COMPARATIVE DYNAMICS OF
BRUCELLA-I-IOST CELL REACTIONS IN
NORMAL AND IMMUNE MDNOCYTES

Thesis for the Degree of M . S.

MICHIGAN STATE UNIVERSITY

JEROLD STEPHEN SH PARGEL
1971

 

 

 

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ABSTRACT

COMPARATIVE DYNAMICS OF BRUCELLA-HOST
CELL REACTIONS IN NORMAL AND
IMMUNE MONOCYTES

BY

Jerold Stephen Shpargel

An in vitgg cell suspension system for measuring
cellular immunity has been devised in order to investigate
some questions concerning host-parasite relationships in
brucellosis. These questions involved the correlation of
virulence of Brucella to the presence of smooth antigens,
and the role of humoral antibody in cellular immunity to
Brucella.

The study involved the in_vit£9 growth of two
antigenically different brucellae, g. ganig and smooth g.
guig 1776, in normal and immune peritoneal cells in cul-
ture.

The results show that g. gangs, which lacks smooth
antigens grows well in normal macrophages and is not elimi-
nated as well as smooth g. EEEE from immune cell cultures.
This suggests that the correlation of virulence with the

presence of smooth antigens in Brucella is not necessarily

Jerold Stephen Shpargel

true. The results also show that there is no significant
role if any for humoral antibody in cellular immunity to

Brucella.

COMPARATIVE DYNAMICS OF BRUCELLA-HOST
CELL REACTIONS IN NORMAL AND

IMMUNE MONOCYTES

BY

Jerold Stephen Shpargel

A THESIS

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

MASTER OF SCIENCE

Department of Microbiology and Public Health

1971

ACKNOWLEDGMENTS

The author wishes to acknowledge the kind assistance
and suggestions for the preparation of this thesis received
from Dr. Norman B. McCullough, Dr. Robert Moon, and

Mr. Brian C. T. Wu.

ii

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . .
LITERATURE REVIEW . . . . . . . . .

Host-parasite Relationships of Classical
Brucella . . . . . . . . . . .

Brucella canis . . . . . . . . .

 

MATERIALS AND METHODS . . . . . . . .
“SULTS . C O C O O O O O O O O 0

Cell Kinetics of Peritoneal Cells in
culture 0 I O O O O O O O O O

Accumulation of Ampicillin within Immune
Peritoneal Cells . . . . . . . .

Growth of Brucellae in Normal and Immune
cell cultures 0 I O O O O O O 0

DISCUSSION . O O C O O O O O O O C

BIBLIOGRAPHY . . . . . . . . . . .

iii

Page

14
17

24

24

32

37
47

53

T—-“'Wm 41:14 a -“ ..

.1-
bro

Figure

1.

LIST OF FIGURES

Cell Kinetics of Normal Peritoneal Cell
Cultures Infected with Brucella and
Ampicillin Added One Hour Later . . .

Cell Kinetics of B. canis Immune
Peritoneal Cell Cfiltures Infected with
Brucella and Ampicillin Added One Hour
Later . . . . . . . . . . . .

Cell Kinetics of E. suis Immune
Peritoneal Cell Cultures Infected with
Brucella and Ampicillin Added One Hour
Later . . . . . . . . . . . .

Intracellular Accumulation of Ampicillin
by E. canis Immune Peritoneal Cell
Cultures Using the Growth of E. suis as
the Test System . . . . . . . .

Intracellular Accumulation of Ampicillin
by E. canis Immune Peritoneal Cell

Cultures Using the Growth of E. canis as
the Test System . . . . . . . .

Intracellular Accumulation of Ampicillin

by B. suis Immune Peritoneal Cell Cultures

Usifig the Growth of E. suis as the Test
system . C O C O O . O . C .

Intracellular and Extracellular Growth of
E. suis and E. canis in Normal Peritoneal
Cell Cultures . . . . . . . . .

Intracellular and Extracellular Growth of
E. suis and E. canis in E. canis Immune
Peritoneal Cell Cultures . . . . .

