JOURNAL OF CLINICAL MICROBIOLOGY, May 1999, p. 1474–1479 Vol. 37, No.

5
0095-1137/99/$04.0010
Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Western Immunoblot Analysis of the Antigens of

Haemobartonella felis with Sera from
Experimentally Infected Cats
A. RICK ALLEMAN,1* MELANIE G. PATE,1 JOHN W. HARVEY,1
JACK M. GASKIN,2 AND ANTHONY F. BARBET2
Departments of Physiological Sciences1 and Pathobiology,2 College of Veterinary Medicine,
University of Florida, Gainesville, Florida 32610
Received 27 October 1998/Returned for modification 19 January 1999/Accepted 8 February 1999

Cats were experimentally infected with a Florida isolate of Haemobartonella felis in order to collect organisms
and evaluate the immune response to H. felis. Cryopreserved organisms were thawed and injected intravenously
into nonsplenectomized and splenectomized cats. Splenectomized animals were given 10 mg of methylpred-
nisolone per ml at the time of inoculation. Blood films were evaluated daily for 1 week prior to infection and
for up to 60 days postinfection (p.i.). Blood for H. felis purification was repeatedly collected from splenecto-
mized animals at periods of peak parasitemias. Organisms were purified from infected blood by differential
centrifugation, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to
nitrocellulose membranes for immunoblot analysis. Serum was collected from nonsplenectomized animals
prior to and for up to 60 days p.i. and was used on immunoblots to identify antigens. The combination of
splenectomy and corticosteroid treatment resulted in marked, cyclic parasitemias without concurrent severe
anemia, providing an opportunity to harvest organisms in a manner that was not lethal to the animals. Several
antigens (150, 52, 47, 45, and 14 kDa) were identified. An antigen with a molecular mass of approximately 14
kDa appeared to be one of the most immunodominant and was consistently recognized by immune sera
collected at various times during the course of infection. These data suggest that one or more of these antigens
might be useful for the serologic diagnosis of H. felis infections in cats.

Haemobartonella felis is an epicellular, hemoparasite of do-
mestic cats that causes a clinical disease known as feline infec-
tious anemia or hemobartonellosis. Haemobartonella spp. are
currently classified in the family Anaplasmataceae, order Rick-
ettsiales. However, Haemobartonella spp., including H. felis,
have recently been shown by 16S rRNA gene sequencing to be
more closely related to Mycoplasma spp. (23). H. felis has a
worldwide distribution and is believed to be one of the most
common causes of hemolytic anemia in domestic cats (8, 10).
fection (p.i.). Common clinical abnormalities include anemia,
depression, weight loss, icterus, pyrexia (in about 50% of in-
fected cats), and splenomegaly. Clinical disease is most often
associated with the acute phase, but organisms can be identi-
fied only periodically during this time and rarely, if at all, in
chronically infected carriers (10, 11). Reported mortality rates
indicate about 33% of the infected animals die, mostly during
the acute phase (6, 10, 20). With appropriate antibiotic therapy

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Even so, many questions regarding the transmission, preva-
lence, and clinical impact of this organism on the domestic
feline population remain unanswered, largely because a reli-
able and readily accessible means of diagnosis is not available.
H. felis may produce clinical disease alone or concomitantly
with other agents such as feline leukemia virus (FeLV) and
feline immunodeficiency virus (FIV) (6, 8, 22). The lack of a
reliable means of diagnosis of H. felis infection makes accurate
assessment of concurrent infections difficult. However, in one
survey, as many as 35% of H. felis-infected cats were FeLV
positive, with even higher percentages of positivity seen among
cats presenting with severe clinical disease (8).
Uncomplicated infection with H. felis occurs in four phases:
the (i) preparasitic, (ii) acute, (iii) recovery, and (iv) carrier
phases (10). The prepatent period for experimentally infected
cats is 2 to 3 weeks. Clinical signs may develop shortly after this
time, but this is quite variable; some experimentally infected
animals did not develop clinical signs until 4 to 6 weeks postin-
* Corresponding author. Mailing address: Department of Physiolog-
ical Sciences, College of Veterinary Medicine, University of Florida,
Box 100103C, Gainesville, FL 32610. Phone: (352) 392-4700, ext. 5858.
