Tropical Canine Pancytopenia: Clinical, Hematologic, and Serologic Response of Dogs to

Ehrlichia canis Infection, Tetracycline Therapy, and Challenge Inoculation

Author(s): William C. Buhles Jr., David L. Huxsoll and Miodrag Ristic
Source: The Journal of Infectious Diseases, Vol. 130, No. 4 (Oct., 1974), pp. 357-367
Published by: Oxford University Press
Stable URL: http://www.jstor.org/stable/30106143
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THE JOURNAL OF INFECTIOUS DISEASES * VOL. 130, NO. 4 * OCTOBER 1974
@ 1974 by the University of Chicago. All rights reserved.

Tropical Canine Pancytopenia: Clinical, Hematologic, and

Serologic Response of Dogs to Ehrlichia canis Infection,
Tetracycline Therapy, and Challenge Inoculation

William C. Buhles, Jr., David L. Huxsoll, From the Division of Veterinary Medicine,
and Miodrag Ristic Walter Reed Army Institute of Research,
Walter Reed Army Medical Center, Washington, D.C.;
and the Department of Veterinary Pathology and
Hygiene, College of Veterinary Medicine,
University of Illinois, Urbana, Illinois

For further elucidation of the pathogenesis of tropical canine pancytopenia

(TCP), 14 German shepherd dogs were evaluated following infection with
Ehrlichia canis, therapy with tetracycline, and challenge inoculation. Nine dogs
developed severe chronic TCP, characterized by severe pancytopenia, hemorrhage,
and secondary bacterial infection. In all dogs antibody titers increased for more
than 80 days after infection, and all dogs developed hypergammaglobulinemia.
Prolonged and incomplete hematologic recovery followed termination of infection
by tetracycline therapy in dogs with severe chronic disease. After treatment levels
of serum y-globulin returned to normal. A second inoculation of treated dogs
with E. canis resulted in reinfection and hypergammaglobulinemia, but with vari-
able increases in serum antibody. Results indicate that aplastic anemia may be
important in the pathogenesis of severe chronic TCP, and that increasing antibody
titers and hypergammaglobulinemia reflect persistent infection with E. canis.
Differences in response to challenge may reflect underlying immunologic dif-
ferences between dogs with severe disease and those that do not become seriously
ill.

In 1968 an epizootic of a highly fatal hemorrhagic
disease occurred among military working dogs in
Southeast Asia [1, 2]. The etiologic agent was
shown to be Ehrlichia canis [3, 4], a tick-borne
rickettsia-like organism first described in dogs in
Algeria in 1935 [5]. The disease was termed trop-
Received for publication October 15, 1973, and in
revised form April 24, 1974.
In conducting the research described herein, we ad-
hered to the "Guide for Laboratory Animal Facilities
and Care," as promulgated by the Committee on the
Guide for Laboratory Animal Facilities and Care of the
Institute of Laboratory Animal Resources, National
Academy of Sciences-National Research Council.
We thank Drs. Herbert L. Amyx and J. J. Kaneko for
advice, Ms. Rita Weisiger for performing the serologic
tests, and L. Beller, R. Branstetter, G. Dalich, D. Eslin-
ger, R. Helt, T. Johnson, and A. Kleiman for excellent
technical assistance.
Please address requests for reprints to Office of the
Director, Division of Veterinary Medicine, Walter Reed
Army Institute of Research, Washington, D.C. 20012.
ical canine pancytopenia (TCP). Further investi-
gations [6-8] revealed that the severity of disease
appeared to be related to the breed of dog infected.
It is now known that experimentally infected Ger-
man shepherds often develop a severe hemorrhagic
syndrome, but that experimentally infected beagles
do not [7]. Nonetheless, in both breeds TCP is
characterized by pancytopenia [7], persistent in-
fection [7], hypergammaglobulinemia [9, 10], and
systemic (primarily perivascular) plasmacytosis
[11-13]. These findings have led to speculation
that some of the changes seen in TCP may have
an immunopathologic basis [10].
Experimentally infected beagles and German
shepherds develop a similar acute-phase disease of
two to four weeks' duration; the acute infection is
characterized by mild clinical signs and a transient
pancytopenia [7]. In beagles and some German
shepherds no further clinical illness occurs, but
the dogs remain infected with E. canis and are
classified as having mild chronic TCP. In contrast,
the majority of German shepherds develop