Intracellular and Extracellular Growth of

E. suis and E. canis in E. suis Immune
Peritoneal Cell Cultures . . . . .

iv

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26

28

3O

34

36

39

41

44

46

INTRODUCTION

Induced immunity is dependent upon the interplay
between host and parasite, and thus is determined by the
characteristics of both the infecting organism, and of the
immunological response.

The members of the genus Brucella are facultatively
intracellular parasites, induce delayed hypersensitivity
in the host and engencer induced resistance believed to be
mainly cellular in nautre. Whether humoral factors are
necessary for a full display of cellular immunity is a sub-
ject of controversy.

The pathogenicity of the classical members of this
genus is correlated with the presence of S antigens. Rough
cultures are avirulent.

E. EEEEE lacks the S antigens of the classical
members of the genus, yet is pathogenic. It is biochem-
ically similar to E. EEE§°

The dissimilarity of these organisms appeared to
offer a means of further clarifying the characteristics of
grucella responsible for virulence and the role of humoral

factors in cellular immunity in brucellosis.

It was decided to devise an E3 vitro system which

would enable the study of the behavior of E. canis and

 

E. suis in parallel systems as follows:

1. Characterize the intracellular growth curves in
monocytes from a mammal (mouse) other than the

preferred host of either species.

2. Similarly, characterize the intracellular growth
curves in cellular immune monocytes of mice

infected with the respective species.

LITERATURE REVIEW

Host-Parasite Relationships of
Classical Brucella

 

 

Antigenic Structure and Humoral Respgnse

An understanding of antigenic structure and
activity of a group of microorganisms is of basic importance
in a consideration of their pathological activities and
elicitation of a host response. The presence or lack of
exotoxins, endotoxins or bacterial protective mechanisms
such as capsular antigen may determine the outcome of an
infective process (41). Early studies dealt with species
differentiation via surface agglutinogens (59). Later
workers studied in more detail internal antigens as well
as external antigens (179 28).

However, perhaps the most important antigenic
characteristic associated with virulence is the smooth-
rough variation. Henry (22) described smooth (S), various
intermediate (I) and rough (R) colonial types. The S type
had full virulence, the rough types were avirulent, and
the I types were of reduced virulence. The antigenic
change is not clearly understood, however there is evidence
it involves the loss of the specific S antigen (60). Non-S
types have not been seen to revert to full 8 virulence

3

(26, 34). Isolates recovered from infected animals are
usually of the S type, but occasionally I and R or mucoid
types are found (26).

Despite the correlation of virulence with S types
of Brucella, E. gyig and E. EEEEE lack the smooth antigens
yet are pathogenic. The limited host ranges of these
organisms may be a reflection of the absence of S antigens
(34).

Several investigators have studied the toxicity of
Brucella endotoxin (14, 29, 30) and found it to be a potent
toxic substance. Generally when smooth strains of entero-
bacteria are extracted with phenol-water procedures, the
biologically active lipopolysaccharides are isolated from
the aqueous phase. However the toxins of smooth strain
brucellae are isolated from the phenol phase (29). The
toxic fractions contain protein-lipopolysaccharide-KDO com-
plexes (29). There are also indications of the presence of
a ribonucleic acid (3). As dissociation progresses S--R,
the endotoxin is lost and rough strains are devoid of the
material (27, 34). However, endotoxin content does not
appear to be a determinant of virulence. Yet, once
infection occurs, it plays an important role in the host
response (34). It could quite possibly account for many of
the symptoms seen in brucellosis, such as the changes in
central and autonomic nervous systems, fever, and effects

on tissue and organs including adrenal glands (55). These

effects may be due to a direct effect of endotoxin and
other products or indirectly due to hypersensitivity.

Antibodies to brucellar antigens appear in all
three major classes of immunoglobulins IgG, IgA, and IgM
(9, 58). In acute cases both low molecular weight (IgG and
IgA) and high molecular weight globulins are present. In
the chronic form of the disease, globulin representation
is mainly confined to the low molecular weight globulins
(9). This antibody activity can be demonstrated as bac-
teriocidins, agglutinins, opsonins, precipitins, and com-
plement fixing antibody (34).

Immunization of animals with killed cells stimu-
lates antibody production, however little protection is
produced to challenge with virulent strains (34). The
multiplication of organisms within the host seems essential
for production of serviceable immunity (34).

