STUDIES ON CANENE, F'ELiNE, AND
MURENE HAEMOBARTONELLOSIS

Thesis for ”19 Degree of M. S.
MECHEGAN STATE UNIVERSETY
Aaron M. Leash

1961

THESIS

 

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STUDIES ON CANINE,
FELINE, AND MURINE
HAEMOBARTONELLOSIS

By

Aaron Me 11.8811

AN ABSTRACT

Submitted to
Michigan State University
in partial fulfillment of the requiremente
for the degree or

MASTER OF SGIHOE

Department of Surgery and Medicine

 

 

1961
Approved: ”£4214”! 574/. [dwem‘d Date: 121%, A531; «4/

//

ABSTRACT

STUDIES on CANINE,
FELINE, AND mam:
HAEMOBARTONEILOSIS

by Aaron M. leash

gaemobartogella 52. is a group of organisas parasitic
on the erythrocytes, and capable of producing a hemolytio

anemia in its hosts. In most instances, splenectoay of the
host is a prerequisite for the production of anemia. The
cat is a notable exception to this rule, since anemia
regularly occurs in the non-aplenectoni sed individual. Vari-
ous studies were conducted on a. m“ of the dog, fl. £21155.
of the cat, and g. m of the rat. Test animals were
proved negative for these parasites by the examination of
Giensa stained blood films daily for a minim of 14 days
before they were inoculated with infective blood.

Oats, when injected with g. m, do not develop a
haenobartonellenia, but their blood becomes infective for
dogs.

Dogs. when injected with g. ELLE: develop a haeao-
bartonellenia, and their blood may be infective for dogs and
cats.

3;. oanis is transferred from bitch to puppies. but

 

whether this occurs in utero or through the milk was not

Aaron M. Leash

determined.

An attempt to prove the mosquito, _C_u_l_e_; M, the
vector of a. Lem was unsuccessful, as was the attempt to
transmit this organism to the laboratory rat, m

0011.

1 intra-

A dose of 2 IIng body weight of Mapharsen
peritoneally, was used to successfully treat g. M
infection in rate, and the MLD of this drug for rats was
found to be between 22 and ens/.5 body weight.

Eabryonated chicken eggs, 8-12 days old, could not be
infected with E. 93241 or 11. 1211i-

Piloidez was used to successfully treat canine haemo-
bartonellosis on a 3-day schedule at the level of 0.20 Isl/lb
body weight, intravenously. n11. drug was unreliable for
the treatment of a. 2.1L! infection in cats at the level of
0.20 al/lb body weight, intravenously, for 10 successive

days.

 

'Parke, Davis and Company, Detroit, Michigan, Brand
of Oxophenarsine Hydrochloride.

amass-Moore Division of Allied Laboratories,
Indianapolis, Indiana, Brand of Arsinobenzanide.

STUDIES ON CANINE,
FELINE, AND MURINE
HAEHOBARTONELLOSIS

by

Aaron M. Leash

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the require-outs
for the degree of

MASTER OF SCIENCE

Department of Surgery and Medicine

1961

ED CA N

his thesis is dedicated to my ever patient wife, Fran.

 

MENTS

the author is deeply indebted to Dr. William v. Dumb,
his former advisor, now at Colorado State university, who
suggested and guided this work. To Dr. Gabel H. Conner,
Department of Surgery and Medicine, sincere thanks for
"adopting" the author and patiently overseeing the writing
of this thesis. The chick embryo work could not have been
done without the helpful suggestions of Dr. Esther Smith,
Department of Anatomy, and Dr. John P. Bowman, Department
of Microbiology and Public Health. Dr. Smith was especially
generous in affording the author the use of her laboratory
and facilities. ‘A special thank you is in order for the
former students, now graduate veterinarians, who performed
many of the routine procedures for the author, and who cared
for the experimental animals: Dr. Theodore Schaub, Dr.
James Resume, and Dr. Ray Jacobs. The efficiency of Miss
Susan Richards in the laboratory was extremely helpful and
greatly appreciated. Many thanks to the Department of
Surgery and Medicine with whose time and facilities most
of this study was conducted, and to the departmental
secretary, Mrs. Betty Jenkins, for*her assistance.

This study was supported by grant no. Eh2718 from
the National Institutes of Health.

111

TABLE OF CONTENTS

INTRODUCTION 0 O O O O O O O O O O O O O O O O

REVIEWOFALITERATURE

CHAPTER.

I.
II.

A.

B.

C.

D.
III.

Bartonella bacilliformis . . . . . . . . .

Haemobartonella muris . . . . . . . . . .

HaemObartgnellé Canie e e e e e e e e e e
Haemobartonella £9113 e e e e e e e e e e

MATERIALS AND METHODS . . . . . . . . . . . . .

A.

B.

C.
D.
E.

F.

G.

H.

I.

J.

Experimental animals a e e e e e e e e e e

Preparation and examination of blood

smears e e e e e e e e e e e e e e e e e e
Haemobartonella grading system . . . . . .
Anti-Haemobartonella drugs . . . . . . . .

Transmission of Haemobartonella canis
t0 the cat e e e e e e e e e e e e e e e e

Transmission of Haemobartonella :elis
t0 the dog e e e e e e e e e e e e e e e e

Maternal transmission of Haemobartonella
Canis to puppies e e e e e e e e e e e e e

Mosquito transmission of Haemobartonella

re 1 e e e e e e e e e e e e e e e e e e

Transmission of Haemobartonella felis
t0 the rat a e e e e e e e e e e e e e e e

Treatment of Haemobartonella muris
infection in rats with oxophenarsine . . .

Determination of the MLD of
oxophenar31ne for rate a e e e e e e e e e

iv

PAGE

d

U1 «P k'

15
15

16
17
17

18

19

19

2O

21

21

23

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . .

REVIEW or LITERATURE . . . . . . . . . . . . .

CHAPTER.

I.
II.

A.

B.

C.

D.
III.

Bartonella bacilliformis . . . . . . . . .

Haemobartonella muris . . . . . . . . . .
Haemobartonella canis
Haemobartonella fe;18 e e e e e e e e e e

MATERIMJS AND MTHODS O O O C O O O C O O C O O

A.

B.

C.
D.
E.

F.

G.

H.

I.

K.

Experimental animals . . . . . . . . . . .

Preparation and examination of blood

smears e e e e e e e e e e e e e e e e e e
Haemobartonella grading system . . . . . .
Anti-Haemobartonella drugs . . . . . . . .

Transmission of Haemobartonella canis
to the cat e e e e e e e e e e e e e e e e

Transmission of Haemobartonella felis
to the dog e e e e e e e e e e e e e e e e

Maternal transmission of Haemobartonella
Canis to puppies e e e e e e e e e e e e e

Mosquito transmission of Haemobartonella

gellg e e e e e e e e e e e e e e e e e e

Transmission of Haemobartonella falls
to the rat e e e e e e e e e e e e e e e e

Treatment of Haemobartonella muris
infection in rats with oxophenarsine . . .

Determination of the MLD of
oxophenarsine for rate . . . . . . . . . .

iv

16
17
17

18

19

19

2O

21

21

23

CHAPTER

IV.

L.

M.

Growth of g, canis and g. falls in
in embryonated chicken eggs . . . . . .

Haemobartonella infection in dogs and
cats treated with arsinobenzamide . . .

RESULTS 0 O O O O O O O O O O O O O O O O O O

A.

B.

C.

D.

E.

F.

G.

I.

Transmission of Haemobartonella canis
to the cat e e e e e e e e e e e e e e e

 

Transmission of Haemobartonella felis
t0 the dos e e e e e e e e e e e e e e e

Maternal transmission of Haemobartonella

canis to puppies . . . . . . . . . . . .

Mosquito transmission of Haemobartonella
fella e e e e e e e e e e e e e e e e 0

Transmission of Haemobartonella gelis to
the rat e e e e e e e e e e e e e e e e

 

Treatment of Haemobartonella muris
infection in rats with 0x0phenarsine . .

Determination of the MLD of
oxophenarsine for rats . . . . . . . . .

Growth of g, canis and g, gelis in
embryonated chicken eggs . . . . . . . .

1. Inoculation of the CA.membrane with
E. canis-positive dog blood . . . .

2. Inoculation of the CA membrane with
E. fella-positive cat blood . . . .

3. Inoculation of the allantoio sac
with g, felis-positive cat blood .

4. Inoculation of the yolk sac with
H. fella-positive cat blood . . . .

Haemobartonella infection in dogs and
cats treated with arsinobenzamide . . .

PAG

23

25
26

26

26

26

27

27

27

28

28

28

29

32

CHAPTER

v. CASE HISTORIES or CLINICAL FELINE
HAEMOBARTONELLOSIS . . . . . . .

A. Case 38623 . . . . . . . . .

B. Case 389A1 . . . . . . . . .

C. Case 40911 . . . . . . . . .

VI. DISCUSSION . . . . . . . . . . .
VII. CONCLUSIONS . . . . . . . . . . .
LIST OF REFERENCES . . . . . . . . . . .

v1

PAGE

42
43
46
49

58
60

LIST OF CHARTS

TABLE PAGE

I. Transmission of Haemobartonella canis to
the cat e e e e e e e e e e e e e e e e e e e e 33

II. Transmission of Haemobartonella falls to
the dog e e e e e e e e e e e e e e e e e e e e 34

III. Treatment of Haemobartonella muris infection
in rats With oxophenarsine e e e e e e e e e e 35

IV. Determination of the MLD of oxophenarsine
for rats e e e e e e e e e e e e e e e e e e e 36

V. Treatment of Haemobartonella infection in
dogs with arsinobenzamide-3 day schedule . . . 39

VI. Treatment of Haemobartonella infection in
dogs with arsinobenzamide-1O day schedule . . . 40

VII. Treatment of Haemobartonella felis infection
in cats with arsinozbenzamide-3 day schedule . 41

vii

CHAPTER I
INTRODUCTION

The genus Haemobartonella1 has been classified most

recently as follows:2 gage; Rickettsiales, Fgmily Barton-
ellaceae.3

Haemgbantonella is extremely polymorphic. Coccoid
forms range from 0.2 to 0.5 microns (u). Rod and violin bow
shaped types range from 0.2 to 1.5 u in width by 1 to over
6 u in length. They stain bluish-red with Giemsa and Wright
stains.

