD method.
Several different short primers designed by Williams et al. (1990) were screened for fingerprinting efficiency of different isolates of the eight Bordetella species. For screening, five different strains of each species were used where possible. The two primers with the best discriminatory potential were chosen to study all of the strains collected: 1247, 5'-AAGAGCCCGT-3', and 1254, 5'-CCCGTCAGCA-3'. Forty-two cycles of denaturation at 94 °C for 1 min, annealing at 36 °C for 1 min and extension at 72 °C for 1 min were applied in the Biometra thermal cycler. Aliquots of amplified PCR products were electrophoresed in 1.5 % agarose gels stained with ethidium bromide. Electrophoresis was carried out in TAE buffer (40 mM Tris/acetate, 1 mM EDTA).
Gels were photographed with the Gel Doc 1000 Gel Documentation System (Bio-Rad). Stored patterns were analysed with the GelCompar Software version 3.1 (Applied Maths) after conversion of the data into TIF format. Electrophoresis gels were normalized according to the external standards contained in each fifth lane (1 kb ladder, Gibco). Dendrograms for cluster analysis were based on similarity matrices calculated from the Pearson product-moment correlation coefficient and the unweighted pair group method using arithmetic averages (UPGMA) algorithm (Everitt, 1993).
Typing by the AFLP method.
In the preliminary screening DNA from, where possible, five isolates from each of the Bordetella species was used to evaluate the potential to obtain distinctive AFLP patterns. Six different previously described AFLP protocols, using HindIII/TagI, ApaI/TagI, MfeI/BglII, HindIII, PstI/TaqI and EcoRI/MseI, based on different enzyme restriction/specific adapter ligation and primer-specific amplification with/without selective bases complementary to nucleotides flanking the restriction sites, were tested (Grady et al., 1999; Huys et al., 1996; Kokotovic et al., 1999; McLauchlin et al., 2000; van Elderle et al., 1999; Vos & Kuiper, 1998). Additionally, new AFLP sets with SpeI restriction and the frequent-cutter enzymes BglII, HindIII, PstI, ApaI and EcoRI, coupled with ligation by newly designed SpeI adapters and primers, were tested (Table 2).
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Briefly, in all tested AFLP sets, 200 ng of Bordetella DNA was digested with the following restriction enzymes according to manufacturer's recommendations: HindIII (BioLabs)/TagI (Gibco-BRL); ApaI (BioLabs)/TagI; HindIII; MfeI (BioLabs)/BglII (BioLabs); PstI (Eurogentec)/TagI; EcoRI (Gibco-BRL)/MseI (BioLabs); SpeI (BioLabs)/BglII; SpeI/HindIII; SpeI/PstI; SpeI/ApaI; and SpeI/EcoRI. Samples were heat-treated to inactivate restriction enzymes. Next, adapters, synthesized by Invitrogen, were ligated to the restriction DNA fragments by T4 DNA ligase (BioLabs) according to manufacturer's recommendations. For rare-cutting enzymes, adapters were used at a concentration of 5 pmol µl–1, and for frequent-cutting enzymes, at 50 pmol µl–1. After completion of ligation, samples were heat-inactivated, 10-fold diluted in distilled water and refrigerated at –20 °C until the start of PCR.
Restriction fragments tagged with specific adapters were selectively amplified with relevant primers. PCR was performed in a 20 µl volume in a Biometra thermal cycler. Each amplification reaction mixture contained 5 µl of a 10-fold diluted ligation reaction sample, 12 ng Cy5-labelled primer, 60 ng non-labelled primer, 120 µM each of dATP, dGTP, dCTP and dTTP, 1.0 U AccuTaq-LA DNA polymerase (Sigma), 3.0 mM MgCl2, 50 mM Tris/HCl, 15 mM ammonium sulphate and 0.1 % Tween 20. Amplification for previously described sets was performed according to recommended conditions (Table 2). In the AFLP amplification step with SpeI primers tested with BglII, HindIII, PstI, ApaI or EcoRI ones, the following conditions were used: cycle 1, 94 °C for 30 s, 65 °C for 30 s, 72 °C for 60 s; cycles 2–13, the same PCR profile as seen in the first cycle except for a stepwise 0.7 °C decrease in the annealing temperature in each subsequent cycle of the 12 cycles; cycles 14–36, 94 °C for 30 s, 56 °C for 30 s, 72 °C for 60 s.