357

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 358 Buhles, Huxsoll,
marked pancytopenia, hemorrhage, peripheral
edema, emaciation, and secondary bacterial infec-
tions 50-100 days after infection. Most of these
dogs succumb as a result of hemorrhage or sec-
ondary infection. This condition, which is termed
severe chronic TCP, was observed in naturally in-
fected German shepherds in Southeast Asia [1].
Some question has existed in the past as to
whether or not severe chronic TCP is merely a
relapse, albeit more severe, of the earlier acute-
phase disease. We now hypothesize that the
changes seen in severe chronic TCP have a differ-
ent pathogenesis than those seen in acute TCP.
To elucidate further the pathogenesis of chronic
TCP, we studied German shepherds with acute
and chronic E. canis infection. We determined the
clinical and hematologic response to tetracycline,
the efficacy of tetracycline in clearing dogs of per-
sistent infection with E. canis, and the suscepti-
bility of dogs cleared of infection to reinfection.
Concomitantly we studied changes in levels of
serum antibody and y-globulin before and after
tetracycline therapy.
Materials and Methods
Animals. Fourteen purebred German shep-
herds (four males and 10 females, aged 1.0-2.5
years) formed the basic experimental group. Pure-
bred beagles of both sexes (one to three years old)
were also used. All dogs were vaccinated for ca-
nine distemper, infectious canine hepatitis, lepto-
spirosis, and rabies. At time of acquisition dogs
had no serum antibodies to E. canis detectable by
the indirect fluorescent antibody test [14]. Dogs
were housed indoors and offered commercial dry
food and water ad lib.
Clinical evaluation and clinical pathology. Dur-
ing critical periods dogs were examined, and rec-
tal temperatures were taken at least once daily;
at other times such examinations were performed
twice or thrice weekly. Venous blood was collected
thrice weekly in sealed vacuum glass tubes (Vacu-
tainer, Becton-Dickinson, Rutherford, N.J.) with
EDTA as anticoagulant. White blood cell (WBC)
and red blood cell (RBC) counts were performed
on an electronic cell counter (Model B, Coulter
Electronics, Inc., Hialeah, Fla.). Platelet counts
were also performed electronically (MK-4 Platelet
Counter, General Sciences Corp., Bridgeport,
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and Ristic
Conn.). Packed cell volumes were determined by
standard microhematocrit methods. One-hour
erythrocyte sedimentation rates were determined
in Wintrobe tubes.
Serology. Blood for serological tests was col-
lected weekly and stored at -20 C until tested.
Serum antibodies to E. canis were determined by
the indirect fluorescent antibody test recently de-
scribed by Ristic et al. [14]. All serial specimens
were tested simultaneously. Sera were screened at
a dilution of 1:10; positive samples were serially
diluted twofold and titrated.
Serum protein fractionation. Serum protein
was fractionated by workers at The Upjohn Com-
pany (Laboratory Procedures, King of Prussia,
Pa.). Protein of selected serum samples was sepa-
rated by electrophoresis with use of cellulose ace-
tate strips (Titan III, Helena Laboratories, Beau-
mont, Texas) and a Tris-barbital-sodium-barbital
buffer. Fractionated protein was stained with Pon-
ceau S (Helena Laboratories), scanned with a
Quick-Scan densitometer (Helena), and quanti-
tated with a Quick-Quant digital computer (Hel-
ena). Total protein was determined using a TS
refractometer. Serum y-globulin was expressed as
a percentage of total serum protein.
Microorganism. E. canis used in this study
was originally recovered from a naturally infected
German shepherd in Southeast Asia and was
maintained by passage in laboratory-maintained
beagles. This isolate had been shown by passage
in splenectomized dogs to be free of Babesia spe-
cies and Hemobartonella species [15]. Initial in-
fection of the 14 dogs in the experimental group
was by iv inoculation of 5 ml of whole, heparin-
ized blood (20 units/ml) from a dog acutely ill
with TCP. Challenge inoculations were similarly
administered.
Determination of infectivity of blood. The E.
canis infectivity of blood was determined by iv in-
oculation of laboratory-maintained beagles with
5 ml of heparinized blood. Beagles were observed
for signs of E. canis infection by thrice weekly
hematologic examination for 30 days and by sero-
logic testing 30 and 45 days after inoculation.
Tetracycline therapy. Tetracycline hydrochlo-
ride was administered orally at a dosage (in two
doses) of 30 mg/lb/day for 14 consecutive days.
No other antibiotic or supportive treatment was
administered to any of the test animals.
 Tropical Canine Pancytopenia 359