In severe infections, Haemobartonella parasitize 90
per cent (fl) or more of the erythrocytes of the affected
animal. Their positioniis epierythrocytic. They rarely
produce disease without prior splenectomy, except in the
cat. They are not easily cultured on artificial media.
Arthropod transmission has been proved for some species.
They are sensitive to organic arsenical therapy.

When splenectomy is performed on a dog which is a

latent carrier of flaemgbgrtogella anis, a marked hemolytic

 

1Tyzzer and Weinman, 1939.

2Robert S. Breed, E. G. D. Murray, and Nathan R.

Smith, gs pg eye sManual of Determinative Bacteriolo , 7th
Edition, 1957.

3Greszoykiewicz, 1939. Revised by Weinman, David,
Department of Microbiology, Yale University, September,
1955.

pr
31
be

be
be

01
3:

5a

anemia develops. Thus, canine haemobartonellosis may become
a,problem to the veterinarian who finds it necessary to re-
move a pathological spleen.

Pbline infectious anemia, caused by ggemgbartonella
Iglig, is a disease better known by veterinarians. In most
cases it presents a clinical picture of a chronic hemolytic
anemia. The acute form of the disease is rarely recOgnised.

The possibility of the dog's being a source of £2352,
hggtgngll§,infection for the cat, and of the cat for the dog,
presented an interesting problem. If transmission from one
species to the other were possible, several questions could

be raised. Are separate species designations, i.e., a. gangs

and g. mvalidt Might exchange of W
between the dog and cat (fighting, blood-sucking arthropods)
be a.mode of natural transmission of the disease?

In an infectious disease, there exists the possibility
of transfer of the etiological agent from mother to off-
spring. The maternal transmission of g, ggnig was investi-
gated in this study, and its role in the perpetuation of the
disease will be discussed later.

Arthropods hare been found to be the vectors of some
species of W. Since the mosquito, 2211i
pipiggg, is common in this area, it was investigated as a
possible vector of 5. 121-1.!-

Unsuccessful attempts have been made to transmit g.

$2.23!. and :1. 121.1,; to laboratory rats. In this study, it
was intended to infect rats with g. fglig, During the
preparation of the rats for injection of feline blood by
splenectomy and examination of blood smears, fl, gurgg, a
fairly common parasite of rats was encountered. This led
to treatment of the flaemObartonellapinduced anemia in rats,
and the determination of the minimum lethal dose (MLD) of
the arsenical used as treatment in this species.

Cultivation of fiartonella MW and W
W 29.11! in incubating chick embryos has been reported.
An attempt was made to cultivate a. 239.11 and g. M in
a similar manner.

Since the veterhnarian‘has few drugs with which to
combat canine and feline haemobartonellosis successfully,

the efficacy of arsinobenmamideI

against these organisms was
investigated.

Finally, 3 case reports of clinical feline haemo-
bartonellosis are presented. hey shed further light on
the natural transmission of the disease and treatment of

advanced feline infectious anemia.

 

1F‘ilcide, Pitman-Moore Company, Indianapolis, Indiana.

CHAPTER II
REVIEW OF LITERATURE
A. Bartonella bacilliformis, Strong et al (1913)

Biffi (1903) and Gastiaburu (1903) reported red cell
granulations in the peripheral blood of a patient suffering
from Carrion's disease (Oroya fever). In 1905 and 1909,
Barton described the bartonella bodies and stated that they
were the etiology of Oroya fever. This was confirmed in

1915 by Strong et a1, and the name Bartogella'b l f i
was proposed. Noguchi and Battistini (1926) obtained growth

of Bagtonella bacilliformis from citrated blood which had
been refrigerated up to 28 days when placed in artificial
media. When the cultures were injected into rhesus monkeys,
skin lesions and bartonellemia were produced. Pinkerton and
weinman (1937) obtained abundant growth in tissue culture
utilizing rat and guinea pig lung, bone marrow, spleen, and
tunioa vsginalis. Jiminez and Buddingh (1940) inoculated 8
to 12 day chick embryos via the chorio-allantoic (CA)
membrane, allantoio sac, amniotic sac, and yolk sac. Growth
occurred at 37° centigrade (C), but better results were ob-
tained at 25 to 28° C, although embryo mortality was higher.
Organisms appeared in the yolk at 48 to 72 hours. Amniotic
fluid remained negative. Better growth on the CA.membrane

resulted from scarification than when it was left intact.

5

Peters and Wigand (1955) considered Bartonella W

to be closely related to the bacteria because of size, form,
growth in cultures, binary fission, unipolar flagella, cell
walls, and behavior in serologioal tests. Griesemer (1958)
noted that W is easily cultured on cell-freemedia,

whereas, nagmgbgrtogellg is not.

B. Haemgpggtonella muril, Mayer (1921), Tyzzer and Heinman
(1939). ‘

Marked anemia in certain rats following splenectomy
has been observed frequently. Holferth (1917) noted that
those rats with splenomegaly developed anemia following
splenectomy; those with normal spleens were unaffected.
Streuli (1918), Lepehne (1918), and Danoff (1919) noted
hemoglobinuria and deathwithin 10 days after splenectomy.

In 1920, Mayor and Zeiss observed a marked anemia
beginning several days after cure of Iznggogoma zhodesiggse
infection with Bayer 205. They observed bartonelliform
bodies on the red blood cells. In 1921, Mayor proposed the
name Baztggglla.ggzig. In 1925, Lauda suggested the name
"infectious anemia of the rat.” This followed his production
of anemia by injecting blood from anemic splenectomized rate
into splenectomized rats which had been normal for 30 days
after surgery. Mayer, Borchardt, and Kikuth (1926) repeated

Lauda's experiements and observed the bartonelliform bodies.

The name Bartonella murig was again proposed.

In 1928, Mayer, Cannon, and McClelland showed the rat
louse, Polzplax spinulosus, to be the vector. This was con-
firmed by Cannon and McClelland in 1926. Eliot (1936) showed
that adult lice removed from the host for several hours did
not transmit the disease, but nymphs did. He thought that
the stronger digestive fluids of the adult destroyed the
organisms. Crystal (1959) demonstrated.that ingestion of
crushed lice from an infected source, or the intraperitoneal
injection of their feces did not produce the disease in rate.
An infection was produced by crushing and rubbing lice into
scarified skin. Lice maintained for 24 hours without access
to an infective source did not transmit the disease; whereas,
lice fed 4 hours previously on an infected rat did transmit
the disease.

Ford and Eliot (1930) postulated that the "virus" re-
produces in the spleen, but becomes vegetative on the '
erythrocytes when the spleen is removed. McCluskie and
Niven (1934) produced a bartonellemia in rats by ligation of
the splenic blood vessels without removal of the spleen.

Tyszer and Weinman (1939) subdivided the genus
Bartonellg into the monotypic genus Bgrtgngllg, with the
species pgpilligggmig, and the genus figgmgbggtgggllg, type
species g. 2923.-

Peters and Wigsnd (1955) did not consider'gggggbggtggr
g;;§,murig to be bacterial because of lack of cellular

structure and resistance to culture. Electron microscopy
showed the position of the organism to be epierythrocytio.

Faulkner and Habermann (1957) reported the circulating
neutrophils and macrophages to contain Barionella.gggig. The
disease was produced in splenectomized or non-splenectomized
white mice when injected with lymph node suspensions from
infected rats.

Infectiveness of the blood was lost after 24 hours at
room temperature or 37° C (lord and Eliot, 1928). Reitani
(1929) reported survival of the organisms for 15 days at
4° C in citrated blood. The incubation period of the disease
was lengthened after 24 hours at 20° C (Alstead, 1938).
Kessler (1942) found no lowering of the death rate when in-
fective defibrinated rat blood was maintained at -7o° C for
11 weeks.

Cultivation on artificial media has been attempted
many times. Sshilling and San Martin (1928) reported
colonies of coccoid organisms on blood agar. Inoculation of
normal and splenectomized rats produced death.“ Marmorstonp
Gottesman and Perla (1932) had growth in 48 hours in semi-
solid serum agar. Mayer, Borchsrdt and Kikuth (1926) found
microsc0pic growth in 5 to 10 days at 306 or 37° C on horse
blood glucose agar. Rat inoculations were negative.
McCluskie and Niven (1934) were unsuccessful in culturing
the organism. Lawkowicz (1938).observed growth on semi-
solid rabbit serum agar'between the sixth and twelfth days.

Injection into bartonellapfree rats produced anemia. Wein-
man (1944) felt that no report of cultivation was completely
satisfactory. He noted that, in pathogenicity tests, infor-
mation excluding the possibility of survival of organisms
from the original inoculum was not offered. Ford and Murray
(1959) stated they had offered such proof in cultivating

fl. mugig on Gisma's medium. Growth was seen in 72 hours at
28° C. The culture was infective for splenectomized rats
carrying Flexner-Jobling tumors and fed a 12% casein diet,
and for rats receiving 4 milligrams (mg) of cortisone per
kilogram (kg) body weight daily.

vogelsang and Gallo (1939) infected 9 to 12 day old
_chick embryos. By the sixth day, all the parasitised eggs
were dead, all without parasites living. Splenectomized,
arsenic-treated rats showed bartonellae in the blood and
died 5 to 8 days after injection with egg material, while
control, arsenic-treated, splenectomized rats remained
normal.

