0095-1137/95/$04.0010
Copyrightq1995, American Society for Microbiology
Prolonged Bartonella Bacteremia in Cats Associated with
Cat-Scratch Disease Patients
DORSEY L. KORDICK,
1KENNETH H. WILSON,
2,3DANIEL J. SEXTON,
2TED L. HADFIELD,
4HERMAN A. BERKHOFF,
5AND
EDWARD B. BREITSCHWERDT
1*
Departments of Companion Animal and Special Species Medicine
1and Microbiology, Pathology, and Parasitology,
5College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606; Division
of Infectious Diseases, Duke University Medical Center,
2and the Veterans Affairs Medical Center,
3Durham, North Carolina 27710; and the Department of Infectious and Parasitic Diseases
Pathology, Armed Forces Institute of Pathology, Washington, DC 20306
4Received 5 June 1995/Returned for modification 16 August 1995/Accepted 12 September 1995
Recent evidence supports a causal relationship between
Bartonella
(
Rochalimaea
)
henselae
, cat-scratch
dis-ease (CSD), and bacillary angiomatosis. Cats appear to be the primary reservoir. Blood from 19 cats owned by
14 patients diagnosed with CSD was cultured. Blood samples from cats owned by veterinary students (
n
5
25)
having no association with CSD or bacillary angiomatosis were cultured as controls. Eighty-nine percent (17
of 19) of cats associated with CSD patients and 28% (7 of 25) of controls were bacteremic with
Bartonella
species
(chi-square
5
16.47;
P
< 0.001). Twenty-three isolates were characterized as
B. henselae
, while one isolate from
the cat of a CSD patient appeared to be a new
Bartonella
species. Thirteen cats remained culture positive during
the ensuing 12-month period. Our results support the conclusion that
B. henselae
is the predominant species
involved in CSD and is transmitted by cats. The incidence of
Bartonella
bacteremia in control cats suggests that
B. henselae
bacteremia is prevalent among the domestic cat population in the United States.
Cat-scratch disease (CSD), cat-scratch fever, or benign
non-bacterial lymphadenitis was recognized by Robert Debre
´ in
1931, but it was first reported in 1950 (5). A presumptive
diagnosis of CSD is made when a patient has an inoculation
wound at the site of a cat bite or scratch, regional
lymphade-nopathy, a positive CSD skin test, and negative test results for
brucellosis, infectious mononucleosis, mycobacteria, syphilis,
and tularemia.
The search for the etiologic agent was intensified after Wear
et al. (40) described the presence of small, silver-staining bacilli
in lymph nodes from 28 CSD patients. In 1990, Relman et al.
(29) used PCR to amplify Bartonella DNA from bacillary
an-giomatosis (BA) lesions of four immunocompromised
individ-uals, including one woman who had recently experienced a
severe cat scratch. The causative agent eluded isolation
at-tempts until concurrent reports by Welch et al. (42) and
Reg-nery et al. (26) described the recovery of small gram-negative
rods from both immunocompetent and immunocompromised
patients by the lysis-centrifugation blood culture technique.
The organisms were determined to be closely related to
Bar-tonella quintana and were subsequently named BarBar-tonella
henselae. The molecular characterization and cultivation of B.
henselae facilitated development of an indirect
fluorescent-antibody (IFA) test which was used by Regnery et al. (27, 28)
to identify a seropositive B. henselae bacteremic cat. Soon
thereafter, Koehler et al. (16) reported the isolation of
Bar-tonella species from the BA lesions of four
immunocompro-mised patients. B. quintana was found in three patients denying
cat or arthropod contact, while B. henselae was cultured from
the blood of the fourth patient who had obtained several
scratches from his flea-infested cat and kitten. In 1993, Dolan
et al. (7) described two immunocompetent patients with B.
henselae-induced lymphadenopathy; both of the patients
owned cats. The most compelling evidence that cats are a
primary reservoir of B. henselae was presented by Koehler et al.
(15) in a study which identified seven of seven Bartonella
bac-teremic cats belonging to or associated with four patients with
BA.