Analysis of hematologic data. The course of
infection was divided into six periods for each
dog: a control period of 30 days immediately pre-
ceding infection; a period of 14 days immediately
preceding onset of outward signs of severe chronic
TCP or initiation of treatment; and four periods
of 30 days each covering the 120 consecutive days
immediately following the initiation of treatment.
Mean platelet, WBC, and RBC counts for each
animal were calculated for each time period.
When applicable, significance of difference from
means calculated before infection was evaluated
by means of Student's t-test. The level of signifi-
cance was set at P < 0.01.
Experimental design. Baseline data were col-
lected for 30 days. The dogs were infected with
E. canis and observed throughout the acute phase
of illness. Treatment was initiated in dogs with
severe chronic TCP at onset of either hemorrhage
or edema. Dogs developing severe chronic TCP
with pancytopenia but without outward signs of
illness or hemorrhage were arbitrarily placed on
tetracycline at 105 days after infection. Dogs with
mild chronic TCP were observed for 145 days
after infection and then treated. To determine sus-
ceptibility to reinfection, three dogs with severe
chronic TCP and three dogs with mild chronic
TCP that survived infection and treatment were
challenged with the homologous strain of E. canis.
Results
Clinical signs. All 14 dogs inoculated with E.
canis developed typical acute-phase TCP with
pancytopenia within 10 days after infection as
described previously [6, 7]. No signs of hemor-
rhage or peripheral edema were apparent at this
time, and all dogs regained a normal physical
appearance. Nine of the 14 dogs developed severe
chronic TCP (table 1). The onset of severe

Table 1. Summary of clinical signs before treatment and clinical response after treatment with tetracycline
in nine dogs with severe chronic tropical canine pancytopenia (TCP).
Signs of severe chronic TCP
Geni- Hemor-
No. Depres- tal rhage
of days sion, tract from Periph-
Dog PI* signs weak- Epi- Hema- hemor- lacera- eral
no. appeared ness staxis toma rhage tions edema Clinical response to therapy
54 65 + + - - - + Improved; edema and hemorrhage stopped.
Worsened 42 days PT* and died 49 days
PT with pancytopenia.
55 76 + - + - - + Developed abscess, bacteremia; died 13
days PT.
57 105t No changes observed.
58 73 + - + - + Worsened; died with pancytopenia one day
PT.

60 95 + - - - + - Improved; hemorrhage stopped; developed

bacteremia and died 32 days PT.

61 89 -+ + + + - - Improved; hemorrhage stopped; remained

emaciated and weak for months.

64 105t - - - - - - No changes observed.

65 56 - + + - - - Improved; hemorrhage stopped; mild hem-
orrhage 90 days PT; regained normal
appearance.

