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Journal of Clinical Microbiology, August 2004, p. 3441-3448, Vol. 42, No. 8
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.8.3441-3448.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

High-Resolution Genotyping of Campylobacter upsaliensis Strains Originating from Three Continents

P. Lentzsch,¹ B. Rieksneuwöhner,² L. H. Wieler,² H. Hotzel,³ and I. Moser^3*

Centre for Agricultural Landscape and Land Use Research, Müncheberg,¹ Institute of Microbiology and Animal Epidemic Diseases, Free University, Berlin,² Federal Research Centre for Virus Diseases of Animals, Institute of Bacterial Infections and Zoonoses, Jena, Germany³

Received 9 January 2004/ Returned for modification 5 March 2004/ Accepted 4 May 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ninety-six Campylobacter upsaliensis strains that originated from Australia, Canada, and Europe (Germany) and that were isolated from humans, dogs, and cats were serotyped for their heat-stable surface antigens. All of them were genotyped by enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) profiling, and 83 strains were genotyped by macrorestriction analysis with the endonuclease XhoI. Eighty-four percent of the strains belonged to five different serotypes (serotypes OI, OII, OIII, OIV, and OVI), with the proportions of strains in each serotype being comparable among the groups of strains from all three continents. Two serotypes, OIII and OIV, were prevalent at rates of 35 to 40%. Serotypes OI, OII, and OVI were detected at rates of 1.5 to 15%. Between 10 and 17.7% of the strains did not react with the available antisera. Analysis of the ERIC-PCR profiles revealed two distinct genotypic clusters, which represented the German and the non-European strains, respectively. XhoI macrorestriction yielded two genotypic clusters; one of them contained 80.2% of the German strains and 34.6% of the non-European strains, and the second cluster consisted of 65.4% of the non-European strains and 19.8% of the German strains. Fourteen strains from all three continents were analyzed for their 16S rRNA gene sequences. Only two minor variations were detected in four of the strains. In conclusion, C. upsaliensis has undergone diverging processes of genome arrangement on different continents during evolution without segregating into different subspecies.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Campylobacter upsaliensis, a catalase-negative or weakly catalase positive thermotolerant Campylobacter species, was first isolated in 1983 from fecal samples of healthy and diarrheic dogs in Sweden (53). A few years later, in 1989, this microorganism was identified in cats (10). Several reports concerning the carriage rates in dogs and cats and the risk that humans can acquire infections from pet animals have been published (2, 5, 6, 15, 18, 33, 34, 39). Cases of clinical disease in humans caused by infections with C. upsaliensis have been reported from different areas of the world. C. upsaliensis was isolated from patients with abortion (17), bacteremia (19, 29, 44), an abscess (11), gastroenteritis (12, 13, 14, 26, 29, 30, 44, 48, 56; W. M. Johnson, D. L. Woodward, R. Khakhria, and L. J. Price, Campylobacter, Helicobacter and Related Organisms, Proc. 9th Int. Workshop, abstr., p. 27, 1998), hemolytic-uremic syndrome (7), and hypogammaglobulinemia (8) and opportunistic infections in immunocompromised hosts (25, 44). However, in contrast to C. jejuni (46), knowledge about the genomic characteristics of C. upsaliensis is limited, and its possible pathogenic capacity for humans and small animals is far from being well defined (4).

Genotypic methods have successfully been applied to accomplish phenotypic approaches to the subtyping of Campylobacter species (32, 45, 49, 54, 63). Previous reports of studies that used pulsed-field gel electrophoresis (PFGE) to characterize C. jejuni (21, 42, 65), C. upsaliensis (3, 34, 44), and C. hyointestinalis (52) at the genomic level demonstrated the usefulness of this method for epidemiological studies. Flagellin gene polymorphism (20, 37, 38) or the combination of macrorestriction analysis and serotyping based on heat-stable or heat-labile antigens have also been performed to distinguish C. jejuni strains from C. coli strains (43, 47, 54). Investigations of the genotypic and serological diversity of canine and feline C. upsaliensis strains originating from two different areas in Germany were previously performed in our laboratories. Human isolates were not available, since C. upsaliensis does not seem to be of major relevance as a human pathogen in Germany. Macrorestriction analysis as well as enterobacterial repetitive intergenic consensus sequence (ERIC) PCR (ERIC-PCR) profiling was used, and a serotyping scheme for heat-stable antigens was developed (34). In general, the study revealed a high degree of genomic diversity but a low degree of serological diversity. The genotypic clusters of the strains did not reflect their geographic origins in Germany or the health status of the carriers. As the vast majority of strains were isolated from dogs and only very few strains were isolated from cats and none was isolated from humans, host-specific differences could not be addressed in this investigation. In the present study C. upsaliensis strains isolated from humans, dogs, and cats in Canada and Australia were genotyped by the same methods described before (34) and compared with the German strains.