Recently, multiphasic studies resulted in the reclassification
of Rochalimaea and Grahamella species into the genus
Bar-tonella and the removal of the family BarBar-tonella from the order
Rickettsiales (1, 3). The spectrum of clinical manifestations
associated with Bartonella infection in people includes classical
CSD (7, 15, 46), Carrio
´n’s disease (30), trench fever (45),
cutaneous BA (12, 16, 22, 25, 29, 38, 42), vasoproliferative
conditions of the liver and spleen (21), lymphadenopathy (7, 8,
10, 29), and endocarditis (6, 9, 13, 36). Reports of
Bartonella-induced neurologic disease (32, 33, 37), relapsing fever (18, 26,
35, 41), granulomatous hepatosplenic syndrome (8, 10, 31, 34,
38, 39), osteolytic lesions (16, 39), pulmonary granulomas (4),
and neuroretinitis (11, 23) are also in the literature.
Exposure to cats is an important risk factor for B. henselae
infection (15, 46). With a population of approximately 60
mil-lion domestic cats in the United States (44), there is a large
potential zoonotic reservoir for introducing B. henselae
infec-tion into the human populainfec-tion. In view of this, we investigated
the incidence of Bartonella bacteremia in cats associated with
CSD patients. Determination of the prevalence and degree of
persistence of Bartonella bacteremia in cats is necessary to
assess the correlation between feline bartonellosis and disease
in humans.
(The study was presented in part at the 34th Interscience
Conference on Antimicrobial Agents and Chemotherapy,
Or-lando, Fla., 4 to 7 October 1994 [17a].)
MATERIALS AND METHODS
Our study compared cats historically associated with patients with diagnosed cases of CSD and healthy cats owned by veterinary students with no related history of disease. All patients were exposed to one or more cats and had a
* Corresponding author. Mailing address: College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough St., Raleigh, NC 27606. Phone: (919) 829-4234. Fax: (919) 829-4336.
3245
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presumptive clinical diagnosis of CSD or serological evidence of Bartonella exposure. The diagnosis of B. henselae infection was confirmed in one patient by PCR amplification of Bartonella DNA from pulmonary granulomas (4).
Cats (n519) owned by patients (n514) with known or suspected CSD were examined and tested at the College of Veterinary Medicine, North Carolina State University. Twenty-five cats owned by veterinary students were used as controls. Control cats appeared healthy, tested negative for feline immunodefi-ciency virus and feline leukemia virus, and had no association with human CSD or BA.
Questionnaires were distributed to all participants in the study. Attending physicians provided specific details relative to each patient’s presentation and course of illness, including criteria for diagnosis, antimicrobial treatment, and patient outcome. Owners of control animals completed questionnaires which included historical details about the cat including whether the cat regularly scratched, bit, licked, or slept with anyone in the household.
Strain sources.The type strains of B. henselae (Houston-1; ATCC 49882) and
Bartonella elizabethae (F9251; ATCC 49927) were obtained from the American
Type Culture Collection (Beltsville, Md.). The B. quintana type strain (Fuller; ATCC VR-358) was provided by one of us (T.L.H.). Twenty-four feline isolates described in this report, in addition to NCSU-F01, a B. henselae isolate used as an antigen for serological testing, were obtained at the College of Veterinary Medicine, North Carolina State University. An avian isolate of Chlamydia psittaci (strain NCSU-165) was provided by Kevin Flammer.
Blood culture isolation of microorganisms.Blood was collected aseptically from the external jugular vein, either 8 ml was injected into Wampole Isolator 10 microbial tubes (Wampole Laboratories, Cranbury, N.J.) or 1.5 ml was injected into Pediatric Isolator 1.5 tubes (Wampole Laboratories), and the tubes were inverted several times. Wampole Isolator 10 tubes were centrifuged at 2,3003g
for 30 min at room temperature, the supernatant was removed, and the concen-trate was applied to Trypticase soy agar (TSA) plates supplemented with 5% defibrinated rabbit blood (BBL, Becton Dickinson, Cockeysville, Md.). The Pediatric Isolator 1.5 tubes were vortexed for 15 s, and the entire sample was streaked onto TSA-rabbit blood plates. Cultures were incubated at 358C in 5% CO2and were examined daily for bacterial growth. Cultures were monitored for
at least 2 months before they were deemed negative and discarded.