66 98 + - - + + - Improved; hemorrhage stopped; regained

normal appearance.
* PI = postinfection; PT = post-therapy.
t Although showing no outward signs of severe chronic TCP, these dogs had been pancytopenic for 30 days and
were treated empirically beginning 105 days after infection.

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 360 Buhles, Huxsoll, and Ristic

chronic TCP was heralded by a recurrence of se- rized in table 1. Clinical signs typical of terminal
vere thrombocytopenia and leukopenia 40-80 severe chronic TCP were noted in all four dying
days after infection (figure 1). Severe chronic dogs. Necropsy performed on three of the dogs
TCP did not develop in five dogs which were clas- yielded a pathologic diagnosis of terminal severe
sified as having mild chronic TCP and in which chronic TCP with massive internal hemorrhages,
severe hematologic changes were not seen (figure evidence of secondary bacterial infections, and
2). Outward signs of illness and hemorrhage oc- generalized perivascular plasmacytosis [11, 12].
curred in seven of the nine dogs with severe Various degrees of depression, emaciation, weak-
chronic TCP 56-98 days after infection (table 1). ness, and fever persisted for 30-60 days after the
Treatment was initiated within 12-72 hr, except start of treatment in surviving dogs with severe
in dog no. 58, which, because of the equivocal chronic TCP. The five dogs with mild chronic
nature of the initial hemorrhage, was not treated TCP appeared to be normal and healthy through
until seven days after the first signs were ob- 145 days after infection. Their clinical status did
served. Dogs no. 57 and 64 did not manifest out- not change following treatment.
ward signs of illness by 105 days after infection; Six dogs were given a second inoculation of E.
however, since they had been severely thromby- canis 236 days after infection (at least 90 days
cytopenic and leukopenic for more than 30 days, after the start of treatment). Results are summa-
treatment was initiated at this time. rized in table 2. One dog died with massive intes-
Clinical responses following treatment of the tinal and serosal hemorrhages 36 days after chal-
nine dogs with severe chronic TCP are summa- lenge; this dog had pathologic lesions typical of

INOCULATED BLOOD NEGATIVE BLOOD NEGATIVE

HEMORRHAGE
WITH E. CANIS FOR E. CANIS FOR E. CANIS

TETRACYCUNE

107

105
oF

103

10
RECTALMP

250

150

50

15

10

2
RBCOUNTWPLAES
milonces/3thuad lm

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190200 210 220) 230 240 250
DAYS AFTER INFECTION

Figure 1. Rectal temperature and platelet, white blood cell (WBC), and red blood cell (RBC) counts of a
German shepherd that developed severe chronic tropical canine pancytopenia after infection with Ehrlichia
canis. Note that hematologic values improved very gradually after treatment, and that cell counts were still
subnormal 160 days after treatment.

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 Tropical Canine Pancytopenia 361

BLOOD POSITIVE BLOOD NEGATIVE

INOCULATED
FOR E. CANIS
WITH E. CANIS FOR E.CANIS

TETRACYCLINE

107

105
oF

103

RECTALMP
101

250

150

50

15

10

8
6

2
RBCOUNTWPLAES
milonces/3thuadmosncel/3

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250

DAYS AFTER INFECTION

Figure 2. Rectal temperature and platelet, white blood cell (WBC), and red blood cell (RBC) counts of
a German shepherd with mild chronic tropical canine pancytopenia. Note that the changes in this dog during
the acute phase of illness are similar to the changes noted for the dog in figure 1.