Mayer, Borchardt, and Kikuth (1927) obtained
”sterilization" of the infection with organic arsenicals.
This has since been confirmed by many workers. Penicillin,
chlortetracycline, and oxytetrseycline were reported effective
'by Griesemer (1958). That oxytetracycline did not alter
<1estruction of the erythrocytes was shown by Rudnick and
Hollingsworth (1959). Parasitized erythrocytes had a life
span of 10 to 12 days compared to the 25 days of

non-parasitized rat erythrocytes.

c. H me to e ganis, Kikuth (1928), Tyzzer and weinman
(1939). ' V "

Kikuth (1928) performed a splenectomy on a dog and 6
.days later noted bartonellaplike structures in blood films.
me name W m was proposed. Rhoades and Miller
.(1935) observed bartonella bodies in splenectomized dogs fed
a‘black-tongue producing (niacin deficient) diet and a
normal diet. The organisms could not be transmitted to non-
splenectomized dogs regardless of diet.

Monaught, Heads, and Scott (1935) depleted the plasma
proteins of dogs by bleeding daily and returning the washed
erythrocytes. Icterus developed and‘flagggngllg‘were found
"in" the red blood cells. Blood transfusions and treatment
with 15 mg of neoarsphenamine per kilogram body weight pro-
duced recovery. Infective blood was injected into splenectom-
ized dogs. After bartonellemias developed they were treated
with neoarsphenamine.

Knutti and Hawkins (1935) transmitted the organism to
13 out of 4 splenectomized dogs by the intravenous injection
of infective blood. An incubation period of 2 to 5 days was
noted. No growth was obtained in Noguchi's leptospira medium

incubated at 30° C. Fifteen mg of neosalvarsan per kilogram

body weight proved curative.

10

Ray and Idnani (1940) found an incubation period of
19 days for dogs inoculated with g, gagig subcutaneously, and
6 to 12 days for dogs receiving intravenous inoculation.

Post mortem findings included emaciation, icterus, pale
emphysematous lungs, ecchymoses in the heart, hepatomegaly,
splenomegaly, and petechiae on the kidneys and intestine.

A spontaneous Haemobartonella infection was found in
a splenectomized dog by Carr and Essex (1944). The infection
was transmitted to 2 other splenectomized dogs.

Ingle (1946) reported a clinical case of canine
haemobartonellosis occurring in a non-splenectomized dog
which he treated with blood, iron, and brewer's yeast.

anb (1958) described a macrocytic, hypochromic
anemia resulting from g. m infection. A leucopenia of
a fluctuating type was also found. Whole citrated blood
stored at 6° to 8° 0 lost its infectivity in 3 days. Treat-
ment was effected by a single dose of 4.5 mg of oxophenarsine
per kilogram body weight intravenously. Spontaneous recovery
conferred immunity, but a dog could be reinfected if the
original infection was terminated by treatment.

Benjamin and Lamb (1959) reported on a case of splenic
llamangioma.in a dog which was treated by splenectomy. The
<dog was returned 2 months later because of polydipsia, anemia,
and listlessness. Organisms resembling LI. 233;; were seen

on examination of blood films. The blood was injected into

11

splenectomized dogs which then developed bartonellemias.
Incubation periods ranged from 15 to 20 days. A non-
splenectomized dog was also inoculated and remained normal
for 1 year, but was then splenectomized with a resultant
bartonellemia.

A case of haemobartonellosis in a non-splenectomized
dog was reported by Donovan and Loeb (1960). The disease
was transmitted to splenectomized puppies but not to a non-
splenectomized puppy by blood inoculation. Transmission did
not occur when splenectomized puppies were housed with in-
fected dogs.

Regendanz and Reichenow (1932) reported that ground
dog fleas were infective, but not the bite or feces of this
insect. The bites of adults or nymphs of ngggggggtg;
W. W W. and Mmo us
1922.91! were similarly non-infective. Serial passage of g.
gggig_in the cat was accomplished.

Rhoades and Miller (1935) were unsuccessful in trans-
mitting fig 359;; to mice, rats, rabbits, guinea pigs, and
puppies. Lamb (1958) found no transmission by contact, lies,
in utero, and through maternal milk. Ingestion of infective
blood produced the disease, and it was postulated that
natural transmission may occur through vaginal secretions,

dog fights, and unclean medical and surgical procedures.

12

D. Winema-

An ”Epcrythggzogg" infection of the cat was first
described by Clark (1942) in which practically all of the
parasites were ring formed and 0.5 to 1.0 u diameter.

Flint and Mess (1953) found ”Anaplasma- ke" bodies
on the erythrocytes of a cat treated for an abscess. The
organisms were transmitted to other cats by the intra-
peritoneal injection of 0.5 milliliters (ml) of blood.
Frozen citrated blood remained infective for 2 weeks.
Splenectomy was not necessary for the infection to manifest
itself. A donor cat which had never shown a positive blood
smear was the source of infection for several recipient
cats.

Flint, McKelvie, and Douglas (1955) observed flhat
most naturally occurring cases of infectious anemia in cats
were in the 1 to 3-year age group. They stated that absence
of organisms on blood smear should not be construed as a
negative diagnosis if symptoms are present. Treatments sug-
gested were: oxophenarsine, 4 mg every fourth day for 4
doses; chloramphenicol, oxytetracycline, or tetracycline,
100 mg twice daily for 18 to 21 days.

Splitter, Castro, and Kanawyer (1956) found the incu-
bation period after intraperitoneal injection of infective
blood into cats to be 3 to 20 days, with an average of 7
7 days. Cultivation attempts on artificial media were

13

unsuccessful. g. Leg}; was non-filterable. Urine collected
during the acute stages of the disease was not infective.
Contact transmission could not be effected. Experimental
transmission to splenectomized rats, mice, swine, cattle,
sheep, and dogs was not accomplished. It was suggested that
biting arthrOpods were the vectors of the disease.

Flint, Rbepke, and Jensen (1958) reported on 30 cases
in cats, 28 occurring in males. They observed that some
rmotor favors deve10pment of the disease shortly after sexual
maturity, possibly fighting. The organism may lie dormant
until a stress situation occurs. Blood transfusion, oxo-
Phenarsine, tetracycline, and chlorsmphenicol were suggested
as treatments.

Schwartzman and Beach (1958) described the bone
narrow of a cat with infectious anemia as containing numerous
¢Dorythrozoon bodies engulfed by macrOphages. his liver was
Yellow and friable. The spleen was 2 to 3 times normal
312s. Flint ( 1959) listed splenomegaly, enlarged lymph
nOdes, severe‘anemia, and icterus (in 50% of the cases) as
‘0 rtem findings.

Flint, Roepke, and Jensen (1959) infected 56 of 76
°lts by injection or oral administration of infective blood.
Transmission by the intraperitoneal injection of splenectomized
dose, white rats, white mice, and brown deer mice with

1directive blood was not accomplished. Brown deer mice could

14

not be infected orally. Contact transmission between cats
did not occur. g, ;g;i§,could be found in the plasma follow-
ing rupture of the infected erythrocytes. Fleas and
mosquitoes were discounted as vectors because of their

scarcity in the Salt Lake City area.

CHAPTER III

(MATERIALS AND METHODS

A. Egggrymental Animals

Experimental dogs were mongrels procured from the dog
pound. They were vaccinated against canine distemper and
infectious canine hepatitis, kenneled individually, and
maintained on a diet of prepared dog food, Fromm Meal.1

Experimental cats were procured either from farms or
private homes. They were vaccinated against feline infectious
enteritis. The cats were kenneled either singly, or in twos
or threes, depending on available space. A.non-infected
animal was never housed with an infected one. Their diet
consisted of prepared cat food, C/D.2

Albino and hooded rats (fiattus nozgegicug) were pro-
cured from a colony maintained by the Department of Micro-
biology and Public Health, Michigan State University. The
rats were caged in groups, depending on whether they were
infected or non-infected. Their diet consisted of Rockland
Rat Diet (Complete) .3

No experimental animal was considered non-infected,

'Federal Foods, Inc., Thiensville, Wisconsin.
2Hill Packing Company, Topeka, Kansas.
3A. E. Staley,Manufacturing Company, Decatur, Illinois.

16

or used for transmission studies, until a minimum of 14 suc-

cessive daily Giemsarstalned blood smears were found to be

negative for haemobartonella. This period followed splenectomy

in the case of dogs and rats. Cats were not splenectomized.
Blood was used only on days when blood films were

positive.

B. Preparation and Examination 2;,Blogg Smears

Blood smears were prepared from each animal daily.
Dogs were bled by piercing the 1abial.mucosa with a lancet.
Cats were bled by piercing the anterior margin of the ear.
Rats were bled with a lancet from the tip of the tail.
Smears were allowed to air dry. The following staining
procedure was then employed:

Methyl alcohol (absolute) ..... 3 minutes

Giesma stain1 ..... 1 hour

Rinse in tap water

Air dry
The stained smears were examined microscopically under 011

immersion using a 100x objective and 10x ocular.

 

1Preparation of Giemsa's stain:

10 ml Giemsa (Fisher Scientific Company, Fair Lawn,
N. J.
5 ml 1/15 molar NaH P04
5 ml 1/15 molar Na2 04
150 ml distilled water

17

G. .flasmahamianallamia.fizaaiaawazaiaa

A grading system was employed to designate the
relative number of Haemobartonella on blood smear.

1 plus ..... less than 1 organism per field.

2 plus ..... 1 to 10 organisms per field.

3 plus ..... 11 to 25 organisms per field.