After completion of the PCR, 5 µl of each reaction tube mixture was mixed with 3 µl loading dye (Amersham Pharmacia), denatured at 90 °C for 3 min and applied to the gel. Selectively amplified fragments were separated through a ReproGel High Resolution (Amersham Pharmacia) in 0.5x TBE buffer on an ALFexpress DNA sequencer. Separation was done at 1500 V, 60 mA, 25 W for 420 min at 55 °C. To evaluate intra- and inter-gel differences and identity levels, a fluorescein-labelled molecular size marker (ALFexpress Sizer 50–500) and an external reference strain were used as external size markers. Stored fluorograms were analysed with the GelCompar Software version 3.1 (Applied Maths) after conversion into TIF format. The track resolution was reduced, excluding the primer front and fragments higher than 500 bp. Dendrograms for cluster analysis were based on similarity matrices calculated from the Pearson product-moment correlation coefficient and the UPGMA algorithm (Everitt, 1993).
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RESULTS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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The overall genetic similarities of all the RAPD patterns obtained using primers 1254 and 1247 within the dendrograms, as defined by the Pearson product-moment correlation coefficient, were 53.9 % and 64.0 %, respectively. RAPD arrays classified Bordetella isolates into several clusters. Yet, exact species-specific differentiation of patterns was not found, however B. avium isolates were found exclusively in single clusters in both of the dendrograms.
Two clusters were found for the RAPD protocol using the 1254 primer: A, containing 72 non-Bordetella avium isolates, and B, containing nine B. avium isolates. Cluster A, at the 63 % overall similarity level, consisted of two subclusters, AI and AII, and a single pattern that was specific for B. trematum no. 14993, defined as AIII. Eleven B. bronchiseptica and four B. trematum isolates clustered at a linkage level of 77 % within the AI subcluster. Subcluster AII patterns clustered at a level of 76 % and contained four clades AIIa–d. The AIIa clade contained groups that were related to each other, including patterns specific for B. pertussis and B. holmesii strains, and a group with five B. parapertussis and a single B. hinzii isolate (no. 14052). Polish isolates of B. parapertussis, B. trematum no. 15543, and B. bronchiseptica no. 3536 formed the AIIb clade. Clade AIIc contained strains of B. hinzii (nos 10980, 16211 and 13494) and B. bronchiseptica no. 3538. The AIId pattern was characteristic of B. petrii.
In the 1247 RAPD set, at the 72 % similarity level, three main clusters were identified. Cluster RA contained patterns of all of the B. pertussis and B. parapertussis strains, and six isolates of B. bronchiseptica (nos 3540, 3543, 1033, 107597, 1943 and 107599). Cluster RB, contained groups of related patterns for nine B. avium isolates and B. petrii (RBa), five B. holmesii isolates (RBc), B. hinzii nos 16211, 13494 and 14052 (RBd), B. trematum nos 5894 and 14993, and B. hinzii no. 10980 (RBb), and B. trematum nos 16877, 15543 and 16652 (RBe). Cluster RC contained seven B. bronchiseptica isolates presenting almost 100 % as the similarity value. The B. petrii-specific pattern has been classified as directly adjacent to the B. avium cluster (RBa). Within the ‘B. bronchiseptica’ cluster, four non-species-specific pattern types were observed. Similarly, for B. hinzii and B. trematum three non-species-specific pattern types were found. For B. holmesii and for B. avium isolates single clusters have been generated (however, the RBa B. avium-like cluster contained B. petrii).
No band variation was observed in RAPD band patterns obtained for DNA isolated from seven passages of a single strain or with DNA content in the range 10–200 ng. Data obtained show that typing with the RAPD 1254 primer can easily identify B. avium from other Bordetella species, as species-specific patterns classified them into a separate branch of the dendrogram. Similarly, typing with 1247 primer may be of value for confirming B. avium and B. holmesii species.
Analysis of RAPD profiling within species
We found that primers used in RAPD typing hardly differentiated B. pertussis, B. parapertussis, B. avium and B. holmesii isolates (Table 3). Relevant isolates of these four species tested with RAPD using primer 1254 or 1247 presented similarity levels near identity value: 87.9/86.7 %, 85.6/91.5 %, 95.5/94.8 % and 95.0/97.4 %, respectively. High homogeneity of patterns was also seen in the case of B. hinzii in RAPD fingerprinting with primer 1247 (91.6 %). Typing allowed differentiation of B. bronchiseptica and B. trematum isolates at the level of 64.3/49 % and 82.2/66.8 % in RAPD profiling using the of 1247/1254 primers, respectively. Some potential for differentiation using RAPD with primer 1254 was seen in the case of B. hinzii (80.8 %). The greatest value for intra-species RAPD typing using these primers was for B. bronchiseptica and B. trematum, since for 13 and five strains tested, seven and three RAPD patterns, respectively, could be differentiated.
