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Journal of Clinical Microbiology, February 2001, p. 743-746, Vol. 39, No. 2
Institut für
Infektionsmedizin1 and Klinik für
kleine Haustiere,2 Freie Universität
Berlin, Berlin, and Hygiene-Institut, Universität
Heidelberg, Heidelberg,3 Germany
Received 28 August 2000/Returned for modification 9 October
2000/Accepted 4 November 2000
Nineteen Bartonella henselae strains and one
Bartonella clarridgeiae strain were isolated from blood
samples of 97 pet cats and 96 stray cats from Berlin, Germany,
indicating prevalence rates of 1 and 18.7%, respectively, for B. henselae and 0 and 1%, respectively, for B. clarridgeiae. Eighteen of 19 B. henselae isolates
corresponded to 16S rRNA type II. Pulsed-field gel electrophoresis (PFGE) analysis revealed seven different PFGE types among the feline
B. henselae strains. Interestingly, all feline isolates displayed low genetic relatedness to B. henselae strain
Berlin-1, which is pathogenic for humans.
Bartonella henselae is
the major causative agent of cat scratch disease (CSD), bacillary
angiomatosis (BA), bacteremia, and other pathological manifestations
(1). Bartonella clarridgeiae has recently been
described in association with CSD (11). Domestic cats are
the main natural reservoir for both species (10, 11). Previous studies have demonstrated differences in prevalence rates among cats from different geographic regions, mostly correlating with
climatic conditions and the type of animal population studied (3,
8-10, 16). The first objective of this study was to determine the prevalence of Bartonella infection among two different
cat populations in Berlin, Germany.
(This paper includes part of A. J. Klose's doctoral thesis.)
Blood samples were collected between June 1999 and May 2000 from 193 cats in Berlin. Ninety-seven of these were pet cats brought to the
School of Veterinary Medicine Teaching Hospital for elective procedures, and another 96 were stray cats from different neighborhoods brought to an animal asylum for neutering. One to 1.5 ml of blood was
collected in a pediatric lysis-centrifugation tube (Isolator 1.5;
Oxoid, Basingstoke, United Kingdom). A 0.1-ml portion was inoculated
onto solid and broth culture media, including Columbia agar with 5%
human blood, chocolate agar (Columbia base) with 10% sheep blood, and
brucella broth supplemented with Fildes enrichment and hemin
(18). Plates were incubated at 37°C in 5%
CO2 for 8 weeks. Broth cultures were plated after 1 and 3 weeks on Columbia agar, and plates were incubated for a further 8 weeks. Preliminary identification of the Bartonella isolates
was based on phenotypic characteristics and biochemical reactions in
the Rapid ID 32 A system (Bio Merieux, Marcy-l'Etoile, France).
Species identification was performed for all Bartonella-like
isolates by partial sequencing of the 16S rRNA gene (approximately 420 bp) (2).
Twenty blood cultures revealed growth of Bartonella spp.; 19 isolates were identified as B. henselae, and 1 was
identified as B. clarridgeiae. The Rapid ID 32A results were
000 005/7 3705 for B. henselae, indicating that some strains
could not cleave proline. The B. clarridgeiae strains
Ber-K185 and ATCC 51734, with the profile 000 001 3705, differed in
that they hydrolyzed neither proline nor leucine-glycine. Growth
characteristics of B. clarridgeiae strain Ber-K185 differed
from those of B. henselae strains. Briefly, the primary
isolate grew only on chocolate agar and in supplemented brucella broth,
and subcultures grew inconsistently or weakly on solid media. These
findings are in accordance with previous reports on the difficulties of
culturing B. clarridgeiae on solid culture media (7,
8). Identification of the Ber-K185 strain was confirmed by
complete 16S rRNA sequencing (1,410 bp), which revealed 100% homology
to published sequences for B. clarridgeiae (8,
11).
One B. henselae strain was isolated from the pet cat
population, whereas 18 B. henselae strains and one B. clarridgeiae strain were grown from the stray cat population.