4 plus ..... 26 or more organisms per field.

D. AntirHaemgbartonella Drugs

Filcide1 is a parenteral solution intended for use in

Diggjilgrig;immitis infections in dogs. Each m1 contains:

Sodium p-bis (Carboxymethylmercapto)
Arsinobenzamide .......... 1% fi/V

(contains arsenic, 1.8 mg)
Water for injection U.S.P.
Benzyl alcohol (Preservative) .......... 0.9%

Hapharsen2 is distributed in ampoules as a powder and
is prepared for use by dissolving in sterile distilled water
or saline.3 It is an effective spirocheticide for treatment

 

1Pitman-Moore Division of Allied Laboratories,
Indianapolis, Indiana, Brand of Arsinobenzamide.

2Parke1- Davis and Company, Detroit, Michigan, Brand
of Oxophenarsine Hydrochloride. .

3Sterile 0.85% sodium chloride solution used as
diluent in this work.

18

of syphilis. Mapharsen (3-Amino-4-Hydroxyphenyl-arsineoxide
Hydrochloride) is supplied as a mixture with sodium carbonate
(anhydrous), sucrose, and ascorbic acid which render the
solution compatible with the blood. It contains 31% tri-

valent arsenic.

E. Transmission 2£_Haemobartonella Canis 32 the Ca

 

 

One ml of heparinized blood from a dog (30) positive
for g, canis was injected intraperitoneally into each of

4 cats (91, 92, 93, 94; see Chart I). These cats had
been proved negative for'fl. felis by 30 to 42 consecutive

days of negative blood smears. On the third, sixth, tenth,
and fourteenth days, blood from the cats was drawn into
heparinized syringes and pooled. One ml of the pooled blood

was injected intraperitoneally into a negative cat, and

intravenously into a negative splenectomized dog (19W).

This dog (19W), after 4 days, became positive and her blood
was inoculated into 2 negative cats (63, 69). When this
same bitch (19W) subsequently whelped, possible infection of
the puppies was investigated by inoculating their blood into
a negative splenectomized dog. All animals remaining
negative were challenged with blood of a known positive

animal of the same species to prove susceptibility.

19

1". W 2; Haemobartonella Felig m the Dog

One ml of heparinized blood from a cat (99) positive
for'fl, fglig,was injected intravenously into each of 4
splenectomized dogs (5, 12, 13, and 953; see Chart II).
These dogs had been proved negative for‘g.‘ga§;g,by 14 con-
secutive days of negative blood smears. On the third, tenth,
and seventeenth days, blood from the dogs was drawn into
heparinized syringes and pooled. One ml of the pooled blood
was injected intraperitoneally into a negative cat, and
intravenously into a negative splenectomized dog. The dogs
developed positive blood smears and their blood was injected
intraperitoneally into negative cats. All animals remaining
negative were challenged with blood of a known positive

animal of the same species to prove susceptibility.

G. Mategng; Transmission 2;,Haemobartonella Canis tg'Puppies

Blood was collected from 4 cats which had been previ-
ously inoculated with blood from a Haemgbartgnellarpositive
dog (30). One ml of pooled cat blood was injected intra-
venously into a gaemobartonellapnegative, splenectomized,
pregnant bitch (19W). The bitch showed blood films positive
for Haemgbartonella 4 days later. One day after whelping,
the puppies were sacrificed and their pooled blood was in-
jected into a Haemobartonellapnegative, splenectomized dog (9).

A flaemgbgtogglla-positive bitch (34) had her puppies

20

delivered by Caesarean section. After 1 day, the puppies
were sacrificed and their pooled blood was injected into a

Hggmobgztonella-negative, splenectomized dog (5).

In both cases, the puppies were allowed to nurse.

H. Mgsguito Transmission 2;,Haemobartonella Felis

An attempt was made to investigate the role of
mosquitoes in the natural transmission of g, iglig, A
mosquito-proof area was constructed in a preeexisting room.
The floor of the room was approximately 6 feet by 6 feet and
composed of terrazzo; the plaster walls were about 10 feet
high. The front wall was constructed of fine mesh wire
screening and a door. A 3-foot space was allowed between
this door and a second door leading to an outside room.

Fbur cages were placed inside the mosquito-proof room. A
figgngngztgggllarpositive cat was placed in each of 2 cages
(36 and 97) and a‘flggmgbggtgggllarnegative cat was placed in
each of the remaining 2 cages (25 and 37). The cats were
clipped over a majority of their body surfaces to allow the
mosquitoes easy access. Pond water was obtained from a
wooded area.heavily infested with gg;25_pipigg§,mosquitoes
and placed in the room in an attempt to hatch mosquitoes.
Plant material was placed in the room as a shelter for the
mosquitoes, and a sugar solution was supplied for supple-

mental feeding. When no mosquitoes emerged from the pond

21

water, adult mosquitoes were captured and released in the
room. When it was found that the mosquitoes could not be
maintained alive for longer than a 3-day period, additional
mosquitoes were captured and released into the room at

frequent intervals.

I. Transmission of Haemobartonella Falls to the Rat

Three days after being treated with 0x0phenarsine,
rats 51, 52, 53, 54, 55, 56, 57, 58, 59, 62, and 63 were
splenectomized.

Giemsanstained blood films from each rat were examined
daily for the presence of Haemobartonella.§g;i§. After 13
days of negative findings, rats 51, 55. 55. 57. 59. and 63
were inoculated intraperitoneally with 0.5 ml of blood from
a cat (29) infected with g, iglig, Eats 52, 54, 56, 58, and
60 served as uninoculated controls. After 48 days, all rats
were challenged with blood drawn from a rat originating in a
colony from which E, gggigrinfected rats had previously been
obtained. The rat was not exhibiting a haemobartonellemia

at the time of bleeding.

J. Treatment 9;,Haemobartonella Muris Infection ig,§ats

With Oxophenarsine

A series of hooded rats (Rattus norvegicus), approxi-

mately 6 months old was prepared for transmission studies

22

of 3. £21.11: by splenectomizing half of them. Giemsa-stained

blood films were examined daily for the presence of blood
parasites which might be confused with.§h Iglig. Rats 76,
.78, 79, 80, 83, and 85 were splenectomized. Rats 82, 84,
86, 87, 88, and 89 served as non-splenectomized controls
(see Chart III).

General anesthesia was induced in the rats with ponto-
barbital sodium.‘ The commercial preparation contains 75 mg
of the anesthetic agent per ml (6.5%). This was diluted
with sterile 0.85% sodium chloride solution to make a 3%
solution. Rats were weighed and injected intraperitoneally
with 30 mg/kg body weight of the pentobarbital solution
through a 27-gauge hypodermic needle attached to a 0.5 ml
tuberculin syringe. Surgical anesthesia was attained in
approximately 5 minutes.

Splenectomy was performed on the rats under aseptic
conditions, except that the same drapes and instruments were
used on each rat, and the Operator did not change gloves.
The splenic vessels were ligated with size A.nylon. Usually
only a single ligature was necessary. The abdomen and skin
were closed in 2 layers, again using size A nylon. It was
subsequently found that the rats chewed out the dermal

sutures, causing a dehiscence of the skin incision. All

 

1Halatal, Jensen-Salsbury Laboratories, Inc., Kansas
City, Missouri.

23

skin closures were then completed with Michel wound clips
which were not removed.

Several days after splenectomy, ring-shaped bodies
were observed on the erythrocytes of the splenectomized rats.
A.macrocytic, hypochromic anemia develOped, with anisocytosis,
poikilocytosis, and polychromasia. The affected rats were
treated with 2 mg/kg body weight of oxophenarsine intra-
peritoneally.

K. e at n 2;_the MLD 91_Oxophena§§ine 32g,fiats

A group of rats (§§§32§,ngrvegicu§) was selected for

use in determining the MLD of oxophenarsine for this species
(see Chart IV). weights ranged from 116 to 524 grams.
Oxophenarsine was diluted to 4 mg/Ml and was injected intra-
peritoneally through a 27-gauge needle attached to a tuber-
culin syringe.

L. Ggwth g; g. Cgig and g. M in Embnongteg Chicken

E

A sterile 10-ml syringe was prepared by aspirating a
sufficient volume of heparin solution‘ to wet the barrel
and ejecting the excess. Ten m1 of blood were drawn from a

known £22m22££1222llérP°81t1V6 dog or cat and transferred to

 

'Heparin Sodium, 10 mg/ml, Upjohn Company, Kalamazoo,
Michigan.

24

a sterile, rubber-diaphragm, steppered vial, to be used as
inoculum. Sterility of the inoculum was tested by inocu-
lating semi-solid medium.1 Viable 8 to 12 day-old embry~
onated chicken eggs were selected. The sites of inoculation
were marked and painted with nitromersol tincture2 or
polyvinylpyrrolidine (povidone).3 These sites were first
opened by drilling with an abrasive disc, later by means of
an egg punch. Sites inoculated were the chorio-allantoic
(CA) membrane, allantoio sac, and yolk sac. Injection was
accomplished through a 27-gauge needle. A 0.2-ml dose of
inoculum was used. After inoculation the eggs were sealed
with paraffin and incubated at a temperature of 37 to 38° C
until death of the embryo occurred or 96 to 140 hours had
transpired.

Fbllowing incubation, the entire contents of the eggs
were removed aseptically from their shells directly into a
Waring blender and ground until the resulting mixture could
be aspirated through a 20-gauge needle. Eggs in which early

death of the embryo occurred were refrigerated at approxi-

mately 4° C until harvested. Maximum refrigeration time was

 

1Bacto Brain Heart Infusion plus Bacto Agar 0.15%,
Difco Laboratories, Detroit, Michigan.

2Metaphen Tincture 1:200, Abbott Laboratories, North
Chicago, Illinois.