The aim of this study was to extend the knowledge of genomic characteristics of C. upsaliensis strains originating from different hosts and different areas of the world.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Collection and cultivation of bacteria. Seventy C. upsaliensis strains were recovered from dogs (n = 63) and cats (n = 7) of different ages and enteric health conditions in two regions of Germany (Berlin and Northrhine-Westfalia) approximately 400 km apart. The isolates were collected from rectal swab specimens obtained by using Culturettes (Becton Dickinson). The isolates were transferred onto selective growth media within 24 h after collection. Three selective media, CAT agar (1), mCCD agar (23), and CSM agar (28), were used in order to obtain as many isolates as possible from the swabs. Further cultivation was performed on Mueller-Hinton agar containing 5% defibrinated sheep blood. The plates were incubated for 48 to 72 h under microaerophilic conditions at 39°C. The bacteria were stored as stock cultures in thioglycolate broth containing 15% glycerol at –70°C. Twenty Canadian C. upsaliensis strains were kindly supplied by F. Rodgers and L. Price, Winnipeg, Manitoba, Canada. Eighteen were of human origin, one was of canine origin, and one was of feline origin. Six Australian C. upsaliensis strains isolated from humans were kindly supplied by G. Hogg, Melbourne, Australia. German C. upsaliensis isolates of human origin were not available.

Species determination. C. upsaliensis isolates were identified by biochemical tests described in the literature (22, 24, 40, 41) by use of the following criteria: gram negative, spiral-shaped rod morphology, motility, requirement of microaerophilic growth conditions, cytochrome oxidase activity, weak or no catalase activity, lack of hippuricase activity, capacity to reduce nitrate, lack of H₂S production on triple sugar iron agar, selenite reduction, and sensitivity to nalidixic acid and cephalothin. Indoxyl acetate activity was tested in some but not all isolates. The results were confirmed by using the species-specific PCR of Eyers et al. (9). Only isolates that reacted by conventional and molecular tests as described in the literature were included in the study.

Indirect hemagglutination. Indirect hemagglutination was performed as described previously (34, 36, 50). Five heat-stable antigens specific for C. upsaliensis were determined by the serotyping scheme developed in our laboratories. In addition, the strains were tested with eight C. jejuni-specific antisera, two C. coli-specific antisera, and one C. lari-specific antiserum available in the laboratory. Serum hemagglutination titers of 1:80 and less were ignored. C. jejuni reference strains were purchased from the Culture Collection University of Göteborg (CCUG). The C. jejuni and C. coli wild-type strains used as reference strains were serotyped at CCUG. The bacterial strains used for antiserum production are listed in Table 1.

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TABLE 1. Bacterial strains used for antiserum preparation

DNA preparation, primers, and PCR amplification. The DNA preparation and PCR amplification procedures were performed as described previously (9, 34, 58). Briefly, for PCR amplification DNA was extracted by heat treatment. The amplification reaction was performed with Ready-To-Go PCR Beads (Amersham Pharmacia Biotech, Freiburg, Germany). Primer sequences were deduced from the sequences of the 23S rRNA and 16S rRNA genes of thermophilic Campylobacter species (9, 31) and ERIC sequences (58), as described before (34). Each PCR included positive and negative controls. Amplified samples were analyzed by electrophoresis on 1.2% agarose gels and visualized by ethidium bromide staining under UV light. The gels were photographed with a digital camera system (Herolab, Wiesloch, Germany).