Prevalence rates for B. henselae were 1% (95% confidence
interval [CI95], 0.98 to 3.04%) in pet cats and 18.7%
(CI95, 10.94 to 26.56%) in stray cats, and those for
B. clarridgeiae were 0 and 1% (CI95, Various typing methods have been used to determine the genetic
relatedness among different B. henselae isolates (5,
14, 15). Bergmans and coworkers have described two types
differing in 3 bp as determined by partial sequencing of the 16S rRNA
gene (4). Type I was the most prevalent variant (75%) in
tissue samples from Dutch patients with CSD, while a minority (28%) of the Dutch cats tested were infected by this variant; therefore, the
authors suggested that type I isolates may be more virulent for humans
than type II variants (3). We determined the 16S RNA type
by partial 16S rRNA sequencing. Eighteen of the 19 feline B. henselae strains revealed sequences identical to that of the BA-TF
strain and therefore corresponded to 16S RNA type II (Table 1). One B. henselae strain
showed the highest homology (99.5%) with the sequence of the type I
strain B. henselae Houston-1 (12). These
findings are in accordance with the data of Sander et al. (17), who found one type I isolate (5.9%) among 17 feline
blood culture isolates in Freiburg, Germany.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.2.743-746.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Genetic Variability and Prevalence of
Bartonella henselae in Cats in Berlin, Germany, and Analysis
of Its Genetic Relatedness to a Strain from Berlin That Is
Pathogenic for Humans
ABSTRACT
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0.99 to
3.07%), respectively. The overall prevalence of Bartonella
spp. was 1% in pet and 19.8% in stray cats. Higher rates of infection
in stray or impounded cats have previously been reported for other
geographic regions (6). Only one B. clarridgeiae strain was isolated from the 193 cats included in
this study. A second isolate with high morphological similarity to the
B. clarridgeiae type strain was lost on first subculture.
Although we used additional broth cultures to achieve optimal growth
conditions for B. clarridgeiae, we cannot exclude the
possibility that some B. clarridgeiae isolates did not grow
under these conditions. Nevertheless, the prevalence of B. clarridgeiae appears to be rather low among domestic cats in Berlin.
TABLE 1.
Characteristics of the B. henselae
isolates studied
Pulsed-field gel electrophoresis (PFGE) has been found to be the most
powerful method for intraspecies differentiation of Bartonella isolates (15, 17). By using this
method, we recently (2) reported that the B. henselae isolate Berlin-1, which was originally isolated from BA
lesions of a human immunodeficiency virus (HIV)-infected patient in
Berlin, is genetically indistinguishable from the B. henselae strain Houston-1 (12). This finding raised the question whether some strains are more likely than others to cause
a particular type of pathology and whether a small number of strains
account for a disproportionate amount of disease (13). Therefore, we determined the genetic relationship among the feline B. henselae isolates from Berlin and compared their genetic
fingerprints to those obtained from the Berlin-1 strain. Because
further human-derived isolates were not available from Berlin or
elsewhere in Germany, the B. henselae strains Houston-1
(ATCC 49882) and Oklahoma (ATCC 49793) (19), which are
pathogenic for humans (human pathogenic), were also included in this
study. Digestion of whole chromosomal DNA with SmaI and PFGE
were performed as previously described (2). Visual
analysis of banding patterns of the feline isolates revealed seven
different PFGE types differing in four or more bands from each other
(Fig. 1A). These PFGE types were
considered to represent genetically unrelated variants, indicating a
considerable genetic heterogeneity among the feline isolates (Table 1).
As previously shown (2), the human-pathogenic B. henselae strains Berlin-1 and Houston-1 were identical and showed
high similarity (88%) to the Oklahoma strain (Fig. 1). Interestingly,
none of the feline isolates revealed a banding pattern identical or
similar to that obtained from the Berlin-1 isolate. Computer-assisted analysis of the fingerprints revealed that the feline isolates displayed only 63% similarity to the human-pathogenic isolates (Fig.
1B).
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Although we studied cats from a limited geographic area during a short time interval, the prevalence of the Berlin-1 strain appears to be low (<0.05%). Our findings are in line with data from the Freiburg area of Germany (17) showing that none of 17 feline B. henselae isolates were identical or similar to the Houston-1 strain, which is genetically indistinguishable from the Berlin-1 strain. It is therefore remarkable that the single human-pathogenic B. henselae isolate available from an immunocompromised patient in Germany is genetically highly related to two B. henselae strains isolated from a similar group of patients from another continent. To date, more human-pathogenic B. henselae strains are not available from Germany. Further investigations should include more human-pathogenic and feline B. henselae isolates from other geographic regions in order to determine whether certain strains possess a specific affinity to humans and/or immunocompromised human hosts.
Nucleotide sequence accession number. The 16S rRNA sequence of the Ber-K185 strain has been submitted to the EMBL nucleotide sequence database (accession number AJ299444).
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ACKNOWLEDGMENTS |
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This work was supported by grant 325-4471-02/50 from the German Federal Ministry of Health.
We thank Dagmar Piske for expert technical assistance and Ralf Ignatius for critical review of the manuscript.
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FOOTNOTES |
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* Corresponding author. Present address: Hygiene-Institut, Universität Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany. Phone: 49 6221 56 7807. Fax: 49 6221 56 5627. E-mail: mardjan_arvand{at}med.uni-heidelberg.de.
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Journal of Clinical Microbiology, February 2001, p. 743-746, Vol. 39, No. 2
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.2.743-746.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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