3Betadine 1% available iodine. Talby-Nason Company,
Inc., Dover, Delaware.

25

3 days. Five-tenths to 1 ml of macerated embryos was inocu-
lated intraperitoneally into Haemobartonellapnegative animals.

Giemsa-stained blood smears were examined daily for

flaggghgrtgggllg. When they remained negative, the test

animals were challenged by inoculation with blood from a
Haemobartonella carrier of the same species to prove
susceptibility.

 

 

M. Haemgbagtgnella Infection in Dogs and Cats Trggted with
Arginobenzamide I

Nine dogs (9, 13, 953, 193, 954, 952. 957. 18, and
19W) exhibiting blood films positive for fiaegobgrtgnella
were selected (see Charts V, VI). Their weights and source
of infection were recorded, and they were divided into 2
groups. Arsinobenzamide was administered intravenously at
various dosages on 3 and 10-day schedules. A.Haemo§§:tonglla-
positive dog (30) served as the untreated control.

Eight cats (5, 11, 12, 49, 62, 68, 71, and 74) were
inoculated intraperitoneally with approximately 1 ml of blood
from a cat (99) positive for 591121;! (see Chart VII). When
a haemobartonellemia occurred, the weight of each cat was
recorded and intravenous treatment with arsenobenzamide was
commenced. Cats 5, 11, 12, 49, 71, and 74 were treated with
various doses on a 10-day schedule. Cats 62 and 68 served

as untreated controls.

CHAPTER IV
RESULTS

A. Transmission of Haemobartonella Canis to the Cat

Chart I is an outline of the experimental procedure.
Cats did not develop positive blood smears after being in-
jected with g, oasis-positive dog blood. The pooled blood

 

of these cats was infective for dogs but not for cats. The
blood of puppies from a bitch which whelped subsequent to
being infected with the cat blood was infective for a

negative, splenectomized dog.

13. wuwmmmm

Chart II is an outline of the experimental procedure.
One of 4 dogs injected with 3;. I21“. positive cat blood
developed a.haemobartone11emia after a 28-day incubation
period. The pooled blood of these dogs was infective for a
cat on the third post inoculation day and for a dog on the
seventeenth post inoculation day. All other attempts to
infect dogs and cats with the pooled blood yielded negative

results.

0. Maternal Trgnsmiggion 2f,Haemobartonella Canis Lg Puppies

A negative, splenectomized dog (9) became positive 5

27

days after injection of pooled blood from puppies of a
positive bitch (19W).

A negative, splenectomized dog (5) became positive 13
days after injection of pooled blood from puppies of a
positive bitch, and died 4 days later.

D. Mgsguito Transmission 2;_Haemobartonella‘ggli§_

Neither susceptible cat (25, 37) exhibited a blood
smear positive for E, fglig. After a period of 45 days, the
negative cats were proved susceptible by challenge with
infective blood.

Since the mosquitoes could not be kept alive for an

extended period of time, this experiment was abandoned.

E. Transmission 2;,Haemobartonella Felis 32,the‘§§t

No positive blood films were demonstrated in any rat,
experimental or control. No blood dyscrasias evidenced by

anisocytosis, polychromasia, or poikilocytosis were observed.

F- Waiwemoba toe awn Wiggle.
mew

The onset, progress, and treatment of haemobarto-
nellosis in rats is recorded in Chart III.

Fbur out of 5 rats were successfully treated with 2
mg/kg body weight of oxophenarsine intraperitoneally. Only

1 out of 6 rats did not develop a haemobartonellemia

28

following splenectomy.

G. Qgtermination 2;,the MLD‘Q; Oxophenarsine for Rate

As recorded in Chart IV, rats injected intraperiton-
eally with up to 22 mg/kg body weight of oxophenarsine lived.
The lowest dosage at‘ which death occurred was 24 mg/kg.

Rats receiving a sublethal dose exhibited varying degrees
of ataxia, salivation, dyspnea, and prostration, from which
they eventually recovered. Rats receiving a lethal dose
died in a minimum of 4 hours, and a maximum of 8 days. One

rat receiving 26 mg/kg lived, as did 1 receiving 30 mg/kg.

H. Grgwth 2;,Haemobartonella ngis and g, Felis‘in
WWW

1. Inoculation g; the _C_A_ Membrane with g. 93;};
Positive Dog B od

a. Examination of several eggs 24 hours after inocu-
lation revealed edematous CA.membranes with some
grayish areas 0.5 cm diameter; livers of the
embryos were hemorrhagic. Blood, liver, and CA

membrane smears were negative for Haemobartonella.

b. Of 13 eggs inoculated, 5 embryos were dead at 24
hours, and 8 were alive at 96 hours. A
suspension was made of the contents of the live

eggs, and a susceptible dog inoculated with it.

0e

29

It remained negative 33 days, was challenged and

became positive in 8 days.

Six eggs served as uninoculated controls. The
CA membranes were dropped and the eggs sealed.
Two embryos were dead at 24 hours, 1 at 48 hours,
1 at 72 hours, and 2 were alive at 96 hours. A
suspension was made of all the control eggs and
a susceptible dog inoculated with it. It re-
mained negative 33 days, was challenged and be-

came positive in 8 days.

The blood vessels on the CA membranes of the
inoculated eggs appeared cyanotic while those

of the controls appeared normal in color.

 

 

 

2. Inoculation 9; the 9A Membrane with g. Felis
Zogitive Cat Biggg. _

8.

0f 13 eggs inoculated, 11 embryos were dead at
48 hours, and 2 were alive at 72 hours. A cat
inoculated with a suspension made from the con-
tents of all the inoculated eggs remained
negative 25 days, was challenged and became

positive in 9 days.

The CA membranes of 13 embryonated chicken eggs

were inoculated with the same embryo suspension

30

(2a). Three embryos were dead at 24 hours, 5 at
48 hours, 1 at 72 hours, and 4 were alive at 96

hours. A cat inoculated with a suspension made

from the contents of all the inoculated eggs re-
mained negative 21 days, was challenged and

became positive in 8 days.

c. Five eggs served as uninoculated controls. The
CA membranes were dropped and the eggs sealed.
One embryo was dead at 48 hours and 4 were alive
at 72 hours. A cat inoculated with a suspension
made from the control egg contents remained
negative 25 days, was challenged and became

positive in 2 days.

d. The CA membranes of 3 embryonated chicken eggs
were inoculated with the same embryo suspension
(2c). All 3 embryos were dead in 24 hours, and
bacterial growth was obtained in semi-solid
medium. A cat inoculated with a suspension made
from the egg contents remained negative 21 days,
was challenged and became positive in 9 days.

3. inocuiation gi,the iiiggigig Sac with g, Fails-
Pogiiigg Cat Biogd

 

a. 0f 11 eggs inoculated, 2 embryos were dead at 92 .

hours, 1 at 116 hours, and 8 were alive at 140

4.

31

hours. A suspension was made of the contents of
the eggs in which embryos were dead at 92 hours,
and 2 cats were inoculated. One remained
negative 54 days, was challenged and became
positive in 12 days. The other remained negative
114 days, was challenged and became positive in

9 days.

Of 5 eggs similarly inoculated with 0.1 ml of
sterile 0.85% sodium chloride solution, 2 were

dead at 24 hours, and 3 were alive at 140 hours.

inoculation g; the Yolk Sac with g, Felis-Posiiive

Cai Biood
a. or 11 eggs inoculated, 3 embryos were dead at 48

b.

hours, 1 at 110 hours, and 7 were alive at 140
hours. A suspension was made from the contents
of the eggs in which embryos were dead at 48
hours, and 2 cats were inoculated. The first
remained negative 55 days, was challenged and
became positive in 12 days. The second remtined
negative 54 days, was challenged and became

positive in 2 days.

Of 4 eggs which were similarly inoculated with
0.1 m1 of sterile 0.85% sodium chloride solution,

2 embryos were dead at 48 hours, and 2 were

32

alive at 140 hours. A cat was inoculated with a
suspension made from the contents of the eggs in
which embryos were dead at 48 hours. It remained
negative 115 days, was challenged and became

positive in 15 days.

c. Giemsapstained blood smears taken from the

embryos which were dead at 48 hours were negative

for Haemgbgrionellg.

I. Haemobartonella Infection 1 Dogs ani Cats Treated with

Arsinobenzamide

 

 

Charts V, VI, and VII record the treatment of experi-
mentally induced haemobartonellosis.

A dosage of 0.20 ml/lb body weight daily was effective
in dogs when used on a 3-day schedule. A dosage of 0.10
ml/lb daily was effective in 1 dog when used on a 3-day
schedule, but not effective in a second dog when the same
dosage was administered on 10 successive days.

A dosage of 0.20 ml/lb daily was not effective in a

(cat when used on 10 successive days.

53
CHART I

TRANSMISSION OF HAEMOBARTONELLA CANIS TO THE CAT

Dog 30 (positive)

Negative cats

91..Negative 36 days; challenged and positive in 8 days.

92..Negative 36 days; challenged and positive in 6 days.
"93..Negative 36 days; challenged and positive in 2 days.

94..Negative 36 days; challenged and negative for 8 days;
rechallenged and positive in 6 days.

Negative cat 95...Negative 33 days; challenged and neg-
ative in 8 days; rechallenged and
positive in 3 days.

Negative dog 19B..Positive in 12 days.

Negative cat 96...Negative 30 days; challenged: suspicious
in 2 days; definitely positive in 18 days.

Negative dog 19w (pregnant)"2..Positive in 4 days.

Negative cat 67...Ne ative 49 days; challenged and neg-
at vs 14 days; rechallenged and remained

10th day negative.

 

Negative dog 18...Positive in 5 days.