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Screening of AFLP sets
Preliminary screening of 12 different AFLP schemes revealed that all of them except the HindIII AFLP set were able to fingerprint Bordetella isolates efficiently (Table 2). Profiles obtained for screened isolates visually appeared to differentiate particular species. For genotyping of all the strains in the study, the EcoRI/MseI and SpeI/ApaI AFLP sets were chosen. The profiles of these sets presented two options of band distribution: higher (EcoRI/MseI, 16–39) and lower (SpeI/ApaI, 6–25) numbers of bands within the profiles.
Overall intra-/inter-gel similarity levels for the external reference strain were 97/92 %, respectively, and thus the number of patterns obtained relates to the identity level evaluated at 92 %. Under the conditions tested, the method was able to discriminate the analysed strains at both the species and the inter-species level. Overall genetic similarities defined by Pearson product-moment correlation coefficient for banding patterns of Bordetella isolates, reached values of 23.6 % and 4.3 % for the EcoRI/MseI and SpeI/ApaI AFLP sets, respectively.
Cluster analysis of the AFLP EcoRI/MseI patterns identified five pattern groups, at the 50 % overall similarity level, each consisting of strains belonging to a single species with the exception of B. pertussis, B. parapertussis and B. bronchiseptica, which all clustered together (Fig. 1). The clusters identified were: E1, nine isolates of B. avium; E2, five isolates of B. trematum; E3, five isolates of B. holmesii; E4, four isolates of B. hinzii; E5, 57 isolates of B. bronchiseptica, B. pertussis and B. parapertussis; and a singly classified B. petrii-specific pattern (E6).
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The closest relationship and lowest diversity were found for patterns clustering at the 67 % level of similarity within E5. Four subclusters were found within E5: E5a, three isolates of B. bronchiseptica; E5b, 10 isolates of B. bronchiseptica and five B. parapertussis; E5c, containing exclusively B. pertussis strains; and E5d, four Polish isolates of B. parapertussis. The subcluster specific for B. pertussis showed a 79 % similarity value. The nine isolates of B. avium showed nine AFLP patterns, which clustered at a linkage level of 74 % into the E1 cluster. The E2 cluster contained five different AFLP patterns for the five B. trematum strains, which associated together at a linkage level of 72 %. The five isolates of B. holmesii, presenting four different patterns, clustered into E3 at a linkage level of 73 %. The four analysed B. hinzii isolates revealed four AFLP patterns which grouped at a linkage level of 78 %. The B. pertussis, B. parapertussis and B. bronchiseptica isolates showed six, five and 12 different AFLP patterns, respectively, and clustered at a linkage level of 69 %.
Dendrograms constructed separately for isolates of eight particular species from the EcoRI/MseI AFLP patterns presented similarity values in the range of 70.9–78.3 % except for B. avium (56.6 %) (Table 3).
The highest number of different patterns was observed within the SpeI/ApaI AFLP set evaluated as the most discriminatory (Table 2, Fig. 2). At the 30 % similarity level, eight clusters (S1–S8) were differentiated. Within the S1 cluster, nine B. avium isolates clustered at a linkage level of 46 %. Two clusters – S2 (three isolates of B. hinzii, nos 10980, 16211 and 14052) and S3 (B. trematum 14993 and 5894) – clustered together at a linkage level of 20 %. S6 (three isolates of B. trematum nos 15543, 16877 and 16652 and B. hinzii no. 13494) and S5 (all B. holmesii isolates in the study) clustered at linkage levels of 69 % and 78 %, respectively. S8 contained B. pertussis strains that were highly related to each other and clustered at a linkage level of 59 %. Within S8 also, isolates of B. bronchiseptica (no. 3538) and B. pertussis (no. 3599/97) were classified as clade S8a and clustered with the B. pertussis clade at a linkage level of of 32 %. B. petrii was classified as a single type pattern (S7) related to the S8 cluster. Cluster S4, with the overall similarity value of 35 %, formed three subclusters: S4a, seven isolates of B. bronchiseptica; S4b, nine B. parapertussis isolates and two B. bronchiseptica, nos 3540 and 3534; and S4c, B. bronchiseptica nos 1943, 3543 and 3536. Within the SpeI/ApaI AFLP set, 16, 10 and six different patterns for the 35, 13 and nine isolates of B. pertussis, B. bronchiseptica and B. parapertussis were found, respectively. For B. trematum, B. hinzii, B. holmesii and B. avium, five, four, four and eight patterns were identified, respectively.