Fax: (352) 392-1769. E-mail: ALLEMANR@MAIL.VETMED.UFL
.EDU.
most animals survive uncomplicated H. felis infection, but
many remain subclinical carriers, probably for life (3, 4, 10, 11).
However, because a reliable means of diagnosis is not readily
available, infection with H. felis often remains undetected and
many animals go untreated.
Presently, the clinical diagnosis of H. felis infection relies on
the positive identification of organisms in peripheral blood
smears with Romanowsky-type or acridine orange stains (5).
Even in acute stages of the disease, however, a definitive iden-
tification of H. felis infection is difficult because of rapidly
fluctuating parasite numbers and a lack of a consistent corre-
lation between parasitemia and clinical signs (10).
Recently, a species-specific PCR assay based on the 16S
rRNA gene sequence of H. felis was developed (19). This assay
may provide workers with a valuable tool with which they can
answer many questions regarding the transmission and preva-
lence of the disease. Reports indicate that the assay detects
acutely ill and relapsing animals but does not consistently de-
tect asymptomatic carrier cats (3, 4). However, PCR-based
assays are labor-intensive, requiring specialized equipment and
highly trained personnel. In addition, these assays are limited
to research and reference laboratories. A sensitive and specific
serological assay based on one or more H. felis antigens would
provide a more accessible means of diagnosis for clinicians

1474
VOL. 37, 1999 WESTERN BLOT ANALYSIS OF HAEMOBARTONELLA FELIS
FIG. 1. PCV and percent parasitemia for each of two splenectomized cats (A and B) from 0 to 60 days postinoculation with an FL isolate of H. felis. Asterisks
indicate periods when the organisms were harvested from the peripheral blood.
presented with cats with suspected hemobartonellosis in which
organisms are not identified in the peripheral blood.
Currently, the Haemobartonella spp. are not amenable to in
vitro cultivation. Therefore, in this study, we used differential
centrifugation to purify a Florida (FL) isolate of H. felis from
the peripheral blood of experimentally infected cats. Purified
organisms were then used with immune sera from experimen-
tally infected animals in Western immunoblot assays to identify
the immunodominant antigens of H. felis and to characterize
the humoral responses of cats to H. felis infection. This was
done to determine the feasibility of a serology-based assay for
the diagnosis of H. felis infection and to identify H. felis anti-
gens which might be targeted for future production as recom-
binant test antigens.
MATERIALS AND METHODS
Experimental animals. A total of six female, domestic short-haired cats bred
for research were purchased from a licensed dealer. These cats were certified to
be free of FeLV and FIV infection by testing for these viruses prior to and 45
days following inoculation with H. felis-infected blood. At preinfection, negative
control serum was collected from each cat. Wright-Giemsa-stained peripheral
blood films were examined daily for 7 days prior to inoculation with H. felis to
ensure that no hemoparasites were present. Two of the four animals to be
infected were splenectomized 21 days prior to inoculation. The splenectomized
cats were each given 10 mg of a single intramuscular injection of methylpred-
nisolone acetate (Depo-Medrol; The Upjohn Company, Kalamazoo, Mich.) per
kg of body weight at the time of inoculation. Two of the four animals to be
infected were not splenectomized. The splenectomized animals were used pri-
marily for the harvesting of organisms and nonsplenectomized cats were used
primarily for the production of immune sera. Two uninfected cats were used as
blood donors for the infected animals.