severe chronic TCP, including generalized plas- treatment) but no longer yielded the organisms
macytosis. Dog no. 64, which originally had se- 60 days after the initiation of treatment. E. canis
vere chronic TCP, and the three dogs with mild was recovered from blood of all six dogs receiving
chronic TCP responded to challenge inoculation a challenge inoculation.
with clinical (and hematologic) changes that were Hematologic changes. Before infection, dogs
milder and of shorter duration than those usually that developed severe chronic TCP had slightly
encountered in German shepherds with acute in- lower mean platelet and WBC counts and slightly
fections. higher mean RBC counts than dogs that developed
Infectivity of blood. Infectivity was deter- mild chronic TCP, but the differences were not
mined in blood of all dogs surviving the treatment significant (P > 0.10). Hematologic changes dur-
period. Blood from dog no. 55, which died 13 ing the acute phase of illness were similar in all
days after the start of therapy, did not yield E. 14 dogs (figures 1 and 2).
canis at the time of death. Blood from dogs no. During the 14-day period before treatment, all
54 and 60, which succumbed after the treatment nine dogs with severe chronic TCP had thrombo-
period, yielded no E. canis organisms 44 and 32 cytopenia (mean + SE, 9.8 - 1.8 X 103/mm3, P
days after the start of treatment, respectively. < 0.001), leukopenia (WBC, 3.57 X 103 +- 4.60
Blood from all dogs surviving severe chronic TCP X 102/mm3, P < 0.001), and anemia (RBC,
yielded no E. canis when subinoculated 44 and 4.54 + 0.33 X 106/mm3, P < 0.01, figures 1 and
104 days after the start of treatment. Blood from 3). As a group, dogs with mild chronic TCP had
all five dogs with mild chronic TCP yielded E. slightly subnormal platelet counts (1.83 X 101
canis 144 days after infection (immediately before t 2.18 X 104/mm3) and WBC counts (1.07 X

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 362 Buhles, Huxsoll, and Ristic

Table 2. Results of challenge inoculation of dogs with Ehrlichia canis.

Development
Clinical signs PC Acute hematologic changes PC of severe
Dog Physical Rectal Platelet WBC RBC chronic
no. Status appearance temper- count count count ESR TCP
ature

64
Transient diarrhea ' , NC NC 4 No
65
Previous severe Typical acute TCP; 4 , 4, 4- 4 Yes
hemorrhage 55 days PC;
chronic TCP
normal 90 days PC
66
Typical acute TCP; 4 4L 4, , 4 Yes
hemorrhage 33 days PC;
died 36 days PC
53 Transient anorexia NC 4, NC NC No
62 Previous mild
Transient conjunctivitis 4, ' NC ' No
chronic TCP and anorexia

63
NC 4 , NC NC NC No
95 Previously Typical acute TCP 4 , , 4 No
uninfected

NOTE. Dogs had previously been treated with tetracycline during chronic infection. After challenge E. canis was
recovered from all dogs. WBC = white blood cells; RBC = red blood cells; ESR = erythrocyte sedimentation rate;
TCP = tropical canine pancytopenia; NC = no change; PC = postchallenge; 4' = increase; 4, = decrease.

PREINFECTION PRETREATMENT POST TREATMENT PERIOD

0 -30 DAYS 31-60 DAYS 61-90 DAYS 91-120 DAYS
325
20 8

300

250
15 6

200 5

10 4
150

RBCmilonces/3 3
WBCthousandcel/m3

100
PLATESthousndcel/m3

5 2

50
I

INOCULATED TETRACYCLINE
WITH E. CANIS

Figure 3. Summary of hematologic changes in 14 German shepherds that developed either severe chronic
or mild chronic tropical canine pancytopenia. Six periods are represented: a 30-day control period before in-
fection, a 14-day period immediately preceding treatment, and four periods of 30 days each after treatment.
Each symbol represents the mean cell count during one period for an individual dog. Mean platelet count:
severely ill dogs (0), mildly ill dogs (0). Mean white blood cell (WBC) count: severely ill dogs (U),
mildly ill dogs (-I). Mean red blood cell (RBC) count: severely ill dogs (A), mildly ill dogs (A).