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DNA isolates obtained from seven subcultures of two different strains repeated over time were used to compare specific pattern reproducibility. The band patterns did not change after seven passages in any of the strains tested (data not shown). Similarly, differing quantities of DNA in the range of 100–500 ng did not induce band variation in the AFLP profile of a single strain.
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DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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In order to overcome the liability of the RAPD results according to the procedures and reagents used (Heersma et al., 2001; Olive & Bean, 1999) and to achieve higher levels of AFLP reproducibility, a standardized methodology, including commercially available kits for DNA isolation and gels, was applied. General approaches for reliable and accurate intra- and inter-gel comparison of banding patterns were included (Desai et al., 1998; Janssen, 2001; Savelkoul et al., 1999). The reproducibility was tested by generating multiple subcultures on which DNA extraction, RAPD and AFLP procedures were performed.
Intra-/inter-gel reproducibility found identity levels for the RAPD and AFLP protocols as 94/90 % and 97/91 %, respectively. The overall genetic similarities, defined by the Pearson product-moment correlation coefficient, were 53.9 % and 64 % for RAPD using primers 1254 and 1247, respectively, and 23.6 % and 4.4 % for AFLP with EcoRI/MseI and SpeI/ApaI protocols, respectively. Although RAPD can provide analysis data very rapidly, the discriminatory power in comparison to AFLP, as predicted, was much lower. Although the limitations of the RAPD method are well-known, it seems that it has the potential to be a quick tool for intra-species differentiation of some species of Bordetella. For B. bronchiseptica, RAPD was found to be highly accurate in tracking differences among isolates coming from different mammalian species; thus in this case it might well provide an additional tool to study genetic diversity. In addition, RAPD can also be useful for confirming B. avium, B. parapertussis and B. holmesii identification.
Previously, AFLP has been found to be useful for both identifying and typing many micro-organisms after defining windows of similarity (Savelkoul et al., 1999). The definition of the similarity value ranges in AFLP genotyping was found to be useful in the taxonomic studies of for example Klebsiella, Mycoplasma, Acinetobacter, Aeromonas and Xanthomonas species (Huys et al., 1996, Kokotovic et al., 1999). Moreover, in some cases, the discriminatory power has been evaluated as equal to or higher than that obtained with the gold standard method of PFGE (van Elderle et al., 1999), and congruent with similar grouping through multilocus sequence typing (MLST; Schouls et al., 2003). AFLP has been widely accepted as an excellent genotyping tool because of its typability and discriminatory power, and good reproducibility; likewise, it is easy to perform and to draw interpretations from (van Belkum et al., 2001).
AFLP fingerprints of the isolates of the Bordetella genus, as revealed by cluster analysis, have retained attributes of species specificity, leading to the conclusion that the method may be used as an additional tool for species-specific identification. In our study, four B. parapertussis isolates from Poland were first incorrectly identified as B. pertussis in the diagnostic laboratory. The AFLP technique clustered them not within the highly related group of B. pertussis but within the ‘B. bronchiseptica’ cluster. By applying PCR (van der Zee et al., 1996a), their identification as B. parapertussis was confirmed. Additionally, because of high reproducibility and easy performance AFLP has value in epidemiological studies of the Bordetella. Slightly different classifications of Bordetella isolates in the two AFLP sets screened might result from the different levels of polymorphism detected. Greater levels of discrimination of fingerprinting in the case of AFLP with the SpeI/ApaI protocol might allow for a more in-depth examination of polymorphism compared to AFLP with the EcoRI/MseI set.