It has generally been accepted that domestic
animals can be protected only by living vaccines. Yet
repeated attempts have been made to immunize laboratory
animals against Brucella infection by using non-living
preparations (15, 50, 51, 53). Various cell wall prepa-
rations, culture filtrates, and chemical fractions have
been used to show mice and guinea pigs can be afforded pro-
tection via immunization. However, non-living vaccines
have not yet been shown to protect the chief hosts against

their respective infecting species.

The bactericidal activity of serum or plasma is
another interesting aspect of the humoral response to
Brucella. Serum or plasma from normal individuals has a
killing capacity for Brucella which requires complement
for Optimal activity (20). Serum or plasma from those with
acute brucellosis had the same killing activity as that of
healthy controls (55). However, the killing ability of
serum from chronic patients was considerably reduced. In
chronic cases, a specific inhibitor appears which prevents
the lethal activity of antibody plus complement (34). When
the serum is diluted, a high bactericidal activity is again
elicited (34, SS). Opsonization of Brucella cells by spe-
cific antibody aids in phagocytosis by polymorphonuclears
and macrophages. Although clearance of Brucella from blood
is rapid, killing is not. Once inside cells, they are pro-
tected from the bactericidal action of blood. Such opso-
nizing activity probably promotes the localization of

infection (34).

Cell Mediated Responses

 

The concepts of cell-mediated versus humoral immu-
nity have been debated since the days of Metchnikoff.
Cellular immunity which is independent of a humoral system
is a concept which over recent years has gained some accep-
tance. The behavior of Brucella within immune mononuclear

phagocytes has added support to some of these concepts.

Phagocytosis of Brucella by polymorphonuclears is
easily demonstrated. However as these cells degenerate,
Brucella are released which are quite viable. The mono-
nuclear phagocytes of the circulation and tissues are
mainly responsible for killing Brucella in normal and
immune animals (34).

Pullinger (44) first demonstrated an increased
resistance to infection with E. abortus in guinea pigs
receiving concomitant tuberculosis infection. It was shown
that the tubercle bacilli stimulate a mononuclear cell
reaction, and these cells, while unable to handle the
tubercle bacilli, phagocytized and destroyed Brucella.

Later in a search for a milder agent capable of
stimulating protective action against Brucella, he found a

low virulence strain of Listeria monocytogenes afforded

 

protection in guinea pigs similar to that stimulated by the

tubercle bacilli (45). By inoculating E. monocytogenes in

 

the same extremity as was inoculated with low doses of

E. abortus a transient localized resistance was established.
Histological studies provided evidence of macrophage
involvement in resistance. Nyka (40) confirmed these
results showing mice injected with E. abortus were more
resistant to tubercle bacilli than controls. He also sug-
gested a strong role for the mononuclear phagocyte in
resistance. Mika (36) showed an increased resistance to

Coxiella burnetti in guinea pigs infected with E. suis.

 

The increased resistance could not be explained on an
immunochemical basis. Elberg (l4) utilized ig_yi££9 tech-
niques to show cross immunity in monocytes. A nonspecific
factor of immune serum was necessary for full monocyte
resistance, as antibody adsorption with antigen had no
effect.

Smooth brucellae were shown by Holland and Pickett
(24) to be ingested and multiply in chick embryo fibro-
blasts in tissue culture. Non-smooth variants and inter-
mediate strain 19 neither multiplied nor survived long
intracellularly unless the cells were massively infected.

These authors (23) found that smooth E. suis, abortus, and

 

melitensis multiply after ingestion in peritoneal monocytes

 

obtained by glycogen stimulation. Non-smooth and mucoid
strains showed slow multiplication. Monocytes from animals
previously immunized with a living vaccine inhibited growth
of all strains. Vaccination with dead brucellae failed to
inhibit intracellular multiplication. The presence of
anti-Brucella antisera had no effect on the growth or sur-
vival of brucellae within immune or normal monocytes.
Pomales-Lebron and Stinebring (43) obtained macro-
phages from normal and immune guinea pigs after glycogen
stimulation. The cells were allowed to phagocytize E.
abortus for two hours. Extracellular growth was controlled
by adding lOug per ml of streptomycin to the tissue culture

medium. The growth of E. abortus in immune cells was not

 

as great as observed in cells from nonimmune animals. The
authors noted variations occurred with respect to intra-
cellular multiplication observed for individual cells.