Organisms and immune sera. An FL isolate of H. felis (10) which had been
cryopreserved in liquid nitrogen was intravenously inoculated into two splenec-
tomized cats and two nonsplenectomized cats. All cats were examined daily, and
peripheral blood was drawn each morning for the monitoring of the packed cell
volume (PCV), total protein, icterus index, and parasite numbers. Infectivity was
quantified with Wright-Giemsa-stained blood films and was recorded as a per-
cent parasitemia. Infected blood was collected from splenectomized cats during
peak parasitemias (when 80 to 90% of the erythrocytes were infected) in
1475
amounts that did not result in mortality (usually 40 ml). An equal volume of
uninfected blood from a donor cat was transfused back to the infected animal.
Immune serum was collected from all infected animals at intervals of 15, 30, 45,
and 60 days p.i.
Antigen preparation. H. felis-infected whole blood was collected in heparin
and centrifuged at 600 3 g for 10 min at room temperature. The plasma and buffy
coat were removed, and packed cells were washed and centrifuged three times in
an equal volume of 0.15 M phosphate buffered saline (PBS; pH 7.2). The wash
solution and any remaining buffy coat cells were removed after each centrifuga-
tion. Wright-Giemsa-stained blood films of washed erythrocytes were examined
for the presence of parasites. The pelleted erythrocytes were then placed in an
equal volume of 0.15 M PBS (pH 7.2)–0.15% (vol/vol) polyoxyethylene-sorbitan
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monolaurate (Tween 20)–1.5% (wt/vol) disodium EDTA and were incubated on
a rocker at room temperature for 2 h to dislodge the H. felis organisms from the
erythrocytic membranes. The preparation was centrifuged at 300 3 g for 10 min
at room temperature, and the supernatant was removed. Wright-Giemsa-stained
blood films were examined to ensure that the parasites were dislodged. The
supernatant containing H. felis organisms was centrifuged at 22,000 3 g for 1 h
at 4°C. The supernatant was removed, and the pelleted organisms were resus-
pended in 5.0 ml of 0.15 M PBS (pH 7.2). The preparation was further purified
from any host cell components by differential centrifugation through 20% di-
atrozoate meglumine and diatrozoate sodium (RenoCal-76; Bracco Diagnostics
Inc., Princeton, N.J.) at 22,000 3 g for 40 min at 4°C. The resulting pellet was
resuspended in 0.5 ml of 0.15 M PBS (pH 7.2) and stored at 220°C. Most H. felis
preparations resulted in 0.5 ml of antigen at a concentration of approximately 1.0
to 2.0 mg/ml, as determined by the Coomassie blue G dye-binding assay (21).
Erythrocytic antigens, prepared from experimental animals prior to infection
with H. felis, were used as a negative control. Briefly, 1 ml of uninfected whole
blood was washed and centrifuged three times in an equal volume of 0.15 M PBS
(pH 7.2), with the wash solution and any remaining buffy coat removed after each
centrifugation. The washed erythrocyte pellet was suspended in 5 ml of 0.15 M
PBS (pH 7.2), freeze-thawed five times in liquid nitrogen, and used as an
uninfected erythrocytic antigen preparation.
Electron microscopy. A portion of the purified H. felis preparation was pre-
pared in McDowell and Trumps fixative for transmission electron microscopy.
The sample was washed in 0.1 M cacodylate buffer, postfixed in 1% buffered
osmium tetroxide, washed several times with distilled water, and dehydrated in a
graded ethanol series and then in 100% acetone. The sample was polymerized in
100% EMbed 812 (Electron Microscopy Sciences, Fort Washington, Pa.) and
then trimmed and ultrasectioned on a Leica Ultracut R ultramicrotome (Leica
Microscopy and Scientific Instruments, Deerfield, Ill.). The sections were stained
in 2% uranyl acetate and then Reynold’s lead citrate.
1476 ALLEMAN ET AL.
FIG. 2. PCV and percent parasitemia for each of two spleen-intact cats (A and B) from 0 to 60 days postinoculation with an FL isolate of H. felis. Asterisks indicate
periods when doxycycline administration was necessary to prevent mortality.