The AFLP set with the EcoRI/MseI protocol seems to be a potential alternative for studying genetic relationships among Bordetella species. The DNA–DNA hybridization of nucleotide sequences of the pertussis toxin operon and MLEE found B. pertussis, B. parapertussis and B. bronchiseptica to be highly related and with limited diversity (Arico et al., 1987; Khattak & Matthews, 1993; Muller & Hildebrandt, 1993; Musser et al., 1986; van der Zee et al., 1997). Genetic differences within the ‘B. bronchiseptica’ cluster were suggested to be difficult to detect because the mechanism of regulation of expression seems to be species-specific. Different strategies in regulating gene expression have been suggested to be more important for the adaptation of particular lineages of the B. bronchiseptica cluster to the particular host than horizontal gene transfer (Gerlach et al., 2001). On the other hand, adaptation to different hosts has been suggested to be a force capable of inducing differences, which could be observed by some tools (Gerlach et al., 2001). Insertion sequence (IS)-dependent rearrangements responsible for different genome organization and size were assumed to be the most important changes possible to observe (Cummings et al., 2004). As for the ‘B. bronchiseptica’ cluster and other Bordetella isolates, the genetic differences generated could be observed through AFLP and the method can be considered as an additional tool to study their relationships.
The different extents of intra-species polymorphism detected seem to confirm the varying degrees of genetic diversity among analysed species (Kokotovic et al., 1999). For B. pertussis, substantial homogeneity was found both in RAPD and AFLP genotyping compared with B. bronchiseptica. As such, in MLEE in the case of B. bronchiseptica as many as 38 polymorphic types have been identified in comparison to the four to five found for B. pertussis and B. parapertussis (van der Zee et al., 1997). Higher genetic variation seen in B. bronchiseptica versus B. pertussis and B. parapertussis has also been found in other studies (Boursaux-Eude & Guiso, 2000; van der Zee et al., 1996b).
The AFLP fingerprinting data are in agreement with previously published data on the close relationship of the B. bronchiseptica cluster, since B. pertussis, B. parapertussis and B. bronchiseptica were strictly classified as a single branch during the clusterization process. Moreover, fingerprints of B. parapertussis and B. bronchiseptica isolates were mixed together in the clade, unlike the highly related group of B. pertussis isolates. Comparative analyses of the genome sequences revealed that B. parapertussis and B. bronchiseptica are more similar in overall genome organization than B. bronchiseptica and B. pertussis (Cummings et al., 2004; Parkhill et al., 2003). Phenotype analysis by in vitro and in vivo assays also has shown that B. parapertussis is more similar to B. bronchiseptica than to B. pertussis, thus the phenotype is related to the genetic species-specific differences (Heininger et al., 2002).
B. pertussis isolates were found to be hardly differentiated by RAPD and AFLP using the EcoRI/MseI protocol compared with AFLP using the SpeI/ApaI protocol. MLEE, PFGE and IS-RFLP studies have shown the population of B. pertussis to be homogeneous or clonal, resulting most probably from high adaptation to humans and limited opportunity for horizontal genetic exchange (Musser et al., 1986; van Loo et al., 1999; van der Zee et al., 1996a). IS sequences are considered to be reasons for genome plasticity of B. pertussis coupled with high-genetic-relatedness (Gerlach et al., 2001; Stibitz & Yang, 1997).
PFGE as a gold standard has been successfully used to study B. pertussis challenges with time, identify outbreak- associated isolates, monitor transmission and evaluate genetic relatedness (Hardwick et al., 2002a; Khattak et al., 1992; Mooi et al., 2000; Peppler et al., 2003; Weber et al., 2001). For strains belonging to the B. bronchiseptica cluster, using PFGE it was possible to differentiate isolates between and within species (Khattak & Matthews, 1993). Moreover, it has been successfully applied for differentiation of isolates of B. parapertussis, B. bronchiseptica and B. holmesii (Hardwick et al., 2002a, 2002b; Khattak et al., 1992; Makinen et al., 2003; Mastrantonio et al., 1998; Mazengia et al., 2000; Peppler et al., 2003; Weber et al., 2001). Although PFGE is the most reproducible method, it is time-consuming and labour-intensive. MLST studies (van Loo et al., 2002), although highly useful for tracing the movements of B. pertussis strains within and between individuals and communities, need sophistically equipped laboratories and highly experienced personnel. We postulate that AFLP using the SpeI/ApaI protocol may be a new, rapid and reproducible option for the genotyping of B. pertussis.