They felt this suggested a heterogeneity in both the mono-
cyte population and bacterial cultures. Braun, Pomales-
Lebron and Stinebring (6) found that smooth strains of
Brucella proliferate in phagocytes while rough strains show
little growth. However phagocytosis of rough strains is
greater than with smooth strains, with the result that all
monocytes were destroyed by 48 hours.

Freeman and Vana (19) used tissue culture techniques
to study the infectivity and growth of Brucella in normal
guinea pig macrophages. With the small inoculums and cul-
ture conditions they used, the presence of streptomycin
for control of extracellular multiplication was needless,
and even harmful, and therefore omitted. The authors
noted intracellular brucellae began multiplication without
a lag phase. The growth rate was independent of the number
of infecting organisms over a wide range and infection need
not be overwhelming for extensive intracellular growth to
occur. No direct correlation between intracellular growth
and animal virulence could be shown in these experiments.
In a study on the cytopathogenic effect of Brucella of dif-
ferent virulence on macrophage cultures, Freeman g3_gl.
(16, 18) found rough, avirulent strains more destructive

than smooth cultures as determined by spectrophotometric

 

 

10

measurements of host cell constituents released into the
medium. It was suggested smooth and rough brucellae may
grow at equivalent rates but the end result of rough bru-
cellae growth is severe damage to the host cell. The freed
brucellae may then be killed by streptomycin in the medium
which could explain divergent reports on growth rates.
Ralston and Elberg (46, 47) showed that monocyte
extracts from immune and normal rabbits contain a lysozyme-
like material capable of acting on the cell wall of E52:
ggllg. Older living cells are extremely resistant to this
material, yet young cultures treated with glycine are sus-
ceptible. With cells of equal ages a gradation of sensi-

tivity is demonstrable. A rough variant of E. melitensis

 

Rev I is most susceptible, Rev I intermediately suscep-

tible, and virulent E. melitensis 6015 least sensitive.

 

Continued growth on Albimi medium caused a change to resis—
tant forms. This suggested cell wall synthesis and struc-
ture is an important factor in controlling the growth and
destruction of intracellular brucellae by monocytes.

Smith and Fitzgeorge (52) examined the behavior
during phagocytosis and the intracellular fate of virulent
and avirulent strains of E. abortus grown ifl.!i££2° Viru-
lent strains of E. abortus resisted the bactericidal power
of bovine phagocytes more than attenuated and avirulent.
strains. The authors noted that cell wall preparations

from E. abortus especially when grown i3 vivo, contain a

ll

protective material capable of interference with the
killing action of bovine phagocytes during and subsequent
to phagocytosis. The antibactericidal action of the cell
walls could be neutralized by pretreatment with specific
antiserum.

Elberg (l3) analyzed much of the data regarding
cellular immunity in his review. He states normal mono-
cytes are lysed by certain ingested virulent bacteria but
not by avirulent ones. Immune monocytes are not lysed by
homologous parasites. Immune monocytes can support the
growth of the homologous parasite temporarily but then it
decreases and stops. Data on persistance of the parasite
in a latent state, as well as from growth experiments,
suggests the parasite may persist as an "elementary par-
ticle" presumably an L form. The presence of serum factors
determine whether the infecting organism persists as an
elementary form or multiplies.

Current concepts of cellular immunity (32) envision
a population of macrophages of altered metabolic capabili-
ties, including the capacity to restrict and limit the
growth of virulent intracellular parasites. The develop-
ment of a population of antimicrobial effector cells seems
to be dependent upon an interplay between the effector and
the immunologically committed lymphocyte. It is postulated

that the lymphocyte of the type which mediates delayed type

l2

hypersensitivity releases a factor which is biologically
active in macrophage activation.