SDS-polyacrylamide gel electrophoresis. The protein concentrations of the
microorganism and noninfected erythrocyte preparations were determined by
the Coomassie blue G dye-binding assay as described previously (21). The pro-
teins were dissolved in one-half their volume of a 33 sample buffer containing
0.1 M Tris (pH 6.8), 5% (wt/vol) sodium dodecyl sulfate (SDS), 50% glycerol,
7.5% b-mercaptoethanol, and 0.00125% bromophenol blue and were heat de-
natured at 100°C for 3 min. Solubilized microorganisms or noninfected erythro-
cytes were applied onto 10% (wt/vol) polyacrylamide-SDS gels at 20 mg per lane
and the gels were electrophoresed. The gels were fixed in 25 mM Tris–191.8 mM
glycine–20% methanol and were electrophoretically transferred to nitrocellulose
membranes (Hybond ECL; Amersham International plc, Little Chalfont, Buck-
inghamshire, England).
Immunoblots with antisera. Nitrocellulose membranes containing transferred
proteins were blocked with 5% (wt/vol) milk in PBS with 0.25% Tween 20 and
washed with 1% (wt/vol) milk in PBS with 0.25% Tween 20 as described previ-
ously (1). The membranes were probed with antisera collected from infected
animals at 14, 21, 30, 45, and 60 days p.i. Serum dilutions of 1/300 or greater were
used. As a negative control, preinfection serum was used with erythrocytic and H.
felis antigens at concentrations equal to or greater than that used for infected
sera. In addition, immune serum was used with erythrocytic antigens to confirm
that the antigens identified in the H. felis preparations were not of erythrocyte
origin. The membranes were washed as described previously (1) and were probed
with horseradish peroxidase-conjugated goat anti-cat immunoglobulin G (whole
molecule; ICN Pharmaceuticals, Inc., Biochemical Division, Aurora, Ohio) at a
dilution of 1/20,000. The membranes were processed for enhanced chemilumi-
nescence with detection reagents containing luminol (SuperSignal Substrate;
Pierce, Rockford, Ill.) as a substrate and were exposed to X-ray film (Hyperfilm-
MP; Amersham International plc) to visualize the bound antibody.
RESULTS
Splenectomy and the administration of methylprednisolone
facilitated the development of recurrent episodes of marked
parasitemias. Parasitemias approaching 100% were seen in
both splenectomized cats, usually without a significant drop in
PCV (Fig. 1). This allowed repeated harvesting of large num-
bers of organisms without the development of severe clinical
disease. Replacement of harvested, infected blood by transfu-
sions with uninfected whole blood maintained a relatively sta-
ble PCV. High parasitemias sometimes persisted for 2 or more
J. CLIN. MICROBIOL.
days in splenectomized animals; however, in some instances
the number of infected erythrocytes went from 90 to ,1%
within a 3-h period. Indirect Coombs’ tests for antierythrocytic
antibodies were negative for both cats.
Neither nonsplenectomized cat developed recurrent, high-
level parasitemias suitable for the harvesting of organisms.
One animal became clinically ill by day 18 p.i., when parasite
numbers reached 9% parasitemia. The PCV decreased from 32
to 19% by day 22 p.i. (Fig. 2A). The cat was Coombs’ test
positive when the test was performed at 37°C with a 1:4 dilu-
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tion. The severity of the disease necessitated a whole-blood
transfusion and the administration (2 days) of doxycycline
(orally at 10 mg/kg twice daily). The animal’s condition was
stable for approximately 2 weeks, but parasite numbers then
increased dramatically and the PCV decreased precipitously to
10%. The severity of the disease necessitated a second trans-
fusion and eventual prolonged treatment with doxycycline at
the same dosage for 21 days. The other nonsplenectomized cat
developed an infectivity which peaked at a 10% parasitemia
(Fig. 2B). Clinical signs were minimal, consisting of a mild
anemia that persisted following the appearance of parasites in
the peripheral blood at day 14 p.i. This cat did not become
Coombs’ test positive.
