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1 Institut für Medizinische Mikrobiologie, Virologie und Hygiene, Universität Rostock, Rostock, Germany
2 Department of Veterinary Clinical Sciences, Purdue University, West Lafayette, IN, USA
3 Sezione Diagnostica di Pavia, Istituto Zooprofilattico Sperimentale della Lombardia, Pavia, Italy
Correspondence
Mardjan Arvand
mardjan.arvand{at}med.uni-rostock.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Jacobs & Schutze, 1998; Raoult et al., 1996). The domestic cat represents the main host and reservoir for B. henselae. Infected cats develop a relapsing bacteraemia that may persist for up to 2 years (Breitschwerdt & Kordick, 2000). Previous investigations have shown that B. henselae induces specific humoral and cell-mediated immune responses in naturally or experimentally infected hosts, including cats, humans and mice (Abbott et al., 1997; Arvand et al., 1998a, 2001a; Chenoweth et al., 2004; Guptill et al., 1999; Regnath et al., 1998). The mechanisms involved in the long-term survival of the bacteria despite vigorous host immune responses are not entirely understood. It has been postulated that Bartonella spp. may persist in an intracellular niche, from where they are occasionally released to invade host cells (Dehio, 2001; Mandle et al., 2005; Schulein et al., 2001). However, the role of other factors contributing to the persistence of bartonellae in the infected host has not been studied in detail.
Generation of genetic and antigenic variants represents another strategy used by different pathogens such as Trypanosoma spp. (Dubois et al., 2005), Helicobacter pylori (Kraft et al., 2006) and Anaplasma marginale (Brayton et al., 2005) to circumvent the host specific immune responses and maintain a persistent infection. Several studies suggest that B. henselae may display a marked genetic and antigenic variability. Kabeya et al. (2002) reported on genetic variability among B. henselae isolates obtained from recurrent bacteraemic peaks of naturally infected cats. However, due to the study design, they could not rule out the possibility of a reinfection of the cats with a different B. henselae strain. Maruyama et al. (2004) detected two different genetic variants among B. henselae cultures obtained from the lymph node of a patient with cat scratch disease. However, it was postulated that the patient was infected by two different B. henselae strains. Kyme et al. (2003) demonstrated that B. henselae may undergo phase variation in vitro, a process that is characterized by the emergence of antigenic variants. More recently, the complete genome of B. henselae was found to contain several regions with numerous short repeats in tandem that are prone to rearrangement and may be implicated in phase variation (Alsmark et al., 2004).
We have recently demonstrated the coexistence of different genetic variants within the community of three primary B. henselae isolates from Germany by PFGE analysis of multiple single-colony-derived cultures (Arvand et al., 2006). No variants were detectable among the cultures obtained from isolates with a long passage history, suggesting that the inhomogeneous structure was restricted to primary isolates. We hypothesized that many, if not all, primary B. henselae isolates may comprise different genetic variants that might emerge in vivo.
The present study was conducted to determine whether (i) all primary B. henselae isolates are composed of different genetic variants, and (ii) the genetic variants originally existed in vivo and were not induced during the procedure of PFGE analysis. To test these hypotheses, 34 primary B. henselae isolates from different geographical regions were evaluated by PFGE analysis of multiple single-colony-derived cultures. The relationship among the genetic variants within a distinct isolate was further evaluated by multi-locus sequence typing (MLST), which has been shown to be an accurate method for the evaluation of genetic relatedness among B. henselae isolates (Arvand & Viezens, 2007; Iredell et al., 2003). In addition, 20 in vitro-generated clones of B. henselae were subjected as single-colony-derived cultures to PFGE analysis in order to determine whether genetic variants might also be detected within the progeny of in vitro-generated clones. Our results suggest that genetic variants occur frequently within primary B. henselae isolates and most likely emerge in vivo.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). All primary isolates were originally conserved as a subculture obtained from a sweep of colonies of the primary growth and stored without additional in vitro passages at –20 °C or –80 °C. The primary isolates were grown on Columbia blood agar with 5 % sheep blood (CBA, Becton Dickinson) for 7–14 days at 37 °C in 5 % CO2. Four or five morphologically different or randomly selected colonies (colonies A–D or E) were picked from each isolate, inoculated to CBA and incubated for 7–10 days to obtain single-colony-derived cultures.
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PFGE.
PFGE was performed as described previously with some modification (Arvand et al., 1998b). Bacteria were harvested from CBA, washed with 0.9 % NaCl and embedded in 1 % SeaPlaque GTG Agarose (BioWhittaker Molecular Applications). Restriction was performed with SmaI (Fermentas) according to the manufacturer's instructions. Electrophoresis was conducted in 1 % Seakem GTG Agarose (BioWhittaker) in a CHEF DR III unit (Bio-Rad) at 5.5 V cm–1 with 3–14 s ramp times at 14 °C for 22 h. Gels were stained with ethidium bromide and photographed with an INTAS Gel Jet imager (Intas Science Imaging Instruments). The lambda ladder PFGE marker (Cambrex Bioscience) was used as molecular mass standard in each gel. Banding patterns were analysed by visual comparison of the restriction patterns. A one-band difference was defined as the presence or absence of a specific band in the PFGE pattern.
MLST.
MLST was performed as previously described (Arvand et al., 2006; Iredell et al., 2003). In brief, the partial sequences of eight genetic loci, including 16S rRNA-DNA, batR, ftsZ, gltA, groEL, nlpD, ribC and rpoB, were analysed. The PCR products were purified and sequenced on both strands with an ABI 3730 XL apparatus (AGOWA, DNA sequencing service, Berlin, Germany). The DNA sequences were analysed by using the DNASTAR Lasergene. Alleles and sequence types (STs) were assigned in accordance with the published data (Arvand & Viezens, 2007; Iredell et al., 2003).