Macrorestriction analysis by PFGE. PFGE was performed with the endonuclease XhoI, as described before (34), by using a CHEF DR III apparatus (Bio-Rad, Munich, Germany). The pulse interval was ramped from 0.3 to 12 s linearly for 24 h. Reference DNA of C. upsaliensis strain DSM 5365 digested with XhoI was run on each gel. Molecular size standard bacteriophage concatamers (molecular size, 48.5 kb; New England Biolabs, Frankfurt, Germany) were run on three lanes (both edges and the middle) of each gel. Finally, the gels were stained with ethidium bromide, viewed under UV light and photographed with Polaroid film.

Computational analysis. The electrophoretic patterns of the ERIC-PCR experiments were analyzed by using GelCompar II, version 2.50, software (Applied Maths BVBA, Kortrijk, Belgium; Herolab). Genetic similarities between isolates based on their positions and relative band areas were calculated by use of the algorithm of Ward (60) and the Pearson correlation coefficient (optimization, 0.0; tolerance, 0.0; for details, see the GelCompar II, version 2.50, manual). The following variable areas of the gel tracks were included in the analysis of genetic similarity: 20.5 to 56.6%, 62.6 to 65.7%, and 70.8 to 100%. Photographs of the gels from the PFGE experiments were scanned; and similarities were calculated by using the same software, algorithm, and coefficient, with a maximum tolerance of 1.0% and optimization of 0.5%. The reproducibility of the profiles was 94%. The jackknife statistical method (GelCompar II, version 2.50, software, for comparative analysis of electrophoresis patterns; Applied Maths) was applied to determine the significance of the defined similarity groups.

16S rDNA sequence analysis. A 1,275-bp DNA stretch of the 16S rRNA-encoding gene (16S rDNA) was amplified by PCR with primers Cups-1 (5'-CCC ATA CTC CTA TTT AGC AT-3') and Cups-2 (5'-GAT TCC ACT GTG GGG GA-3'), as described by Linton et al. (31). The DNA sequences of the PCR products were analyzed by AGOWA Gesellschaft für molekularbioligische Technologie (Berlin, Germany) for the German strains and by the Federal Research Centre for Virus Diseases of Animals (Bundesforschungsanstalt für Viruskrankheiten der Tiere, Jena, Germany) for the Canadian and Australian strains. Briefly, the 16S rDNA fragments were amplified with primers Cups-1 and Cups-2. Bands were excised from 1% agarose gels, and DNA was extracted by using the QIAquick Gel Extraction kit (Qiagen, Hilden, Germany). Sequencing was performed as cycle sequencing with a BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Darmstadt, Germany), according to the instructions of the manufacturer. Primers Cups-1, Cups-2, Cups-4 (5'-GCA GTA GGG AAT ATT GCG-3'), Cups-5 (5'-CGC TAA GGC GCG AAA GCG-3'), and Cups-6 (5'-CAA ATC AGC CAT GTT GCG G-3') were used for sequencing. Nucleotide sequences were determined with an ABI Prism 310 genetic analyzer (Applied Biosystems). The processing of the sequence data was conducted with the Vector NTI Suite, version 8.0, program (Informax Inc., Oxford, United Kingdom).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Distribution of heat-stable antigens. Ninety-four of 96 C. upsaliensis strains isolated in Germany, Canada, and Australia were typed on the basis of their heat-stable antigens by use of the serotyping scheme described previously (Table 2; 34). Two serotypes, serotypes OIII and OIV, were present in all three geographic groups at rates of 37.2 and 36.2%, respectively, and were present at the same orders of magnitude in the Canadian and the German strain collections (35.0 and 40.0%, respectively, versus 35.3 and 36.8%, respectively). The number of Australian strains tested (n = 6) was too small to calculate the proportion. Among the other serotypes, 6.4, 1.0, and 3.2% of the strains were serotypes OI, OII, and OVI, respectively. Of 94 C. upsaliensis isolates, 16.0% did not react with any C. upsaliensis antiserum. Beyond this, heat-stable C. upsaliensis antigens did not react with antisera recognizing C. helveticus OV; C. jejuni O:1, O:2, O:4, O:13, O:16, O:37, O:40, or O:43 (Penner); C. coli O:20 or a non-cross-reacting C. coli strain; or one untyped C. lari strain available in the laboratory (34, 35, 36).