Collection of infected erythrocytes in tubes containing hep-
arin as an anticoagulant prevented dislodging of the parasites
from the erythrocyte membranes, which may occur when
EDTA is used as an anticoagulant (10). Pelleted erythrocytes
maintained original levels of parasitemia after gentle washes in
PBS. This was evaluated with Wright-Giemsa-stained smears
prepared from erythrocytes following each wash in PBS (data
not shown). However, blood films prepared after incubation of
infected erythrocytes in 1.5% EDTA for 2 h at room temper-
ature revealed that the vast majority of the organisms were
dislodged from the cell membranes. Slow-speed centrifugation
VOL. 37, 1999
FIG. 3. Electron micrograph of H. felis bodies purified from infected whole blood by differential centrifugation. Bar, 1 mm.
allowed easy separation of uninfected erythrocytes (pellet)
from H. felis organisms (supernatant). A solution of 1.5%
EDTA resulted in mild to moderate hemolysis of erythrocytes;
however, lower concentrations (1.0% EDTA for 2 h) left sig-
nificant numbers of organisms remaining on the erythrocyte
membranes (data not shown). Host cell membranes remaining
in the H. felis-rich supernatant after slow-speed centrifugation
were subsequently removed by differential centrifugation with
20% diatrozoate meglumine and diatrozoate sodium. This re-
sulted in an H. felis preparation suitable for evaluation for
antigen. The purity of the preparation was evaluated by elec-
tron microscopy (Fig. 3), revealing minimal host cell mem-
brane contamination.
Western immunoblot analysis of purified H. felis organisms
was done to detect individual antigens. Uninfected erythrocytic
preparations were used as negative controls to ensure that the
identified antigens were of parasite origin. Serial dilutions of
immune sera collected at 21 days p.i. identified five major
antigens with molecular masses of 14, 45, 47, 52 and 150 kDa
(Fig. 4). Three of these (14, 52, and 150 kDa) appeared to be
the most immunodominant antigens. We have designated
them major antigen 1 (MA1) (14 kDa), major antigen 2 (MA2)
(52 kDa), and major antigen 3 (MA3) (150 kDa). A 56-kDa
antigen was also recognized by immune sera at various dilu-
tions. An antigen of similar size, however, was identified in
uninfected erythrocyte preparations and when H. felis prepa-
rations were used with preinfection sera (Fig. 4). This antigen
was assumed to be of erythrocytic origin.
In order to evaluate the temporal immune response of in-
fected cats to H. felis, purified organisms or uninfected eryth-
WESTERN BLOT ANALYSIS OF HAEMOBARTONELLA FELIS 1477
rocytes were used with either preinfection sera or sera col-
lected at 14, 30, 45, or 60 days p.i. Antibodies to some H. felis
antigens were detected with immune sera collected as early as
14 days p.i. (Fig. 5). Bands of 14-kDa and possibly 52-kDa
antigens were recognized by immune sera at dilutions of
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1/1,000. Several antigens were recognized by sera collected at
30, 45, and 60 days p.i., and the antibody response to H. felis
antigens intensified during the course of the infection (Fig. 5).
These data suggest the 14-kDa antigen of H. felis is not only
one of the most immunodominant antigens but is also consis-
tently recognized by immune sera at various times during the
course of infection.
DISCUSSION
Parasite numbers have been known to fluctuate rapidly in
the peripheral blood of cats infected with H. felis, resulting in
a poor correlation between the clinical signs and parasite num-
bers in the peripheral blood (10). There is substantial evidence
implicating the spleen as the major organ involved in the re-
moval of the organisms from infected erythrocytes (17, 18). H.
felis organisms were removed less readily in splenectomized
cats, resulting in parasitemias that lasted approximately twice
as long as those in nonsplenectomized cats (16). Splenectomy
performed on recovered, carrier cats resulted in the transient
reappearance of parasites, in most cases without the develop-
ment of clinically significant anemia (11). The administration
of methylprednisolone has also been shown to increase circu-
lating parasite numbers in chronically infected carriers (11).