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 Tropical Canine Pancytopenia 363

104 + 1.20 X 103/mm3) before treatment, but Table 3. Time of seroconversion in 14 German

these differences were not significant (P > 0.10, shepherds infected with Ehrlichia canis.
figures 2 and 3). Days after No. of dogs No. of dogs
infection seronegative seropositive
Pancytopenia persisted in dogs with severe
Before infection 14 0
chronic TCP in spite of tetracycline therapy (fig-
7 12 2
ures 1 and 3). Dogs surviving severe chronic TCP 13 5 9
remained thrombocytopenic and leukopenic for at 20 1 13
least 120 days after therapy was begun, and two 26 1 13
42 0 14
remained anemic for that period. Anemia abated
in the other three dogs between 30 and 90 days
after commencement of treatment. Irrespective of Titers in all dogs increased from seroconversion
persistent pancytopenia, platelet, WBC, and RBC to a maximal titer at 42-98 days after infection.
counts in all five surviving dogs with severe chronic Evaluation of the geometric mean titer (GMT) of
illness gradually improved during the 120-day all dogs before treatment revealed that this value
period after therapy was begun (figure 3). All continued to increase until approximately 80 days
four dogs that died with severe disease had severe after infection (figure 4A).
pancytopenia. Several dogs with mild disease The GMT of antibody to E. canis decreased
(moderate thrombocytopenia or leukopenia) im- from 1:110 (value before treatment) to 1:56 at
proved hematologically following treatment. 100 days after treatment with tetracycline was be-
Hematologic changes following reinoculation of gun (figure 4B). Individual titers during the 81-
six dogs are summarized in table 2. Dog no. 64 100-day period after the start of treatment ranged
and the three dogs with mild chronic TCP that from 1:20 to 1:160. Titers in four treated dogs
were challenged had milder changes during the that could be evaluated for an additional 100 days
acute phase of illness than were seen during the remained essentially unchanged during this period.
period following initial infection. The titer of indirect fluorescent antibody in-
Serology. All dogs were seronegative for anti- creased in four dogs by six days and in one dog
bodies to E. canis prior to infection. The earliest by 14 days after challenge (figure 4C). In one
positive conversion occurred seven days after in- dog the titer had increased by 33 days after chal-
fection in two dogs, and all but one dog were lenge; this dog developed severe chronic TCP and
seropositive by 20 days after infection (table 3). died 36 days after challenge. The GMT for dogs

A B C
160 160 160
TETRACYCLINE
INFECTION WITH E. CANIS CHALLENGE WITH E CANIS

120 (7) 120 120 SCTCP

(13)
(1o) (5)
(13) (2) (2)
(12)
80 80 80
(II)
(5) (2)
RETIAF
(9)
(10)
40 40 40 NCTCP

NAEMCIRTOGLP
0 20 40 60 80 100 150 0 10-20 21-40 41-60 61-80 81-100 0 20 40 60 80 100

DAYS AFTER INFECTION TIME PERIODS AFTER DAYS AFTER CHALLENGE

TREATMENT (DAYS)

Figure 4. Reciprocal geometric mean titers of antibody to Ehrlichia canis in experimentally infected German
shepherds. A, after initial infection with E. canis (mean -t SE of 14 dogs unless noted in parentheses);
B, after treatment with tetracycline; C, after challenge of six dogs with the homologous strain of E. canis.
Dogs are classified on the basis of response to the original infection; SCTCP = severe chronic tropical canine
pancytopenia; NCTCP = mild chronic tropical canine pancytopenia (mean of three dogs unless shown in
parentheses).

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 364 Buhles, Huxsoll, and Ristic

with severe or mild chronic disease approximately rapid decreases in y-globulin were observed in the
doubled by 14 days after challenge. Between 21 two dogs that died with severe chronic TCP after
and 33 days after challenge, the GMT for the treatment.