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TABLE 2. Heat-stable antigen specificities of C. upsaliensis strains originating from Germany, Canada, and Australia

ERIC fingerprint analysis. All C. upsaliensis strains serotyped (n = 94), with six of the German strains tested in duplicate, were characterized by ERIC-PCR (Fig. 1). Two main clusters that differed considerably with respect to band pattern similarity were generated (–80% on the Ward scale of –100% through +100%). One cluster contained the German strains, and the other one contained the non-European strains. With an internal similarity of –50%, the German strains, the most numerous group in this study, exhibited remarkable intragroup heterogeneity. The two geographic regions of Germany, approximately 400 km apart, from which the strains were isolated were not reflected by the dendrogram. The non-European cluster consisted of the Canadian strains, which exhibited an internal similarity of –20%, and the Australian strains were integrated into this group at a +40% similarity. Only one German canine strain was included in the non-European cluster. With respect to host association, the most numerous German cluster contained canine and feline strains, whereas the non-European cluster consisted of all human strains, two canine strains, and one feline strains.

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FIG. 1. Genetic similarity based on ERIC-PCR profiles of C. upsaliensis strains from humans, dogs, and cats in Germany, Canada, and Australia.

The results of statistical calculations assessing the stabilities (significance) of the defined groups are shown in Table 3, which presents the percentages of correct identifications for members of a group. German strains were grouped in the German cluster at a rate of 98.7%. Canadian strains were assigned to the Canadian cluster at a rate of 94.7%, while 5.3% of Canadian strains exhibited a higher degree of similarity to Australian strains. Of the Australian strains, 83.3% were classified in the Australian group.

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TABLE 3. Internal stability (significance) of the defined strain clusters by geographic and host origin

Macrorestriction analysis. For DNA macrorestriction analysis, 50 of 63 canine strains of German origin were selected to ensure that the most prevalent serotypes and the untypeable strains were represented in comparable numbers. Isolates from all the other sources were included in the total. Therefore, 83 C. upsaliensis strains were subjected to macrorestriction analysis with the XhoI endonuclease.

In general, most of the isolates possessed a number of recognition sites for XhoI, yielding between 10 and 20 DNA bands (molecular sizes, up to approximately 250 kb; Fig. 2). Every isolate exhibited a unique restriction pattern, as shown in the dendrogram in Fig. 2. Two major PFGE groups that reflected each of the geographic and the host origins of the strains were differentiated. With regard to geographic origin, one group contained German strains at a rate of 73% and non-European strains at a rate of 11.5%. The second group consisted of non-European strains at a rate of 89.5% and German strains at a rate of 27%. With regard to host origin, one group contained strains of animal origin at a rate of 78.0% and strains of human origin at 37.5%. The other group consisted of strains of animal origin at a rate of 22.0% and strains of human origin at a rate of 62.5%. There was no association of PFGE groups with serotypes. The stability of the defined groups (significance) is shown in Table 3. On the basis of geographic origin, 96.5% of the German strains were grouped in the German cluster and 85.0% of the Canadian strains were grouped in the Canadian cluster, but only 16.7% of the six Australian strains were classified in the Australian cluster. On the basis of host association, 75.0% of the human strains were grouped in the human cluster, 88.2% of the canine strains were assigned to the canine cluster, and 25.0% of the feline strains were classified in the feline cluster.

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FIG. 2. Genetic similarity based on the macrorestriction profiles of C. upsaliensis strains from humans, dogs, and cats in Germany, Canada, and Australia generated with the restriction endonuclease XhoI.

Five strains (Fig. 2, diamonds) were isolated from the blood and stool of a newborn girl in Canada and two pet animals living in the same household. The results for these five strains are presented separately in Fig. 3. Two strains, one isolated from the baby's stool and a canine strain, exhibited identical band patterns and serotypes (serotype OI). A second strain from the child' stool and a strain from the child's blood were characterized by identical serotypes (serotype OIV) that differed from those of the first two strains and similar but not identical band patterns. The differences were perhaps due to methodological problems during strain preparation. The feline strain was unique. Two other German feline strains (Fig. 2, dots) were isolated from littermates, and two canine strains (Fig. 2, left-pointing triangles) were isolated from animals living in one household.