Microscopic and biochemical analyses.Bartonella organisms were visualized
by light microscopy following Gram or Gimenez staining. Commercial bacterial identification systems which assay for preformed enzymes and carbohydrate utilization (UniScept 20E, An-IDENT, and API-ZYM; Analytab, Sherwood Medical, Plainview, N.Y.), the presence of oxidase (Dryslide; Difco Laboratories, Detroit, Mich.), and urease production (Urea agar; BBL, Becton Dickinson) were used in conjunction with standard microbiological methods to characterize the isolates (19).
Cellular fatty acid composition.The organisms were grown on rabbit blood agar at 358C in 5% CO2for 4 to 9 days. Cells were harvested from an average of
three plates and were processed as follows. Briefly, the cells were saponified in methanol-NaOH with heat. The liberated fatty acids were methylated, and the methyl esters were extracted into an ether-hexane mixture. One microliter of the fatty acid methyl ester mixture was injected onto the column. Fatty acid profiles were determined with the Microbial Identification System (Microbial ID, Inc., Newark, Del.). The system consists of an HP5290 gas chromatograph equipped with a flame ionization detector, 5% methylphenyl silicone fused-silica capillary column, auto sampler, integrator, computer, and printer. The run begins at a temperature of 1708C and increases by 58C/min to 2708C. Fatty acids are iden-tified and relative proportions are determined by computer analysis.
Serological analysis.Sera were analyzed by microimmunofluorescence assay for immunoglobulin G (IgG) reactive for B. henselae Houston-1 and NCSU-F01,
B. quintana Fuller, and C. psittaci NCSU-165. Bartonella organisms were
culti-vated in Vero cells and were harvested when the cells were.80% infected (3 to 5 days postinoculation). Antigen for IFA testing was prepared by pelleting and resuspending the microorganisms in 0.5% bovine serum albumin in phosphate-buffered saline (PBS). Five-microliter aliquots of crude antigen were applied to 24-well Teflon-coated slides (Cel-line Associates, Newfield, N.J.), air dried, ac-etone fixed, and frozen until they were used. The C. psittaci organisms were similarly processed following culture in McCoy’s cells, a mouse kidney fibroblast cell line.
Slides used for the IFA test were blocked in 5% skim milk prior to the application of diluted test sera. Twofold dilutions of serum in PBS ranging from 1:16 to 1:1,024 were applied to slides in 10-ml aliquots; this was followed by 30-min incubation (378C) and wash (PBS) steps. Fluorescein isothiocyanate (FITC)-conjugated goat anti-cat IgG (heavy and light chains; Cappel, Organon Teknika Corp., Durham, N.C.) was diluted 1:200 in 0.5% BSA-PBS and was applied to each well containing feline serum in order to detect IgG-reactive antibodies in the cats. In some cases, the reactivity of human serum to B. henselae Houston-1 and B. quintana Fuller was determined in our laboratory by using FITC conjugated goat anti-human IgG (heavy and light chains; Cappel) at a working dilution of 1:20 in 0.5% BSA–PBS. The slides were incubated for an additional 30 min, washed in PBS, and examined at340 magnification with a fluorescence microscope. Human sera were assayed by the IFA test in our laboratory or at the Centers for Disease Control and Prevention, Atlanta, Ga., by previously reported methods (28).
All feline sera were tested for the presence of feline leukemia virus antigen and feline immunodeficiency virus antibodies by using the CITE Combo enzyme-linked immunosorbent assay test kit (IDEXX, Portland, Maine).
DNA extraction.Subcultures of the blood isolates were grown on TSA-rabbit blood plates and were recovered in filter-sterilized PBS with a cell scraper. The harvested bacteria were frozen at2708C until extraction of the DNA. DNA extraction for PCR amplification and subsequent sequencing was performed by a standard phenol-chloroform protocol following agitation with glass beads in a mini-beadbeater (Biospec, Bartlesville, Okla.). Dedicated equipment and re-agents were used to assemble the PCR mixtures in a biological containment hood, and extreme care was taken to prevent cross-contamination of samples.
High-molecular-weight chromosomal DNA for restriction endonuclease diges-tion was obtained by an alternative method. Bartonella colonies were recovered from agar plates in glucose-Tris-EDTA and were kept on ice until they were subjected to proteinase K digestion. The bacterial lysate was purified with hexa-decyltrimethylammonium bromide-NaCl at 658C, and genomic DNA precipi-tated with isopropanol.