three dogs with severe chronic TCP continued to
increase, whereas titers in all mildly ill dogs
dropped to the level noted before challenge.
Serum protein fractionation. Changes in se-
rum protein were serially evaluated in four dogs
with severe chronic TCP and in two mildly ill
dogs. Five of the six dogs became moderately
hypoproteinemic between 20 and 42 days after
infection, but by 63 days after infection, values
for total protein were normal. The percentage of
albumin decreased in five dogs by 20-26 days
after infection and in the remaining dog by 42
days after infection. The magnitude of these
changes was similar to that reported by Burghen
et al. [10]. No significant change occurred in al-
globulin, but four of six dogs had moderate de-
creases in percent a2-globulin and increases in (3-
globulin during the acute phase of infection. All
six dogs were hypergammaglobulinemic by 20
days after infection (figure 5A). Levels of y-glob-
ulin increased twofold from a mean (_ SE) of
10.5% + 0.72% before infection to 21.7% ++
3.03% 26 days after infection. Hypergammaglob-
ulinemia persisted in all but one dog until treat-
ment was initiated (figure 5, A and B).
After tetracycline therapy, y-globulin levels in
all five dogs with hypergammaglobulinemia de-
creased to values noted before infection, although
this return to normal took place more quickly in
some dogs than in others (figure 5B). The most
In the four dogs evaluated after challenge inoc-
ulation with E. canis, the percentage of y-globulin
increased from a mean (+ SE) of 10.8 + 1.4 be-
fore challenge to 26.3 _+ 3.6 at 21 days after chal-
lenge (figure 5C). However, hypergammaglob-
ulinemia persisted in only two of the four chal-
lenged dogs; two mildly ill dogs had normal y-
globulin levels by 97 days after challenge.
Discussion
Amyx et al. [15] reported that tetracycline given
at the peak of the acute phase of experimental
TCP resulted in the remission of clinical and he-
matologic signs within 14 days. In contrast, we
found that dogs surviving severe chronic TCP
showed very gradual clinical improvement after
tetracycline treatment and that pancytopenia per-
sisted for months. This difference in response to
tetracycline supports the hypothesis that severe
chronic TCP has a different pathogenesis from
that of acute TCP. It would appear that the tran-
sient pancytopenia of acute-phase TCP may be
similar to the pancytopenia seen in kala-azar and
in some acute bacterial, rickettsial, and viral in-
fections [16, 17]. In these diseases cell counts
decrease with a normal or hypercellular bone mar-
row, indicating increased sequestration or destruc-
tion of blood cell elements rather than decreased
production. That this is the case in acute TCP is

A B C

INFECTION WITH E. CANIS TETRACYCLINE CHALLENGE WITH E. CANIS

30 30 30

20 20 20

10 10
I0

NILUBOGAMRESTCP
0 20 40 60 80 150 0 20 40 60 80 180 0 20 40 60 80 100

DAYS AFTER INFECTION DAYS AFTER TREATMENT DAYS AFTER CHALLENGE

Figure 5. Percentage of y-globulin in sera of dogs infected with Ehrlichia canis. A, after initial infection
with E. canis (mean + SE Of six dogs); B, after treatment with tetracycline (arrows indicate that dogs died);
C, after challenge inoculation with the homologous strain of E. canis (mean -+ SE of four dogs).