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FIG. 3. PFGE band patterns (XhoI) of C. upsaliensis isolates from a newborn child, a dog, and a cat living in the same household in Canada.

16S rDNA sequence analysis. Among the 83 C. upsaliensis strains investigated by macrorestriction analysis, seven German, three Australian, and four Canadian strains (Fig. 2, right-pointing triangles) were selected for 16S rDNA sequence analysis. According to Linton et al. (31), the major part of the 16S rRNA-encoding DNA sequence was amplified. A DNA stretch of 1,169 nucleotides of each of the 14 strains beginning at nucleotide position 225 (64) (GenBank accession number L14628) was analyzed. This corresponded to 80.1% of the entire 16S rRNA gene of C. upsaliensis CCUG 14913, which comprises 1,460 nucleotides. The strains possessed identical nucleotide sequences compared to the sequence of strain CCUG 14913, with two minor variations. At position 377, the C in one Canadian strain, two Australian strains, and one German strain was replaced by G; and the G at position 382 was replaced by C in the same strains.

Top Abstract Introduction Materials and Methods Results Discussion References

 
C. upsaliensis is a microorganism that is widespread on all continents and that is primarily isolated from the intestinal environment of dogs. Its relevance as a pathogen that causes enteric diseases in animals is not clear. However, on the basis of a number of reports from different countries, it is recognized as a human pathogen. The different frequencies of isolation from humans may be due to the methodological approaches, the need for supplements or intolerance to the antibiotics present in growth media, or different socioeconomic conditions in different countries. It may also be related to the development of genomic characteristics of the microbe which cause differences in virulence in different areas and continents. The analysis of the C. upsaliensis strains from Germany, Canada, and Australia revealed a considerable degree of genomic heterogeneity. These data are in accordance with those from previous reports and may be due to the properties of Campylobacter strains which enable them to take up DNA from the environment and integrate it into the genome or undergo changes within the genome by rearrangement of DNA (4, 16, 20, 59, 61, 62). Heat-stable antigen typing revealed that, in contrast to C. jejuni and C. coli, which are divided into more than 60 heat-stable serotypes, as described by Penner and colleagues (50, 51), C. upsaliensis seems to possess only a small number of different heat-stable antigens. This is true not only for strains isolated in Germany but also for strains isolated in Canada and Australia. The difference in heat-stable (Penner-type) antigen diversity between C. jejuni-C. coli and C. upsaliensis may perhaps be due to different biochemical structures of the respective antigens. Karlyshev et al. (27) reported evidence that the antigens of C. jejuni accounting for the Penner serotype specificity are capsular structures. It must be determined whether the heat-stable antigens of C. upsaliensis are capsular or lipooligosaccharide-lipopolysaccharide in nature. Eighty-four percent of the strains belonged to five different serotypes, and the ratio of serotypes was comparable for all three continents. Two of the serotypes, serotypes OIII and OIV, were prevalent at rates between 35 and 40% in Germany and Canada. The number of Australian strains available for this study was too small to calculate percentages. However, the tendency for a serotypic prevalence was also seen among the Australian strains, with four of the six strains belonging to serotype OIII. Among the German and Canadian strains, serotypes OI, OII, and OVI were detected at rates of 1.5 to 15%. These serotypes were not identified among the few Australian strains. These data point toward the clonal expansion of C. upsaliensis and indicate a high degree of conservation of heat-stable antigens of the species during evolution. Between 10 and 17.7% of all strains analyzed were untypeable with the available C. upsaliensis-specific antisera. Reactivity with antisera recognizing a number of heat-stable antigens of other Campylobacter species was not detected for any of the C. upsaliensis strains isolated in Germany, Canada, or Australia. Antisera against C. helveticus OV (34), nine heat-stable C. jejuni antigens mainly representing serotypes prevalent worldwide, two C. coli antigens, and one C. lari antigen were included. As reported previously (29), South African C. upsaliensis strains reacted with antiserum against C. coli O:28. It would have been interesting to determine whether the strains investigated in this study also shared antigenic determinants with C. coli O:28. Unfortunately, the strain or the respective antiserum was not available. Therefore, this question could not be answered. Geographic restrictions perhaps contribute to the different results.