Negative cat 31...Negative 30 days; challenged and
suspicious in 3 days; definitely positive
14th day in 9 days.

Negative dog 10...Positive in 23 days.

 

1Evidence of maternal transmission of gaemobggtoneilg infection
to the puppies is presented elsewhere in this paper.

Negative cat 69 was inoculated with positive blood from dog 19W.
One blood smear positive 63 days post inoculation was attributed to
mislabeling of slides. Smears were negative for the next 9 days. The
cat was challenged and remained negative 14 days; rechallenged and
positive in 3 days. '

Negative cat 63 was inoculated with positive blood from dag 19W;
remained negative 45 days. The cat was challenged and remained neg-
ative 14 days; rechallenged and positive in 9 days.

CHART II

34

TRANSMISSION OF HAEMOBARTONELLA FELIS TO THE DOG

Cat 59 (positive)

Negative dogs

5oooN°sat1ve 65 days; challenged and positive in 12 days;
died 4 days later.

12..Positive

Pooled
13..Negative
953.Negative
Negative cat

3rd day

Negative dog

Negative cat
10th day

Negative dog

Negative cat
17th day
Negative dog

 

1

in 28 days and died 38 days later.1

75 days; challenged and positive in 4 days.

75 days; challenged and positive in 4 days.

32...Positive in 11 days and died 13 days

later.

954W.Negative 75

positive
11...Negative
positive
9e e e e Negative
positive
55...Negative
positive

952..Positive

in 4

39
in

53
in

53
in

in

days; challenged and
dues

days; challenged and
5 days.

days; challenged and
5 days.

days; challenged and
12 days

19 days.2

Negative cat 64 was inoculated with blood from dog 12, 22 days

after the initial positive blood smear. Cat 64 remained negative.

2Negativecat 66 was inoculated with blood from dog 952, 5 days

after the initial positive blood smear.

Cat 66 remained negative.

35

.Uaodh hon mamamewao mm some one: ++++
.caeau eon manamsmae mm|—. +++
.caoau sen namamswao oat. ++

 

 

 

 

 

 

 

 

 

 

.caoau non amusemao . men» smog +
.haaeesOpaaoamaaea mamas: moon wx\ws N .omamaemonnoxo and: possess
.nanphooaaxdoa .nquphooudms .sdmeaoanohaom m
.eanesoanohaom a
.msaeaa ma soon uoasmsaw n
.ovaam mo meeheoanahmo 39h w
.ocdam so soon nophooanphao oz .
.aeoam pecan cemaspnlmmaoaw so soon «flammdmmmmmmmém oz 0
.conaaoaoesoanmimoz mz
.ponaaoaoocoaam m
o o o o o o o o o o o o mz .b— 9 mm
o o o o o o o o o o o o mz e—m 2 mm
o o o o o o o o o o o 0 m2 mm. a rm
0 o o o o o o o o o o 0 m2 R. .m mm
o o o o o o o o o o o 0 ma mm. a em
0 o o o o o o o o o o 0 m2 «pm x mm
o n n o .o o +++ ++++ +++ + o m mm. 2 mm
o o n mo 0 o ++++ ++++ ++++ +++ +++ m m—N x mm
o n n no 0 o + ++ +++ +++ ++++ o m onm 2 cm
0 m n o .o .0 ++ ++++ ++ ++++ ++++ o m mum x mm
o m mm. a 2.
o m ma. 2 we
. msoupm mmflaw New on
ma .ym new

 

 

 

 

 

 

 

 

MZHmm4zmmeNo mHHS madm 2H onBUHHZH mHmbxflddqflzoam4moxH<m ho Bzmxfi<mma
HHH ammmo

 

CHART IV
DETERMINATION OF THE MLD OF OXOPHENARSINE FOR RATS

 

Ear Weight Dose in

Sex in k m m As m As k Result
51 M .448 2 0.28 0.62 Lived
52 M .449 2 0.28 0.62 Lived
53 M .479 3 0.48 1.07 Lived
54 M .465 3 0.43 0.90 Lived
55 M .524 4 0.65 1.24 Lived
56 M .434 4 0.54 1.25 Lived
57 M .390 5 0.60 1.53 Lived
58 M .311 5 0.48 1.54 Lived
59 F .296 6 0.55 1.86 Lived
60 F .286 6 0.53 1.85 Lived
62 M .310 7 0.67 2.16 Lived
63 F .298 7 0.65 2.18 Lived
64 M .128 7 0.28 2.19 Lived
65 M .116 7 0.25 2.16 Lived
66 F .113 8 0.30 2.45 Lived
67 M .129 8 0.32 2.49 Lived
69 M .116 9 0.31 2.67 Lived
70 F .126 9 0.35 2.79 Lived
71 M .141 10 0.44 3.12 Lived
72 M .152 10 0.47 3.10 Lived
74 F .138 11 0.47 3.41 Lived
75 M .128 11 0.44 3.44 Lived

37

CHART IV (continued)

 

Ear Weight Dose in

t Sex in k m m As As Result

76 M .154 12 0.57 3.70 Lived

77 M .132 12 0.49 3.73 Lived

78 F .155 14 0.67 4.32 Lived

79 M .155 14 0.67 4.34 Lived

80 M .123 16 0.61 4.98 Lived

81 M .179 16 0.89 4.97 Lived

82 F .160 18 0.89 5.56 Lived

84 M .161 18 0.90 5.59 Lived

85 M .138 20 0.86 6.23 Lived

87 M .157 20 0.97 6.18 Lived

88 M .145 22 0.99 6.83 Lived

89 F .160 22 1.09 6.81 Lived

6 M .176 24 1.31 7.44 Died in 48 hours

31 F .125 24 0.93 7.44 Died in 24 hours
1 F* .330 24 2.46 7.45 Died in 5 hours
2 F‘ .370 24 2.75 7.43 Died in 4 hours

29 M .160 26 1.29 8.06 Lived

34 M .147 26 1.18 8.03 Died in 48 hours
3 F* .291 26 2.16 7.42 Died in 4 hours
4 F* .331 26 2.67 8.07 Died in 4 hours

47 .128 28 1.11 8.67 Died in 6 hours

43 M .208 28 1.81 8.70 Died in 6 hours

 

* Castrated

CHART IV (continued)

Ear Weight Dose in
ex in k m As
5 F* .304 28 1.64
6 F* .302 28 2.62
46 F .110 30 1.02
39 F .126 30 1.17

 

* Castrated

m As k
8.68
8.68
9.27
9.29

38

esu t

Died in 5 hours
Died in 6 hours
Died in 8 days
Lived

39

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swam on an: enema .aaoeean one: commune menace neon ernnmeaum ascended canooeoaonx
.epnvemem sew nopna can no men neon noemnem
.ununasenomsa .cnsan use new man: unennepmo use unannpmmw onwennnosem «mmonmoa macaw .m
.mdnannnno on

sonpeenmn «Adummmmmmomumm no son-ensmmsna Hsmneass no somecnpe ns hansonpeam ecumeuoam .—

 

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e menace canes-ca assessensaeenn o o o o a . .Hoasnoo 0
fine .m. n3:
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ace-mm e menace essence escapees him eném 44.9 3.0 an «a no e83 ceases me.

m.o>nannon wmnsooen asomnnm aqua
lemon» ansa ona nouns ease n pong mm._ ee.mm me.m on.o mm e on men nmm

.monnepnonno no conned

 

27% e sedans 3:83 sequence mo; made 86 8.0 em e on men n.
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ass-mm e 323 .539: pennant: {To Tm. me 26 mm m woe no spasm m
mu m" .tri
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m. N. N. W a. U. h a. O u
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a. n J a
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32%
f

m h a a

 

 

 

 

 

 

 

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nevime s mannso opnpswom ccsnmsem m.n m..m m_.m
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menu .mhec hm opnaswem vomnsaem m.— w.m¢, ww.¢
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heuim¢ s wannsu e>nasmea comnmsmm m.o n.¢m m¢.m
.mnswm senannon
wensooop psonpnz nopsa hand 0 dong
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mama” am sen:
ceasasoomn nwou
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p. .t
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u

 

41

.mnmenonoaon anmaazcmasnpxo one

.ommnnnoaon .conpmowcoo cozonm manage any
.meoaam one an memoshnoom

.mnwmo

”meonmoa enaoomononz
unmanned mmonw

.—

 

 

 

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.nounmonenp
cananeoa nascenpnaneenn “are aseaneeneupeoa esp so +n m0._ 0.» en.0 00.0 a n m.
.500 anospsenpipmoa fine. so mason
fines: caneneoa nasneesnsnoesn «nee successes sen so +n ma.0 e.n 0n.0 40.0 m m .0
.hsu asospsenp
-9000 show no peace fines: censneoa anssoesnsnosnn
sass essaeecnsueeoa en._ 00 +e «nee escapees» nan no +m 0n.0 0.. m..0 «0.0 m m. m
a m. n m m. chum. w
8 1. 1. ”W 7. D.m n+
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1. ll .+.£ e m
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9 S 0 v. I. O.
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HH> Bm<mo

 

 

 

 

 

 

 

 

 

CHAPTER V

THREE CASE HISTORIES OF CLINICAL FELINE HAEMOBARTONELLOSIS
PRESENTED AT THE MICHIGAN STATE UNIVERSITY VETERINARY
HOSPITAL FOR DIAGNOSIS AND TREATMENT

43

A. Case 3862:

Feline, domestic, male 10 months old, 8 pounds.

History: The cat had been castrated 3 weeks previously; 10
ml Normal Feline Serum1 was administered at that time; he had
been in a fight in which the skin had been broken 2 weeks be-
fore being presented at the clinic; lethargy present for 4
days prior to examination; anoretic for the past 24 hours;

he had been treated with sulfas.

fihzgiggi'exggingtion: Temperature 94.70 F; mucous membrane
white.