PCR amplification.PCR amplification of the 16S rRNA gene was accom-plished with P0-C and PC-5A primers, which are modifications of previously described eubacterial primers (43). Reactions were performed in a Gene-Amp 9600 thermal cycler (Perkin-Elmer Cetus, Norwalk, Conn.) by a protocol re-ported elsewhere (2).
DNA sequencing of 16S rRNA gene.Sequencing reactions of the purified PCR products were performed in a PE 9600 apparatus in preparation for use in an automated DNA sequencer (model 373A; Applied Biosystems Inc., Foster City, Calif.) as described previously (2). Published data confirm the existence of a ‘‘signature region’’ in the 16S rRNA gene between nucleotide positions 1101 and 1109 (Escherichia coli numbering) that is species specific for B. henselae, B.
elizabethae, and B. quintana (16). The inclusion of sequence data from nucleotide
positions 976 through 990 can further distinguish between Bartonella vinsonii and
Bartonella bacilliformis. Primer PC4 (59-TTGACGTCATCCCCACCTTCC TC-39) initiates the reaction at position 1175 and generates the sequence in the desired region of residues 976 to 1109. The nucleotide sequence of each isolate in this signature area was compared with GenBank data for the Bartonella organisms B. henselae, B. quintana, B. elizabethae, B. vinsonii, B. bacilliformis, B.
talpae, B. peromysci, B. grahamii, B. taylorii, and B. doshiae.
Restriction digest of chromosomal DNA.B. henselae chromosomal DNAs
obtained from 10 isolates from felines were subjected to digestion with restric-tion endonucleases MspI and XhoI according to the manufacturer’s recommen-dations (New England Biolabs, Beverly, Mass.). DNA was digested for 2 h at 378C, and the products were separated by electrophoresis through a 0.65% agarose gel to assess strain differences.
RESULTS
CSD patients.
The ages, sexes, immune status, clinical signs,
criteria for diagnosis, duration of illness, and treatment
regi-mens for CSD patients are summarized in Table 1.
Recovery of
Bartonella
species from cat blood.
The
signal-ment of the cats (age, breed, sex), date of initial culture, and
relationship to the onset of CSD are summarized in Table 2.
Lysis-centrifugation blood cultures were examined daily for
growth. Bartonella colonies appeared within 7 to 31 days of
incubation. Most isolates (19 of 24) appeared within the range
of 7 to 13 days, 3 of 24 isolates required 17 to 20 days of
incubation, and 2 of 24 isolates did not show evidence of
growth until 31 days had passed. Two colonial forms were
recovered and occasionally, for some strains, coexisted within
the same culture. One form consisted of rough, very adherent,
cream-colored colonies and the other was mucoid,
nonadher-ent, and opaque to cream in coloration. Both colonial forms
ranged from
,
1 to 2.5 mm in diameter. Subcultures of all
isolates on TSA-rabbit blood plates yielded colonies of one
phenotype within 3 to 6 days. The rough, adherent form
ap-peared to be dominant. Hemolysis of the rabbit blood was not
observed with any isolate.
Persistent bacteremia was documented in 13 cats from which
blood was cultured for various periods of time (Fig. 1). Blood
samples from six cats belonging to a patient with pulmonary
bartonellosis were initially cultured, and the cats were found to
have Bartonella bacteremia 6 months after the patient became
noticeably ill. The cats remained bacteremic for an additional
year. Another patient in our study experienced classical CSD
shortly after acquiring two kittens during January 1993.
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tures of blood from both cats were positive for B. henselae in
October 1994, representing a presumable period of bacteremia
spanning 22 months. Interestingly, 7 of 25 (28%) of the control
cats were bacteremic. No persistent skin lesions, febrile
epi-sodes, lethargy, or lymphadenopathy had been reported in any
household resident in the control group within the previous 18
months. Serology was not performed on the veterinary
stu-dents.
Microscopic and biochemical analyses of the isolates.
[image:3.612.55.559.83.289.2]Pleo-morphic gram-negative rods ranged in size from 0.5 to 2.0
m
m.