For these reasons, animals used for propagation of organisms
1478 ALLEMAN ET AL.
FIG. 4. Immunoblot of an FL isolate of H. felis and noninfected, feline
erythrocytes (RBC) with preinfection serum (Pre) or immune serum (Post)
collected from an experimentally infected animal 21 days postinoculation. The
fractions (1/300 to 1/3,000) indicate the dilutions of serum used. Molecular size
standards (in kilodaltons) are given on the left.
were splenectomized and administered methylprednisolone
prior to inoculation. This resulted in consistently high para-
sitemias without the concurrent development of severe ane-
mia. PCVs remained stable in infected animals when collected
blood was replaced with an equal volume of uninfected blood
from donor cats. Parasitemias of 80% or more sometimes
persisted for up to 3 days, allowing ample time for collection.
However, as reported previously (10), parasite numbers often
declined rapidly from .90 to ,1% in 3 h or less, even though
the animals were splenectomized. Thus, parasite numbers need
to be monitored closely to determine the optimal time for
harvesting. The mechanism of parasite removal in splenecto-
mized animals is speculative, but macrophages in the lung,
liver, and bone marrow are thought to play a significant role
(12).
A 1.5% (wt/vol) concentration of disodium EDTA in PBS
was effective in releasing the majority of the parasites from
infected erythrocytes. This concentration of EDTA has been
previously shown to cause the release of a closely related he-
moparasite, Eperythrozoon suis, from infected swine erythro-
cytes (9). However, the infectivity of the purified E. suis or-
ganisms was markedly reduced when the organisms were
inoculated into susceptible pigs. The investigators concluded
that this method of dislodging E. suis from infected erythro-
cytes produced a nonviable preparation but that the technique
was suitable for the preparation of antigens for use in immu-
nological assays. Although we did not attempt infectivity trials
with purified H. felis organisms, on the basis of the reported
findings with E. suis, we suspect that the viabilities of the
purified organisms would be reduced.
J. CLIN. MICROBIOL.
FIG. 5. Immunoblot of an FL isolate of H. felis and noninfected, feline
erythrocytes (RBC) with preinfection serum (Pre) or immune serum collected
from an experimentally infected animal at 14 days (Post 14), 21 days (Post 30),
45 days (Post 45), and 60 days (Post 60) postinoculation. The fraction (1/1,000)
indicates the dilution of serum used. Molecular size standards (in kilodaltons)
are given on the left.
Incubation of H. felis-infected erythrocytes for 2 h in a so-
lution containing 1.0% EDTA resulted in significant numbers
of organisms remaining attached to erythrocytes. A previous
study (9) found that lower concentrations of EDTA (0.75%)
were not as effective in releasing E. suis organisms from in-
fected erythrocytes. However, unlike E. suis-infected erythro-
cytes, a 1.5% concentration of EDTA resulted in a mild to
moderate lysis of H. felis-infected erythrocytes. Although it is
possible that feline erythrocytes are inherently more sensitive
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than swine erythrocytes to the effects of EDTA, we speculate
that attachment of H. felis to the erythrocytic membranes re-
sults in increased erythrocyte fragility, more so than with the
attachment of E. suis organisms. Previous studies have dem-
onstrated the production of erosions and pockmarks on eryth-
rocyte membranes in areas where H. felis organisms were at-
tached (13, 24). These lesions were considered to be important
in the development of the hemolytic anemia associated with H.
felis infection (13).
A method of differential centrifugation with 20% diatrozo-
ate meglumine and diatrozoate sodium was necessary to fur-
ther separate organisms from the erythrocytic membranes
lysed during incubation in EDTA. This method has been
shown to be effective in separating rickettsia from host cells in
cell culture preparations (7). The concentrations of diatrozo-
ate meglumine and diatrozoate sodium were reduced to en-
hance recovery of the mycoplasma-like H. felis organisms. The
resulting preparation was shown by electron microscopy to
contain large numbers of H. felis bodies with minimal erythro-
cytic membrane contamination.