Hemogram:
Hemoglobin 2.1 grams %

Hematocrit 10 vol. % (packed cell volume)
WBC (corrected for normoblasts) 15,800 leukocytes per
cubic mm of blood
Differential, percentages
Neutrophils 86
Segmented 18
Non-segmented 62, metamyelocytes 6
Lymphocytes 14
Normoblasts 381/100 WBC

 

1Fromm Laboratories, Inc., Grafton, Wisconsin.

44

Polychromasia, anisocytosis
Haemobartonella felis seen on Giemsa-and Wright-

stained smears.
Stooi Examination: Ascarid and hookworm ova.

Treatment was begun with 35 ml of whole cat blood
intravenously. Additional therapy included Normal Feline
Serum, 8 ml subcutaneously; Daribiotic,1 0.5 ml intras
muscularly (discontinued when diagnosis made); Injectoplex2
and Covifol3, 1 ml of each intramuscularly. After the
figgngbgzigggiia were seen, 2 mg of oxophenarsine (0.5 mg/kg)
was administered intravenously.

The following day the temperature was 103.80 F. Treat-
ment was continued with oxOphenarsine, vitamin B—complex, and
vitamin B-12 injections. The cat was force-fed with Sustagen.4
The same regimen was followed for a total of 10 days. The

temperature fell to normal on the third day and color returned

to the mucosae after 5 days. Force feeding was discontinued

 

1S. E. Massengill Company, Bristol, Tennessee.

2Atlas Pharmaceutical Laboratories, Detroit, Michigan.
Contains thiamine HCL, nicotinamide, pyridoxine HCL, panto-
thenic acid, and riboflavine.

3Atlas Pharmaceutical Laboratories, Detroit, Michigan.
Contains folic acid, vitamin B-12, cobalt gluconate, liver
injection.

4Mead Johnson and Company, Evansville, Indiana. De-
hydrated, powdered, therapeutic food containing all known
essential nutrients.

45

after the fifth day. A.hematologic examination was performed
on the ninth treatment day.
Hemoglobin 8.5 grams %
Hematocrit 26 vol. % (packed cell volume)
WBC 8,200 leukocytes per cubic mm of blood
Differential, percentages
NeutrOphils 59
Segmented 23
Non-segmented 36
Lymphocytes 32
Monocytes 7
Eosinophils 2
Giemsa—stained blood smear negative for‘fiagggr

bartonelia.

Two weeks later a blood smear was negative for Haemo-

gggtoneii .
The cat remained normal until he succumbed to a

streptococcosis 1 year later.

46

B. Case 38241

Feline, domestic, male, 1 year old, 11 pounds.

Histogz: The cat had been castrated 3 weeks previously; he
had been in a fight since that time, with skin being broken;
he had been losing strength, appetite, and weight for 10
days, refusing all food but milk.

Physical Examination: Temperature, 1050 F; mucous membranes,
white.

Hemogram:
Hemoglobin. 5.2 grams %

Hematocrit, 19 vol. % (packed cell volume)
WBC (corrected for normoblasts), 14,300 leukocytes
per cubic mm of blood
Differential, percentages
Neutrophils 52
Segmented 21
Non-segmented 31
Lymphocytes 38
Monocytes 7
Eosinophils 3
Polychromasia, anisocytosis
Some neutrophils show slight toxic granulation.

Blue cytoplasmic inclusion bodies in some neutrOphils.

ngmgbgrtoneiia felis seen on Giemsa stain.

47

Stogi Examination: Ascarid ova.

 

Treatment was begun with 5 mg oxophenarsine intrae
venously (1 mg/kg). Supportive treatment consisted of Ferro-

B', 1 tablet, and Tonamine2

, 1 ml orally daily.

The next day the temperature was 103.40 F. The same
treatment was continued. Five ml Normal Feline Serum3 were
administered subcutaneously. The cat drank a small amount
of Sustagen4.

The following day the temperature was not recorded.
Treatment was altered by the reduction of oxophenarsine to
2.5 mg (0.5 mg/kg).

Temperature was normal the next day and remained so
during the 10-day course of treatment. 0n the tenth day the
hematocrit was 32 vol. %. A Giemsa-stained blood smear was
negative for ngmobgrtoneiia. The cat was discharged,

apparently normal.

The animal was returned 1 month later for a hematologic

 

1Pitman-Moore Division of Allies Laboratories, Indiana-
polis, Indiana; iron and ammonium citrate, manganese citrate,
thiamine HCL, yeast extract.

2Strassen‘burgh Company, Rochester, New York; B-complex
appetite stimulant.

3Fromm Laboratories, Inc., Grafton, Wisconsin.
4Mead Johnson and Company, Evansville, Indiana. De-

hydrated, powdered, therapeutic food containing all known
essential nutrients.

examination:
Hematocrit, 41 vol. % (packed cell volume)
WBC, 19,250 leukocytes per cubic mm of blood
Differential, percentages
Neutrophils 49
Segmented 14
Non-segmented 35
Lymphocytes 41
Monocytes 4
Eosinophils 6
Giemsa-stained blood smear negative for Haemobarigneiia

There have been no recurrences of symptoms up to this

writing (13 months).

49

 

Feline, domestic, male, 4 years old, weight 6.5 pounds.

Histogxi Barn cat which was presented because of inactivity,

inappetence, and loss of weight.

ghysical Examination: Temperature, 101.5o F; mucous membranes,

pale; lice present.

Hemogrgm:
Hematocrit, 14 vol. % packed cell volume

WBC (corrected for normoblasts), 16,280 leukocytes per
cubic mm of blood
Differential, percentages
Neutophils 57
Segmented 13
Non-segmented 44
Lymphocytes 35
Monocytes 4
EosinOphils 4
Nbrmoblasts 59/100 WBC
Polychromasia, anisocytosis
Haemobartoneila igiig seen on Giemsa-stained smears
on the fifth day.

Stooi Examination: Ascarid, hookworm, and irichuris ova.

TREATMENT SCHEDULE

50

 

Observations

 

 

T. 101.50; blood smear

blood smear

active; appe-
blood smear

T. 102.2°; appetite poor;
blood smear positive.

irsinobenzamide 0thgr
1 Parabomb-M‘é
Injectoplex , 1 ml, negative.
intravenously
2 Parabomb-M; Injectoplex T. 102.50;
1 m1; anti-feline dis- negative.
temper serum}, 5 m1;
whole cats' blood, 20
m1, intravenously
3 Injectoplex, 1 ml T. 102.4°;
tite fair;
negative.
4 Injectoplex, 1 ml
5 2.6 ml IV Injectoplex, 1 ml

(0.4 ml/lb)

6 2.6 ml IV

 

 

Injectoplex, 1 ml; 5%
Dextrose in 1 anrter
Strength Saline , 200
ml, subcutaneously

Injectoplex, 1 ml;
Dextrose in Saline,
ml

100

 

T. 102.0°; anorectic, de-
pressed, mucous membranes
pale; blood smear
positive.

T. 100.90; listless,
appetite poor; blood
smear positive.

r. 101. °; active; blood
smear negative.

 

1Haver-Lockhart Laboratories, Kansas City, Missouri.

siticidal spray containing malathion.

2Atlas Pharmaceutical Laboratories, Detroit, Michigan.

Ectopara~

Contains

thiamine HCL, nicotinamide, pyridoxine HCL, pantothenic acid, and

riboflavine.

3Fromm Laboratories, Inc., Grafton, Wisconsin.

For intravenous or intramuscular injection.

Feline origin,

produced from cats exposed to panleucopenia virus and inoculated with
feline distemper vaccine.

4

Abbott Laboratories, North Chicago, Illinois.

5Jensen-Salsbery Laboratories, Inc., Kansas City, Missouri.

TREATMENT SCHEDULE (continued)

51

 

Day
8

1O

11

12

13

14

15

Arsinobenzamide
W

 

Other

Obseryations

 

1.3 ml IV
(0.2 ml/lb)

1.3 ml IV

1.3 ml IV

0.6 ml IV
(0.1 ml/lb)

0.6 ml IV

Oe6 1111 IV

 

Dextrose in Saline, 100
ml; Injectoplex, 1 ml

Dextrose in Saline, 100
ml; Injectoplex, 1 ml

Dextrose in Saline, 100
ml; InjectOplex, 1 ml

Dextrose in Saline, 100
ml; Injectoplex, 1 m1

Dextrose in Saline, 100
m1; Injectoplex, 1 ml

Dextrose in Saline, 100
ml; anti-feline dis-
temper serum, 4 ml

Injectoplex, 1 ml

Injectoplex, 1 ml

 

T. 103.00; appetite fair;
blood smear negative.

T. 102.00; active, appe-
tite good; blood smear
negative.

T. 102.00; improving;
blood smear negative.

T. 10t.1°; mucous membranes
pink; blood smear negative.

T. 100.8°; appetite good,
very active; blood smear
negative.

T. 100.80; condition good;
blood smear negative.

T. 100.8°; condition good;
blood smear negative.

T. 101.20; condition good;
blood smear negative.

 

Hemogram 4 days after termination of arsinobenzamide therapy:

Hemoglobin 7.5 grams %

Hematocrit 23 vol. % (packed cell volume)

NBC,

Differential, percentages

NeutrOphils 63

Segmented 24

Non-segmented 39

Lymphocytes 32

17,150 leukocytes per cubic mm of blood

52

Monocytes 4
EosinOphils 1
The cat continued to eat and the condition gradually
improved. A slough occurred along the femoral vein where
some of the arsinobenzamide had been extravasated. The cat
was wormed, and when the sloughed area was well granulated,
the cat was discharged.
Two months later the owner reported that the cat was

back to its normal activity and had gained weight.