TABLE 1. Clinical characteristics of CSD patientsCharacteristic Patient Summarya
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Sexb F M M M F F M F M M F M M F 6F, 8M
Age (yr) 9 13 51 23 19 8 36 9 36 27 39 28 20 23 Median, 23
Immune compromised 2 2 2 2 1 2 2 2 2 2 2 2 2 2 11, 132
Cat contact 1 1 1 1 1 1 1 1 1 1 1 1 1 1 141, 02
Clinical signs
Fever 1 1 1 1 1 2 2 1 1 1 1 1 2 1 111, 32
Myalgia 1 2 1 1 2 2 2 2 2 2 2 2 2 2 31, 112
Hyporexia 1 2 1 1 1 2 2 1 2 2 2 2 2 2 51, 92
Weight loss 1 2 1 1 1 2 2 2 2 2 2 2 2 2 41, 102
Inoculation wound 1 1 1 2 1 1 1 1 1 1 1 2 1 1 121, 22
Lymphadenopathy 1 1 1 1 2 1 1 1 1 1 1 1 1 1 131, 12
Parenchymal 1 2 2 1 1 1 2 2 2 2 2 2 2 2 41, 102
Encephalopathy 1 2 2 2 2 2 2 2 2 2 2 2 2 2 11, 132
Biopsy resultc 2 2 2 1 1 1 2 1 2 2 2 1 2 2 51, 92
Serology titer
B. henselae Houston-1 64 128 64 1,025 1,025 NDd ND ND 256 32 128 ,64 128 ND
B. quintana Fuller 128 64 128 1,025 1,025 ND ND ND ND ,16 128 ND 2,048 ND
Antimicrobial treatmente cefd, cefp nt naf pen dox ceph ery amcl, tris dox dox nt cip nt nt
Duration of illnessf 4w 6w 8w 4w 5m 4w ND 7w 4w 2w 12m 2w 4w 4w
aNumbers indicate numbers of patients. bF, female; M, male.
cPatient 5, lung tissue; other patients, lymph node. dND, not determined.
ecefd, cefadroxil; cefp, cefproxadine; naf, nafcillin; pen, penicillin; dox, doxycycline; ceph, cephalexin; ery, erythromycin; amcl, amoxicillin-clavulanate potassium; tris
trimethoprim-sulfamethoxazole; cip, ciprofloxacin; nt, not treated.
fw, weeks; m, months.
TABLE 2. Blood culture and serology results for cats associated with CSD cases
Patient Cat
Identification no.
Date of onset of illness (mo/yr)
Identification letter
Signalment (age/ sex/breeda)
Initial culture date (mo/yr)
Culture result
Serologyb
Bh F01 Bq Cp
1 11/92 A 1.5 yr/M/DSH 5/93 1 64 64 128 ,16
2 9/93 B 6 mo/M/DLH 11/93 1 256 ,16 64 ,16
3 10/93 B
4c 3/93 C 1.5 yr/F/DSH 5/93 2 ,16 64 ,16 ,16
5 9/92 D 3.5 yr/F/DSH 3/93 1 64 64 64 128
5 E 2 yr/F/DSH 3/93 1 64 64 32 64
5 F 9 mo/M/DSH 3/93 1 128 256 128 128
5 G 2 yr/M/DSH 3/93 1 64 64 256 1,024
5 H 2 yr/M/DSH 3/93 1 64 32 256 512
5 I 2 yr/F/DSH 3/93 1 128 128 256 256
6 1/93 J 5.5 yr/M/DSH 4/93 1 64 ,16 ,16 ,16
7 12/91 K 6.5 yr/F/DSH 4/93 2 64 64 128 ,16
8 12/92 L 2.5 yr/F/DSH 5/93 1 128 128 128 ,16
9 4/93 M 1.5 yr/M/DSH 6/93 1 128 256 128 ,16
10 10/93 N 8 wk/F/DSH 1/94 1 1,024 ,16 ,16 ,16
11 1/94 O 2 yr/F/DLH 2/94 1 1,024 ,16 64 ,16
12 10/93 P 6 mo/F/DSH 2/94 1 128 ,16 ,16 ,16
13 12/93 Q 9 mo/M/SiamX 3/94 1 ,16 ,16 512 ,16
14 1/93 R 1 yr/M/DSH 9/94 1 256 ,16 256 ,16
S 6 mo/F/DSH 9/94 1 32 ,16 ,16 32
a
Age at onset of illness; M, male; F, female; DSH, domestic shorthair; DLH, domestic longhair; SiamX, Siamese crossbred.
b
Data are reciprocal endpoint titers. Bh, B. henselae Houston-1; F01, B. henselae NCSU-F01; Bq, B. quintana Fuller; Cp, C. psittaci NCSU-165.
c
Only one of three cats in contact with the patient was made available for blood culture.