Nevertheless, despite the negative results obtained in the present study, it is not surprising that some cross-reactivity exists between C. upsaliensis and other closely related bacterial species.

In contrast to serotyping, genotypic methods (ERIC-PCR and PFGE) revealed a high degree of genomic heterogeneity within the species C. upsaliensis. ERIC-PCR profile analysis demonstrated the discriminatory potential of this method, as described before (34, 57, 58). Beyond this, it divided the strains into two clearly distinct genotypic clusters, a Canadian-Australian (non-European) cluster and a German (European) cluster, whereas the local origins of German strains were not reflected by the dendrogram. The jackknife calculations confirmed the validity of the defined groups, with the limitation that the assignment of Australian strains to the respective group might not have been sufficiently valid, since only six strains were available for the study. With respect to host origin, it could be noticed that the non-European strains, regardless of animal or human origin, were assembled in one group and that the German strains, both canine and feline, with one exception (a canine strain), were assembled in the other. The serotypic groups did not correlate with the genotypic clusters. The results give rise to the suspicion that diversification of the species into serotypes developed before dispersal of the species to different continents. In comparison to a collection of 88 German C. jejuni strains characterized previously (35), the C. upsaliensis strains formed a separate phylogenetic cluster, despite their intraspecies heterogeneity.

In the tree determined by XhoI macrorestriction analysis, intraspecies clustering was also detected. Two clusters were generated: one of them contained 73% of the German strains and 11.5% of the non-European strains, and the second cluster consisted of 88.5% of the non-European strains and 27% of the German strains. With regard to host origin, one cluster contained 78.0% of strains of animal origin and 37.5% of strains of human origin. The second cluster consisted of 22.0% of animal origin and 62.5% of strains of human origin. Subclusters reflecting the local origins of the German strains were, again, not detected, and an association of genotypic clusters with serotypes was also not found. The significance of the defined groups was again confirmed by jackknife calculations, with the limitation that the assignment of Australian and feline strains to the respective groups might not have been sufficiently strong, since only six and seven strains, respectively, were available for the study. The findings of Stanley et al. (55), who identified genotypic differences between human and canine strains by 16S rRNA ribotyping, could not be confirmed by the methods used in the present study.

The high degree of 16S rDNA sequence similarity among the 14 C. upsaliensis isolates sequenced was in marked contrast to the high degree of heterogeneity of the whole genome and confirmed the idea that all the strains belonged to one species. Among the whole 16S rDNA length of 1,169 nucleotides, only one nucleotide was changed at each of two positions, and these changes were identical in the four strains from three continents. With these exceptions, all 14 strains exhibited 16S rDNA sequences identical to that of C. upsaliensis strain CCUG 14913 supplied in the database (64).

Investigating C. upsaliensis isolates originating from different continents and hosts, we intended to analyze the differences in the distributions of genomic characteristics in bacterial populations. The methods used in this study enabled us to detect and describe differences in structural but not functional aspects. The phenomenon that bacterial populations from different continents possess similar antigenic characteristics points toward a common source early in evolution. The Canadian and Australian strains assembled in one genotypic cluster more or less strictly separated from the German strains and may have been distributed to their continents from a common source at a later time.

The possible significance of C. upsaliensis in human disease is increasingly realized. The findings that strains from different continents exhibit significant differences in their genomic characteristics may point toward the possibility of differences concerning pathogenicity or host adaptation. Further studies are necessary to confirm the significance of these results.

 
We gratefully acknowledge the donation of C. upsaliensis strains from Canada and Australia by F. Rodgers and L. Price and by G. Hogg, respectively. We thank P. Schwerk for excellent technical assistance.

 
* Corresponding author. Mailing address: Bundesforschungsanstalt für Viruskrankheiten der Tiere, Institut für bakterielle Infektionen und Zoonosen, Naumburger Str. 96a, D-07743 Jena, Germany. Phone: 0049 3641 804 328. Fax: 0049 3641 804 228. E-mail: i.moser{at}jena.bfav.de.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, August 2004, p. 3441-3448, Vol. 42, No. 8
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.8.3441-3448.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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