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 Tropical Canine
suggested by the prompt return of blood cell
counts to normal after administration of tetracy-
cline during the early phase of illness.
In contrast, the persistence of pancytopenia
after treatment of our chronically infected dogs
appears to be related to impaired production of
blood cells during severe chronic TCP. Hilde-
brandt et al. [11, 12] have observed bone marrow
hypoplasia in severe chronic TCP. In man, pancy-
topenia with marrow hypoplasia, or aplastic ane-
mia, may be idiopathic [18, 19] or may result
from an idiosyncratic reaction to drugs [18-21],
exposure to radiation [19], or infections such as
viral hepatitis [22, 23]. Regardless of etiology,
pancytopenia is often irreversible; patients die with
severe hemorrhage due to thrombocytopenia or
with secondary infections due to leukopenia [18,
19]. Patients surviving aplastic anemia may have
depressed cell counts for years. This long-term
effect is believed to be due to severe depletion
of hematopoietic stem cells or irreversible changes
in the vascular microenvironment of the bone
marrow [19, 24-27], which persists in the absence
of the original causative agent. Accordingly, per-
sistence of pancytopenia in severe chronic TCP
after removal of the etiologic agent suggests that
aplastic anemia is important in the pathogenesis
of the severe disease.
Tetracycline was apparently effective in clearing
persistent E. canis infection from dogs, although
it is possible that the dogs remained infected and
that either the numbers of circulating infectious
units of E. canis were too low to be detected or
viable organisms were harbored in host tissues
other than the blood, as is believed to occur in
another rickettsial disease, recrudescent typhus
[28]. The fact that all dogs challenged with E.
canis became reinfected is further evidence that
dogs were completely freed of the original infec-
tion after treatment with tetracycline.
A humoral antibody response clearly occurs in
dogs infected with E. canis, but this response does
not prevent persistent infection. The continued
increase in antibody titer for 80 days after infec-
tion, and the development of hypergammaglob-
ulinemia and plasmacytosis probably reflect per-
sistence of the antigen and may represent an
excessive production of immunoglobulins, such as
that which occurs in several viral diseases [29-33].
That the immune response itself may be a factor
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Pancytopenia 365
in the development of severe chronic TCP is in-
dicated by the fact that the height of antibody
response, hypergammaglobulinemia, and plasma-
cytosis in dogs experimentally infected with TCP
follows the acute-phase illness. The chronic phase
of illness does not become apparent for at least
40-60 days after infection, when levels of serum
antibody and y-globulin are markedly elevated.
However, other factors must be involved, since
dogs with mild chronic TCP also have elevated
levels of antibody and serum y-globulin.
Titers of indirect fluorescent antibody persist
after termination of infection with tetracycline,
but hypergammaglobulinemia does not persist.
The apparent correlation between the level of y-
globulin and infectivity may be relevant to the
clinical diagnosis of TCP. Levels of y-globulin
may be useful as a diagnostic aid in differentiating
carrier dogs from dogs from which infection has
been cleared.
Differences between serum antibody response
to challenge in severely ill dogs and the response
in mildly ill dogs, as well as differences between
clinical and hematologic responses of these two
groups after challenge, serve to suggest (within
the limitations of sample size) a resistance to E.
canis in mildly ill dogs but not in all severely ill
dogs. This resistance, however, was not reflected
in the titers of indirect fluorescent antibody before
challenge. Severely ill dogs reacted to challenge
infection almost as if they had not previously been
exposed to E. canis. This finding may indicate
some degree of immunologic unresponsiveness in
dogs with severe chronic TCP, which again is not
reflected in changes in titers of indirect fluorescent
antibody.
Changes in levels of antibody and y-globulin
during TCP are similar to those in Aleutian mink
disease, where y-globulin levels may increase
three- to fourfold by 30 days after infection, and
titers of indirect fluorescent antibody may reach
> 1:100,000 [29, 30]. Titers of antibody during
TCP do continue to increase well into the chronic
phase of infection, as occurs in Aleutian mink
disease, and severe disease in both TCP and Aleu-
tian mink disease usually occurs two months or
more after infection [6, 7, 29]. Further study of
immunologic changes in TCP may reveal more
similarities with Aleutian mink disease. Unlike
the virus-induced immunopathologic diseases, in-
 366 Buhles, Huxsoll,
fection with E. canis can be readily terminated,
a fact that facilitates the study of the host-parasite
interaction.
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Pancytopenia 367
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Addendum
Since this manuscript was completed, additional
serological tests on the chronically infected dogs
in this study provided evidence that titers of indi-
rect fluorescent antibody continue to increase.
After an 18-month observation period, titers of
indirect fluorescent antibody in chronically in-
fected dogs ranged from 1:2,560 to 1:5,120. The
magnitude of these titers is consistent with our
findings of hypergammaglobulinemia.