Hemogram 2 months post treatment:
Hemoglobin,8.5 grams%
Hematocrit, 27.5 vol. % (packed cell volume)
WBC, 12,400 leukocytes per cubic mm of blood
Differential, percentages
Neutrophils 67
Segmented 45
Non-segmented 22
Lymphocytes 30
Monocytes 1
EosinOphils 2
Giemsa-stained blood smear negative for Haemobartonella.
One year later the owner reported that the cat was

apparently normal.

CHAPTER VI
DISCUSSION

Haemobartonella muris and‘g. canis have been known
for many years. There is adequate justification for their

inclusion in the genus Haemobartonella. Such is not the

 

case with H, igiig, This organism is not included in the
7th Edition.of Bergey's Manual. The application of the name
Haemopgrtonelig‘igii§,to this organism is apparently the re-
sult of a letter from Dr. David Weinman, a recognized
authority on the Bartonellaceae, published by Flint and Moss
(1953). Dr. Weinman felt that the feline parasites were
either Haemobartonelig or Epegythrozoon, on the basis of
morphology. He also believed it unusual for these organisms
to produce disease without splenectomy. Whether the feline
organism will remain in the genus Haemopartonella remains to
be seen.

In any transmission study, it is first necessary to
prove the test animals negative for the organism which is to
be transmitted, so that the experimental results can be
properly evaluated. When Regendanz and Reichenow (1932) re-
ported that'H.lgani§,had been transmitted to a cat, they were
unaware of the existence of H. igiig, and consequently their

experimental results might be questioned. In this study,

54

great care was taken to prove the test animals negative for
Haemobartonelia before transmission was attempted.

The incubation period of haemobartonellosis is that
time between the introduction of infectious material, or the
splenectomy in the case of a latent infection, and the appear-
ance of Haemobarionella in the peripheral blood. Based on
published incubation periods, the minimum pro-inoculation
observation period was set at 14 days. Most animals desig-
nated Haemobartoneila-negative were observed for a longer
time.

Kikuth (1932) reported the post-splenectomy incubation
period of‘g. gggig in dogs to be 6 days after surgery. Knutti
and Hawkins (1935) found the incubation period of H. ggnig
to be 2 to 5 days. Ray and Idnani (1940) reported the incu-
bation period to be 19 days in subcutaneously inoculated
dogs, and 6 to 12 days in intravenously inoculated dogs.
Benjamin and Lumb (1959) made observations on 4 dogs with
incubation periods of 15 to 20 days.

Splitter, Castro, and Kanawyer (1956) reported the
incubation period of‘fl.‘£gii§ in cats to be 3 to 20 days,
and averaging 7 days.

Flint, Rbepke, and Jensen (1959) reported an average
incubation period of 15.7 days, and ranging from 9 to 34
days after intraperitoneal inoculation. After intravenous

inoculation, the incubation period averaged 16.4 days,

55

ranging from 8 to 23 days. Their results with splenectomized
cats were approximately the same.

In this study, the average incubation periods should
be regarded in groups according to the source and route of

administration of infective blood.

1. Canine to canine, intravenously (11 dogs):

range 4 to 28 days, average 9 days.

2. Canine to feline, intraperitoneally, and back
to canine intravenously (4 dOgs): range 4 to

23 days, average 11 days.

3. Feline to feline intraperitoneally (18 cats):

range 2 to 15 days, average 7 days.

4. Feline to canine intravenously, and back to

feline intraperitoneally (1 cat): 11 days.

While cats did not develop positive~blood smears,‘fl.
gggig remained viable in this foreign host, and the cat
blood was infective for dogs. When blood from these dogs
was inoculated into cats, haemobartonellemia did not occur
in the cats. H, igiig, on the other hand, produced a haemo-
bartonellemia in a dog, and pooled blood of dogs, inoculated
with H, igiig was infective for a dog and a cat. This indi-
cates that the cat, while not showing a haemobartonellemia,

may be a carrier for g. ganig. Dogs develop a

56

haemobartonellemia when injected with H, igiig. It is con-
ceivable, therefore, that cross infection occurs in nature,
due to exchange of blood in fighting, or perhaps due to
parasitic arthrOpods. H. gggig and H. igiig appear to be
separate entities.

Maternal transmission oflfi.‘gggi§ to puppies was
demonstrated inc2 instances. In both cases, the puppies
were allowed to nurse. It, therefore, remains to be deter-
mined whether the infection of the puppies occurred in
utero, or whether it was passed through the bitchs' milk.
Maternal transmission may be an important factor in the
perpetuation of the disease.

The attempt at mosquito transmission of fi,{igii§'was
inconclusive.

The inability to produce an infection in splenectomized
rats with H. i2ii§,confirms the reports of other workers.
0x0phenarsine proved to be effective in the therapeutics of
‘H.‘gggi§ infection in rats, and should be considered for use
if it is ever necessary to splenectomize rats for experi-
mental use. The MLD of this drug for rate was found to be
between 22 and 24 mg/kg body weight. This coincides with the
published MLD for rats of greater than 23 mg/kg body weight
(Spector, 1956).

Inoculation of incubating chick embryos via the chorio-
allantoic membrane, allantoio sac, and yolk sac, with‘H. gagig
andIH.‘igii§ did not yield viable organisms. Gross

57

examination of the embryos and their membranes suggested
that infection had occurred but that the organism did not
survive. It was impossible to infect dogs or cats with
harvested egg material. Further work in this area should
include the intravenous route of embryo inoculation as well
as variation of the incubation temperature.

Arsinobenzamide is commonly used as a treatment for
canine heartworm infection. A 3-day treatment with this
drug at 0.20 ml per pound of body weight is suggested as
therapy for canine haemobartonellosis. The same dosage, on
a 10-day schedule, is unreliable in the cat, although it may
be effective in certain instances (see case reports).

OxOphenarsine has been recommended for treatment of
H. igii§_infection. Two case reports are presented in which

it was used successfully.

3.

7.

CHAPTER'VII
CONCLUSIONS

Cats may be carriers of Haemobartonella canis, and thus '

potential sources of infection for dogs.

Dogs can be infected with Haemobartoneiia felis, showing
a.haemobartonellemia. The dog blood is then infective

for dogs and cats.

Maternal transfer of Haemobartonella canig to puppies
occurs either in utero, through the milk, or possibly
through contamination at birth. This may play an im-

portant part in the natural transmission of the disease.

Mosquitoes were not proved to be vectors of Haemo-

bartoneila felis.

Rats could not be infected with Haemobartonella ielis.

Haemobgrigneila guris infection in rats, which becomes.
manifest after splenectomy, can be successfully treated

with oxophenarsine, 2 mg/kg body weight intraperitoneally.

The MLD of oxophenarsine for laboratory rats is between

22 and 24 mg/kg body weight.

Embryonated chicken eggs, 8 to 12 days old, could not be

9.

10.

59
infected with Haemobartonellg canis or‘H. felis.

Arsinobenzamide can be used successfully to treat
canine haemobartonellosis on a 3-day schedule at the

level of 0.20 ml/lb body weight.

The use of arsinobenzamide at a level of 0.20 ml/lb

body weight for 10 days was unsuccessful in the treat-
ment of feline haemobartonellosis. Results in 1
clinical case, however, suggest that it may be effective

in certain instances.

LIST OF REFERENCES

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Acta path. et microbial. Scandinav., Suppl. 37:37.

Barton, A. L. 1909. Descripcion de elementos endo-globulares
hallados en los enfermes de fiebre verrucosa (Articulo
preliminar). Cron. med., Lima, 26:7.

Bayon, H. P. 1928. Bartonella muris: Its pathogenic action
in the progressive anemia following rat splenectomy and
its resemblance to‘g. bacilliformis of Carrion's disease.
J. TrOpical Med., 31:29.

Benjamin, M. M., and W. V. Lumb. 1959. Haemobartonella
canis infection in a dog. J. Amer. Vet. Med. Assoc.,
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Biffi, U. 1903. Sobre las hemoaglutininas de la sangre
humans y la hematologia de la "enfermedad de Carrion."
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Breed, R. S., E. G. D.Murray, and N. R. Smith. 1957.
Bergey's manual of determinative bacteriology. 7th ed.,
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Cannon, P. R., and P. H. McClelland. 1928. Role of
ectoparasites in bartonella infection of albino rats.
Proc. Soc. Exper. Biol. and Med., 26:157.

Cannon, P. R., and P. H. McClelland. 1929. The transmission
of bgrtonella infection in albino rats. J. Infect. Dis..
44:5 .

Carr, D. T., and H. E. Essex. 1944. Bartoneila canis in
splenectomized dogs. Proc. Soc. Exper. B101. and Med.,
57:44.

Clark, R. 1942. Eperythrozoon felig (sp. nov.) in a cat.
J. South African Vet. Med. Assoc., 13:15.

Crystal, M. M. 1959. The mechanism of transmission of
Haemobartonella muris (Mayer) of rats of the spined rat

louse, Polyplgi spinulosa (Burmeister). J. Parasit..
44:603.

61

Danoff, U. 1919. Der Einfluss der Milz auf der respirator-
ischen Stoffwechsel. Biochem. Ztschr.. 93:44.

Donatien, A., and F. Lestoquard. 1937. Etat actual des
connaissances sur les rickettsioses animales. Arch.
Inst. Past. Algerie., 15:142.

Donovan, E. F., and W. F. Loeb. 1960. Hemobartonellosis in
the dog. Vet. Mede, 55:57e

Eliot, C. P. 1936. The insect vector for the natural trans-
mission of Eperythrozoon coccoides. Sci., 84:397.

Farris, E. J., and J. Q. Griffith. 1949. The rat in
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