The epidemiology of B. bronchiseptica is still not well enough elucidated, especially in terms of the possibility of host inter-species transmission. Some studies have documented co-species transmission between dogs and cats (Binns et al., 1998; Dawson et al., 2000; Foley et al., 2002), from rabbits to humans (Gueirard et al., 1995) and from rats/cats to pigs (Switzer et al., 1966). Available genotyping tools involve restriction enzyme analysis (REA) of chromosomal DNA, ribotyping, PFGE and RAPD (described for canine isolates) (Keil & Fenwick, 1999; Sacco et al., 2000b). Genetic diversity of B. bronchiseptica isolates from 12 different host animals measured by REA was considerable in the study by Sacco et al. (2000b), with similarity ranges of 68–97 % for HinfI and 46–96 % for AluI. Thus AFLP using SpeI/ApeI might present a better alternative because of its higher discrimination level (30.1 %). In our study, we found similar heterogeneity of B. bronchiseptica strains isolated from different host animals using the AFLP SpeI/ApaI protocol and even using as simple an approach as RAPD. Further studies are expected to show a degree of variation detected by AFLP in a higher number of B. bronchiseptica strains exclusively isolated from particular hosts, e.g. swine and dogs. In addition, AFLP SpeI/ApaI fingerprinting might be a new option to the previously described alternatives to follow the genotype trends for vaccine and clinical strains in the population of B. bronchiseptica in dogs, similarly as it is possible for B. pertussis in humans (Keil & Fenwick, 1999; Mooi et al., 1998, 1999; van der Zee et al., 1996a; Fry et al., 2001; Gzyl et al., 2001).
Among nine B. parapertussis isolates, four and six different patterns in the AFLP EcoRI/MseI and SpeI/ApaI sets were observed, respectively, proving its high capability for differentiation. A higher number of B. parapertussis isolates is expected to be studied and compared with AFLP results with previously described data on limited genetic variability (Heininger et al., 2002; Khattak & Matthews, 1993; Makinen et al., 2003; Mastrantonio et al., 1998; Porter et al., 1996).
For 32 B. holmesii isolates recovered from nasopharyngeal specimens up to five pulsofield types were identified, which supports restricted diversity of this species (Mazengia et al., 2000). In our study, although only five isolates of B. holmesii were available for testing, four different patterns in both AFLP protocols were observed.
REA and ribotyping have been described as useful tools for discriminating between B. avium and B. hinzii species and for epidemiological purposes (Register et al., 2003; Sacco et al., 2000a). Similarly, in our study we found AFLP and RAPD to be useful tools for identification of B. avium identification among other species of the Bordetella genus; for the nine strains in the study, seven and six different AFLP fingerprints obtained with EcoRI/MseI and SpeI/ApaI protocols were observed, respectively. In the case of B. hinzii, for both AFLP sets tested, the four isolates presented four different patterns.
It has been proposed that the evolution of Bordetella is associated with the adaptation to particular hosts of environmental bacteria (Gerlach et al., 2001). Isolation of B. petrii as the first environmental Bordetella of anaerobic metabolism (von Wintzingerode et al., 2001) has further supported the evolutionary relationship of Bordetella with its environmental ancestor. In the AFLP EcoRI/MseI protocol, with the highest potential for taxonomic discrimination of the two studied, the B. petrii isolate came last in the relationship with other Bordetellae.
Our study has described the potential of AFLP for discrimination of isolates of the Bordetella genus. However, a higher number of strains is expected to be analysed for a precise confirmation of these preliminary data. The newly designed AFLP protocol with SpeI/ApaI seems to possess the highest discrimination potential, and especially as regards B. pertussis might become a first-choice method after exact standardization with gold-standard methods. Furthermore, it seems that AFLP has some potential for the genetic phylogenetic studies of Bordetella; however, studies with higher numbers of isolates and standardization using tools such as 16S rDNA, PFGE, MLEE, MLST and IS-RFLP should first be performed for exact confirmation. Finally, AFLP data obtained with two different sets of restriction enzymes should be compared to well-established methods such as MLEE, MLST and PFGE (Mooi et al., 2000; van Loo et al., 2002; van der Zee et al., 1997; Advani et al., 2004) in order to establish the most-probable valid evolutionary pathway. Additionally, some other different AFLP procedures, tested in the preliminary screening on only a limited number of strains and not on the whole strain collection used here, remain available for assessment of their value for genotyping of isolates of different species of Bordetella.
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ska for help with English version of the manuscript and to Ms Barbara Husejnow for her excellent technical assistance. |
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