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[image:3.612.61.556.461.693.2]Organisms were also visualized with Gimenez stain. Eight- to
13-day-old subcultures were evaluated in triplicate for various
phenotypic characteristics. Twitching motility, assessed by the
hanging drop technique and with motility agar (M Medium;
bioMerieux Vitek, Inc., Hazelwood, Mo.), was observed in
most instances (20 of 24 isolates). All isolates were catalase
negative when they were analyzed by the An-IDENT,
Uni-Scept 20E, and 3% hydrogen peroxide tests, urease negative
when they were streaked onto urea agar, and Voges-Proskauer
negative. Tests for indole production, carbohydrate utilization,
and nitrate reduction were negative in all cases. Most isolates
displayed variability when tested for oxidase by using 1%
tet-ramethyl-p-phenylenediamine and the Dryslide technique.
Biochemical analysis of 24 isolates yielded two discrete
phe-notypic clusters. The biochemical profiles of 21 isolates were
nearly identical, while 3 isolates formed a different group. In
the larger cluster, which consisted exclusively of B. henselae,
the following aminopeptidases were detected: alanine,
argi-nine, glycine, histidine, leucine, phenylalaargi-nine, and valine. The
three isolates in the group that was different did not contain
histidine, leucine, phenylalanine, proline, tyrosine, or valine
aminopeptidase and, in addition, possessed indoxyl-acetate,
esterase, and esterase lipase and utilized arginine.
Cellular fatty acid analysis.
Nineteen feline isolates and two
American Type Culture Collection Bartonella type cultures
were analyzed by fatty acid methyl ester analysis. Six to nine
fatty acids were detected in each of the isolates. The
predom-inant fatty acids were 18:1
v
7c, 18:0, 16:0, and 17:0, which
made up more than 97% of the fatty acid content of the cells.
Serological findings.
Sera from 17 of 19 CSD-associated cats
had reactivity to B. henselae Houston-1 and NCSU-F01 in
addition to B. quintana Fuller, with reciprocal titers ranging
from 64 to 1,024 (Table 2). In 12 of 25 control cats,
seroreac-tive IgG reciprocal titers ranged from 64 to 512. Three B.
henselae bacteremic animals were seronegative for both
IgM-and IgG-specific antibodies. One cat has been bacteremic but
seronegative on repeated occasions. In order to evaluate
sero-logical cross-reactivity between Bartonella species and C.
psittaci, sera from CSD-associated cats and 3
specific-patho-gen-free cats experimentally infected with B. henselae as a
component of a separate study were analyzed for C.
psittaci-specific antibodies. Six of 19 CSD-associated cats, all from the
same household, were seroreactive to both B. henselae
Hous-ton-1 and C. psittaci NCSU-165, while the remainder were
seronegative for chlamydia-specific antibodies. Sera from three
experimentally infected cats demonstrated Bartonella-reactive
IgG titers of 1,024, and the cats were seronegative when their
sera were tested against C. psittaci.
Sequence of the 16S rRNA gene.
Partial sequencing of the
16S rRNA gene was used to identify the species of the
Bar-tonella isolates obtained in blood culture. Of 24 isolates, 23
were identified as B. henselae following analysis of the
signa-ture regions between residues 976 and 1109. The identity of
one isolate could not be determined because of substantial
differences in the nucleotide sequence of this hypervariable
region, which may be indicative of a novel Bartonella species or
subspecies. Additional genotypic characterization of this
iso-late is in progress.
Restriction digest of DNA.
The patterns produced by MspI
and XhoI cleavage of chromosomal DNAs from 10 isolates
from felines, including 6 isolates from felines in one household,
matched the general digestion profile of B. henselae Houston-1
and differed substantially from the profiles of B. quintana
Fuller and B. elizabethae F9251. Strain differences among the
isolates were suggested by the further definition of several
subgroups. This is best illustrated by the restriction patterns
derived from isolates obtained from six cats belonging to
pa-tient 5 (Fig. 2).