Hypersensitivity in brucellosis is a phenomenon
which is well established (34, 55). Delayed hypersensi-
tivity develops usually after one or more weeks of
infection and may last up to thirty years as demonstrated
by cutaneous reactivity to brucellar antigens. Heilman
EE_2l° (21) used an lg 31359_migration technique to study
the response of spleen cultures from normal and infected
animals to killed E. EEEE antigen. The antigen was more
effective in inhibiting migration from infected than normal
spleen cells. Macrophages were more sensitive than leuko-
cytes to the toxic action of the antigen. Delayed hyper-
sensitivity to Brucella appears to be a generalized response
with several types of tissue cells entering the reaction
(34). The marked damage of sensitized cells in the presence
of antigen probably has a bearing on the pathology and
symptomology of brucellosis, as well as playing a role in

the defense mechanisms against it.

A Speculative Model

 

McCullough (34) has given a speculative model for
the pathogenesis and host-parasite relationship in brucel-
losis. Once the bacteria pass the primary barrier they
enter lymphatics and are transported to regional lymph
nodes, often within polymorphonuclear leukocytes. These

cells are ineffective against the bacteria. In the

13

regional lymph node multiplication occurs within the
mononuclear phagocytes. Death of some host cells occurs
with the release of both bacteria and host cell components.
This stimulates an intense mononuclear cell proliferation
and activation. Cell adapted bacteria are more resistant
to bactericidal action of normal blood, are readily phago-
cytized and are well adapted for intracellular life.
Release of some antigenic material stimulates antibody
forming mechanisms and specific hypersensitivity. If anti-
bodies are present, their Opsonic action would promote
phagocytosis. The develOpment of specific hypersensitivity
increases the local inflammatory response. After general
dissemination concurrent to endotoxin release there is a
strong stimulus for further generalized development of
humoral and cellular immune responses along with manifes-
tations of systemic disease. If infection proceeds beyond
generalized systemic disease, release of bacteria from
necrotic cells occurs and extracellular multiplication
becomes prominent. Here, humoral factors may play a role
in limiting the infection. The opsonic activity of anti-
body promotes phagocytosis and may play some role in intra-
cellular killing by macrophages. Hypersensitivity of
tissues aids in localization and granulomas may develop.

As humoral and cellular immune mechanisms bring the

infection under control, a predominant intracellular phase

14

returns. After termination of the infection, a relative

immunity is present.

Brucella canis

 

Outbreaks of abortion in Beagle dogs and the sub-
sequent isolation of a gram negative organism which resem-

bled members of the genus Brucella was first reported by

 

Carmichael (1). Carmichael and Bruner (8) studied the
morphological, biochemical, cultural and antigenic charac-
teristics of the organism. These workers emphasized the
morphological and biochemical relationship of the canine
abortion agent to E. ERIE! and the antigenic relationship
to E. gyig. Since it was not identical with any recognized

Brucella species, the name E. canis was suggested.

 

Further examination of the antigenic character of
the canine abortion organism strongly suggested the organ-

ism was a member of the genus Brucella (11). The agent was

 

found similar in structure to rough E. abortus, E. meliten-

sis, and E. ovis, but different from smooth Brucella. The

 

organism was found to lack the 1ipopolysaccharide-endotoxin
associated with the agglutinogen of smooth brucellae.

There is still some controversy over the naming of
the organism. Meyer (35) in studying ten strains of the
organism, concluded that the overall metabolic character of
the organism resembled that of E. §2i§° She argued that

antigenic considerations play no role in the definition of

15

a species. She suggests the organism be made a new biotype
within the species E. ggig.

Howe, Morisset, and Spink (25) studied the biolog-
ical properties of the antigens of E. 33235. It was found
that very large numbers of organisms were required to kill
a chick embryo. E. EEEiE soluble antigen "induces" lethal-
ity, but Boivin and EDTA endotoxin preparations do not.

The soluble antigen was shown to have a ribonucleic acid
moiety associated with it. Thin layer chromatography and
enzymatic digestion revealed the ribonucleic acid necessary
for the biological manifestations of the E. Eggig antigen.

Deyoe compared the pathogenesis of E. EEEEE to that
of E. EEEE in dogs and other domestic animals (10). It was
found E. EEEEE infection was characterized by a high multi-
plicity of organisms ig_yiyg. A prolonged incubation was
necessary before development of pathologic alterations.