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RESULTS |
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). Two distinct variants were detected within the progeny of 18 isolates, whereas three variants were found within the community of two isolates (Table 2). The PFGE patterns of the genetic variants of an isolate displayed one to three band differences. In detail, a one-band difference was observed within the single-colony-derived cultures of 10 isolates, while a two- and three-band difference was detected among the progeny of 9 and 2 isolates, respectively (Table 2). No genetic variants were detectable among the single-colony-derived cultures of 14 (41.2 %) isolates.
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), isolates displaying one to three band differences are considered to be closely related and represent a unique strain. Therefore, we hypothesized that the PFGE variants among the single-colony-derived cultures, which displayed one to three band differences and were each isolated from a unique host, most probably represented genetic variants within the community of a distinct strain, rather than a co-infection of the host by two or more different B. henselae strains. To test this hypothesis, two different single-colony-derived cultures from 10 primary isolates were subjected to additional typing by MLST, which is a robust, sequence-based typing technique for the analysis of genetic relatedness among B. henselae isolates. Both single-colony-derived cultures of each primary isolate studied displayed an identical sequence type, indicating that they belonged to the same strain (Table 3).
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). Genetic variants could not be detected within the progeny of any experimental clone (Table 4), indicating that the variants were not induced in vitro during the preparation of subcultures for PFGE analysis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The close relatedness of the variants was confirmed by a second typing method, MLST, which is discriminatory for B. henselae to the strain level. The frequency of genetic variants within the community of a unique isolate appears to be high, since two or three different variants were detectable among the progeny of approximately 59 % of the isolates by screening four or five single-colony-derived cultures of each isolate. Although genetic variants were not found among the single-colony-derived cultures of 41 % of the isolates studied, we can not exclude that they might have existed within those isolates but remained undetected because of the limited sensitivity of our screening method. Most primary isolates included in this study were of feline origin. Only one human isolate was available as a primary isolate and evaluated. Different genetic variants were observed within most feline isolates and the only human-derived isolate analysed, suggesting that a mosaic-like genetic structure is a feature shared by feline and human B. henselae isolates. It is noteworthy that all primary isolates investigated here were originally conserved as a subculture from a sweep of colonies of the primary growth, which seems to be crucial for the conservation of the mosaic-like structure of B. henselae isolates in vivo.
We further analysed the prevalence of genetic variants within the progeny of in vitro-generated clones of B. henselae in order to rule out that the genetic variants observed above were induced by one or two in vitro passages or a freeze-and-thaw cycle, which are prerequisites for PFGE analysis. We could not detect any variants among the progeny of the in vitro clones, indicating that the genetic variants within primary isolates must have originally emerged in the mammalian host and were not artificially induced in vitro.
Generation of genetic and antigenic variants may contribute to the persistence of a pathogen in the infected host or in the community in different ways. Emergence of genetic variants may lead to a more effective adaptation to an individual host and specific niches within a host, as shown for Helicobacter and Bacteroides (Kraft et al., 2006; Cerdeno-Tarraga et al., 2005). Antigenic variation may alter surface-coat proteins and contribute to the evasion of specific immune responses such as B cell recognition (Dubois et al., 2005), which can result in prolonged bacteraemia and a more effective transmission to other hosts. Our results suggest that B. henselae undergoes genetic and therefore may undergo antigenic variation in the mammalian host. They are in accordance with recent data of Lindroos et al. (2006) that revealed evidence for extensive rearrangements within the B. henselae genome. It is likely that the variations observed in our study are caused by genetic rearrangement. Further investigation is required to identify the genetic loci and antigenic structures that are predominantly affected by these events.
The fact that primary B. henselae isolates are composed of different genetic variants might be of particular importance for the interpretation of future typing studies on B. henselae. We found up to three different genetic variants within a unique isolate. A careful interpretation of the PFGE data by using the guidelines for interpretation of PFGE typing results was necessary to obtain a reliable classification of different PFGE patterns to the variant, isolate or strain level. Different genetic variants within the community of B. henselae isolates might also be detectable by other typing techniques, especially those based on analysis of repetitive elements or hypervariable genomic regions such as multi-spacer typing or variable number of tandem repeats typing. Therefore, a careful interpretation of the latter typing results and comparison with standard typing methods such as PFGE or MLST is strongly recommended to ensure a correct classification of different patterns to the variant, isolate or strain level.
Consideration of the mosaic-like structure of primary isolates might also be important for studies on pathogenicity of B. henselae, since different variants could vary in production of virulence factors. Furthermore, it is possible that a distinct variant may eventually overgrow the other variants within the community of an isolate under certain environmental conditions, giving rise to a high-passage isolate with different genetic or antigenic properties than the original one. This could explain the observation of several investigators who found that the copy of the Houston-1 isolate used in their laboratory was different from the previously described Houston-1 isolate (Alsmark et al., 2004; Kyme et al., 2003; Riess et al., 2007). Further studies are required to test this hypothesis, e.g. by monitoring the genetic alterations within the progeny of a primary B. henselae isolate upon serial in vitro passages.
In summary, our results indicate that different genetic variants may coexist within the community of most primary B. henselae isolates. They probably emerge in vivo and could serve as an escape mechanism to circumvent the host specific immune responses and maintain a chronic infection. Consideration of the coexistence of genetic variants within primary isolates is essential when interpreting typing studies on B. henselae.
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ACKNOWLEDGEMENTS |
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Edited by: P. H. Everest
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 1 February 2007;
revised 14 March 2007;
accepted 20 March 2007.
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, Lambda ladder PFGE marker.