H. felis antigens of various molecular sizes ranging from 150
to 14 kDa were identified in Western blots. The immune re-
sponse to some antigens was recognized as early as 14 days p.i.
and persisted throughout the course of this study (60 days p.i.).
VOL. 37, 1999 WESTERN BLOT ANALYSIS OF HAEMOBARTONELLA FELIS
Cats infected with H. felis and mice infected with Haemobar-
tonella muris have been shown by indirect fluorescent-antibody
testing to mount an immune response to surface antigens on
the organism (10, 23). However, the onset, duration, and spe-
cific targets of the immune response have not been previously
identified. The detection of H. felis antibodies from 14 to 60
days p.i. indicates that it is possible to diagnose natural infec-
tions both during the acute phase of the disease (when clinical
hemobartonellosis is most often seen) and during the recovery
and carrier phases.
We identified antigens of 150, 52, 47, 45, and 14 kDa in an
FL strain of H. felis. One of these antigens, the 14-kDa antigen
(MA1), appears to be one of the more immunodominant an-
tigens and is consistently recognized by immune sera during all
phases of infection. Previous analysis of antigens of H. muris
found antigens of 118, 65, 53, 45, and 40 kDa (23). That study,
which used 16S rRNA gene sequence analysis, found H. muris
to be most closely related to the FL strain of H. felis (89%
sequence similarity) (23). The 52- and 45-kDa antigens of H.
felis are similar in size to the 53- and 45-kDa antigens of H.
muris; however, the antigenic relationships are unknown.
Given the close phylogenetic and sequence similarities in the
16S rRNA gene, antigenic cross-reactivity between these two
species would seem likely.
There are obvious antigenic differences between these two
species, and one of the most immunodominant H. felis antigens
(the 14-kDa antigen) was not recognized in H. muris prepara-
tions (23). In fact, on the basis of the findings of Rikihisa et al.
(23), differences between isolates of H. felis are likely. When
the rRNA gene sequences of three strains of H. felis were
compared, it was found that an Ohio isolate of H. felis shared
an identical gene sequence with an FL isolate; however, a
California isolate of H. felis shared ,85% sequence similarity
(23). Less sequence similarity was identified between these two
strains of H. felis than was identified between the FL strain of
H. felis and H. muris (23). On the basis of these findings, the
investigators concluded that more than one species of Haemo-
bartonella may infect cats. It is possible, therefore, that the five
antigens identified in the FL isolate of H. felis may not be
conserved in other strains of H. felis.
Although speculative, there is some concern that antigenic
variation may play a role in the development of the cyclic
parasitemias identified in our experimentally infected animals
and those reported previously (10). Antigenic variation has
been a proposed mechanism for the development of cyclic
parasitemias with other hemoparasites such as Anaplasma
marginale (2, 14). This would have a significant impact on the
selection of a diagnostic antigen since the use of a conserved
antigen would be more beneficial in a serologic test.
We have successfully developed methods for propagation,
harvesting, and purification of H. felis organisms from experi-
mentally infected cats. In addition, we have identified five
major antigens from an FL isolate of H. felis. We have also
shown that the immune response to experimental H. felis in-
fection begins as early as 14 days p.i. and continues throughout
the course of the infection. This suggests that it is possible to
develop a serologic assay based on one or more of these anti-
gens for the detection of H. felis-infected cats. However, the
sensitivity and specificity of particular antigens in detecting
infected animals remain unknown. Further work must be done
to determine which antigens are conserved and recognized by
cats experimentally infected with H. felis isolates from different
geographic areas and which antigens are recognized by im-
mune serum from animals infected with closely related organ-
isms.
1479
ACKNOWLEDGMENTS
We thank Heather Sorenson for technical support in performing
Western immunoblot experiments and Karen Vaughn and the Elec-
tron Microscopy Core, ICBR, University of Florida, for electron mi-
croscopic technical support.
This work was supported by the Division of Sponsored Research
grant 2805773-12 from the University of Florida.
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