DISCUSSION
[image:4.612.129.484.71.278.2]Fourteen patients from North Carolina and Virginia with
known or suspected CSD were studied. Six patients were
ex-amined by members of the Division of Infectious Diseases at
Duke University Medical Center. The remaining patients were
examined by family physicians, and the case records were
re-viewed by the authors. Bartonella bacteremia was documented
in 17 of 19 (89%) cats historically associated with 14 cases of
CSD, thereby suggesting exposure to Bartonella-infected cats
in 12 instances. Aside from a renal transplant patient, all other
patients in our study involved previously healthy,
immunocom-petent people. Two of the cats whose blood was cultured may
FIG. 1. Persistent Bartonella bacteremia in cats. Blood samples for culture were obtained at various time points following the onset of patient illness. *, data derived from the patient with the earliest onset of illness.
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not have been the source of infection. In patient 7, illness
occurred 17 months prior to culturing of blood of the
associ-ated cat. It is possible that these cats were bacteremic at an
earlier date but spontaneously eliminated the organism or that
the degree of bacteremia was below the level of detection by
lysis-centrifugation. Our laboratory has detected relapsing
bac-teremia of prolonged duration in cats experimentally infected
with B. henselae (17b). Cats A and B appear to have eliminated
their bacteremia. Cat A initially had a reciprocal titer of 64 to
B. henselae Houston-1 that persisted unchanged for 17 months,
while cat B maintained reciprocal titers to B. henselae
Hous-ton-1 of between 256 to 512 during a 12-month period. In
contrast, bacteremia was detected in some cats for periods of
up to 18 months following the diagnosis of CSD.
Bartonella bacteremia was detected in 7 of 25 (28%) control
cats, which were not associated with any known instances of
human disease. The incidence of B. henselae bacteremia in
CSD-associated cats compared with that in cats not associated
with CSD was significant (P
,
0.001). Interestingly, within the
control cat population, six of seven bacteremic cats
occasion-ally bit, licked, scratched, or slept with their owners, yet clinical
signs of CSD were not reported in those households. Since
serological evaluation of the owners of control cats was not
performed, it may be that they were immune to Bartonella
infection or, alternatively, that the strains infecting these
par-ticular cats were nonvirulent for humans. Recent studies report
that approximately 3% of healthy human controls are B.
henselae seropositive and that 12 to 29% of veterinarians are
CSD skin test positive (20, 28, 46). Koehler et al. (15) reported
Bartonella bacteremia in 25 of 61 (41%) pet and pound cats in
the San Francisco area, and Olson et al. (24) cultured B.
henselae from 15 of 61 (25%) pet and stray cats in southern
California. Given the current domestic cat population in the
United States of approximately 60 million, 15 million to 25
million cats may potentially be infected with B. henselae.
Re-gardless of the actual numbers of bacteremic cats, recent
re-ports suggest that the potential for zoonotic transmission of B.
henselae is considerable. However, it seems important to
rec-ognize that the infectivity of B. henselae strains isolated from
healthy cats for people has not been established
epidemiolog-ically.
In the present study, 3 of 15 (20%) B. henselae
Houston-1-seronegative cats were bacteremic. In addition to the potential
for false-negative results, the IFA test is not species specific
(10, 17). Because of cross-reactivity among the various
Bar-tonella species, we elected to test sera against B. quintana
Fuller as well as two strains of B. henselae strains Houston-1
and NCSU-F01 (Table 2) (39). Serological cross-reactivity is
best illustrated by the responses of patient 13 and his cat (cat
Q); serum samples from both the patient and his cat contained
higher IgG levels against B. quintana Fuller, even though B.
henselae, which is directly implicated in the transmission of
CSD, was cultured from his cat. These data indicate that IFA
serological testing of cats and people is reliable only to the
genus level. Knobloch et al. (14) and Drancourt et al. (9) have
described human serological cross-reactivity between
Bar-tonella and Chlamydia species. Evaluation of feline serum
failed to demonstrate cross-reactivity between these two
or-ganisms. In addition to 13 of 19 CSD-associated cats whose
sera had variable titers against B. henselae Houston-1,
experi-mentally infected specific-pathogen-free cats with reciprocal
IFA test titers of 1,024 were seronegative when their sera were
tested against C. psittaci NCSU-165. The six C.
psittaci-sero-positive cats were from the household of patient 5. It is quite
likely that these cats are chronically infected with chlamydia,
resulting in persistent conjunctivitis and rhinitis.
The diagnosis of Bartonella infection is best accomplished by
direct isolation of the organisms from blood or tissue
speci-mens. Feline blood cultures readily grow bartonellae when the
cultures are processed and incubated as described above,
al-though in the present study, periods of culture as long as 31
days have passed before colonies were visualized. In our
expe-rience, Bartonella species are considerably more difficult to
isolate from humans. Whether this observation is due to a
more prolonged bacteremia or a higher bacterial titer in the cat
is unknown. Determining the etiologic species is also difficult.
Following isolation, Bartonella organisms cannot be identified
to the species level by conventional methods used in clinical
microbiology laboratories. Biochemically, Bartonella species
are quite inert. Cellular fatty acid analysis, although useful for
many bacterial organisms, may not be a reliable method for
determining species of this genus since the fatty acid content of
Bartonella species is predominantly composed of only four
fatty acids. Presently, 16S rRNA gene sequencing, DNA
hy-bridization, and immunocytochemistry provide the most
accu-rate means of species identification. The increasing use of
molecular techniques to detect bacterial DNA will
undoubt-edly enhance the ability to diagnose these fastidious organisms.
There is considerable variation in the clinical presentations
and lesions associated with Bartonella infection in people. This
variability may be attributable to bacterial strain differences as
well as differences in the immunological response of the
in-fected individual. Among our B. henselae isolates, we detected
differences in the biochemical constituents, cellular fatty acid
contents, and restriction endonuclease digestion patterns of
the B. henselae DNAs. These results suggest that several B.
henselae strains might contribute to the variations in clinical
presentation or cellular inflammatory response.
[image:5.612.102.250.70.314.2]Kittens have been implicated as a risk factor in CSD, yet we
FIG. 2. Analysis of restriction endonuclease digests of Bartonella DNA by
MspI. Chromosomal DNAs from six B. henselae isolates from cats and three Bartonella type strains were digested with MspI, and the products were separated
by electrophoresis through a 0.65% agarose gel. Isolates D through I were obtained from cats residing in the same household (patient 5). B. henselae strain variations are suggested by differences in the digestion profiles.
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did not find a disproportionate number of kittens involved in
cases of CSD in our study. Most of the cats associated with our
CSD patients were older than 6 months of age (14 of 19 cats)
and, therefore, would be classified as adults. Similar to
previ-ous reports, most of the bacteremic cats in our study were
clinically healthy. The medical histories of CSD-associated cats
were unremarkable. Most received routine vaccinations and
had only otherwise been seen by a veterinarian for declawing,
neutering, and management of bite wounds sustained in cat
fights. Six CSD-associated cats originating from one household
had conjunctivitis most likely of chlamydial etiology. Another
cat presented with prominent generalized lymphadenopathy
that gradually resolved without treatment during a 3-month
period. This cat also experienced a brief (2-day) episode of
mild, nonlocalizing neurologic dysfunction, as might be
ex-pected with a metabolic encephalopathy. Since cats can
main-tain a subclinical bacteremia for extended durations in the
presence or absence of circulating antibody, establishing a
causal relationship to unusual or rare manifestations of
bar-tonellosis will be difficult.
ACKNOWLEDGMENTS
This work was supported by a grant from SmithKline Beecham Animal Health, Exton, Pa., the U.S. Department of Veterans Affairs, and the Department of Companion Animal and Special Species Med-icine Research Fund.
We thank Carol L. Lemons for providing clinical microbiology sup-port and Barbara C. Hegarty for technical assistance in the develop-ment of a microimmunofluorescence assay. We are also indebted to Hal Hills, Jenny Simchok, Rhonda Blitchington, and Gary Anderson for generously sharing their expertise in molecular techniques and Vincent T. Andriole and Talmadge T. Brown for critical review of the manuscript. CITE Combo ELISA test kits were a gift of the IDEXX Corp.
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