E. EELE had the opposite characteristics. Serum antibody
titres and bacteremia in E. EEBEE infection reached high
levels throughout the experiments. These parameters of

E. E232 infected dogs increased until three weeks post
exposure and declined. E. Eflflié seemed to be of low viru—
lence for other domestic animals and guinea pigs.

Spink (54) has studked the globulin types found in
Beagles after E. Eggig infection. His group isolated a 198
agglutinating antibody, and at least three different 75

antibodies. However it appears there is minimal

16

bactericidal activity in the serums of infected and control
animals. Spink believes that it is likely that the immune
response to E. EEEEE is not unlike that of other animals
naturally infected with brucellae. Carmichael suggests
that cellular immunity is of the greatest significance in

the immune response to infection with E. canis (7).

MATERIALS AND METHODS

Bliss

Eighteen to twenty gram, female, Carworth Farms
mice (CF-1) from Carworth Inc. (Portage, Michigan) were
used in all experiments. Purina Laboratory Chow (Ralston
Purina Co., St. Louis, Missouri) and water were provided
39 libitum. Mice were kept in groups of five, in cages

containing wood shavings as litter.

Microorganisms

Both species of Brucella used in the following
experiments were obtained from the collection of Dr. Nor-
man B. McCullough. Stock cultures of E. EEEE 1776 were
isolated from frozen infected guinea pig spleens. This

strain was wholly smooth with an ID for the guinea pig of

50
two organisms. The culture was periodically checked for
purity and for maintenance of smooth characteristics by
staining procedures and the oblique lighting method of
Henry (22). E. EEEEE RM-666 originally from L. E. Car-
michael's laboratory was isolated from a frozen stock cul-
ture stored on Trypticase Soy agar. This organism lacks

the antigenic relationship with smooth Brucella cultures

when tested by reciprocal agglutination (ll). Periodic

l7

18

checks for purity and identity of this organism were made,
using staining procedures and a tube agglutination test
with a known positive guinea pig serum for E. canis. Bac-

teria used for immunization and i3 vitro infection were

 

removed from agar slants, rather than broth cultures so as
to maintain antigenic character. Slants were rinsed with
normal saline plus 0.1% peptone (to preserve viability).
Initial enumeration of bacterial numbers was made by
observing the per cent light transmittance on a Bausch and
Lomb Spectronic 20, and determining the value on a standard

9 bacteria.

curve on which 78% transmittance equaled lxlO
All dilutions were made in normal saline plus peptone. All
plates used for bacterial enumeration were Trypticase Soy

Agar (BBL, Cockeysville, Maryland). Plates and slants were

incubated at 37C.

Immunization

 

Mice were immunized with live bacteria 21 days
prior to the harvest of peritoneal cells. Injections were
made by way of the intraperitoneal route. To assure main-
tenance of the antigenic character, 48 hr. slants of the
bacteria were washed in normal saline plus peptone and
diluted to the immunizing dose. The immunizing dose for
E. ggig was 2x106 organisms per mouse. E. gggig was
administered in a dose of 2x107 organisms per mouse. These
doses were chosen as a reflection of comparable spleen size

increase. Since mice are relatively resistant to brucellae,

19

splenomegaly is one of the few detectable signs of

infection.

Peritoneal Cell Culture

 

Animals were prestimulated to give reproducible,
maximum, peritoneal macrophage cell yields. Four days
prior to harvest, a 10% solution of Proteose peptone #3
(Difco, Detroit, Michigan) was used. An injection of 1.5
ml was given intraperitoneally, following the procedure of
Unanue and Cerottini (57).

Tissue culture media was used for both harvesting
and culture of the cells. Experience in our laboratory
suggested Medium 199 (39) (Microbiological Associates,
Bethesda, Maryland) was the medium of choice. This medium
was further supplemented with 1% of a 200mM solution of
L-glutamine (Microbiological Assoc.), 10% fetal bovine
serum (Microbiological Assoc.) which had been virus
screened and immunoelectrophoretical1y checked for absence
of gamma globulins, and 5 units per ml of heparin (Sher-
wood Medical Industries, St. Louis, Missouri). The pH was
adjusted to 7.0-7.2 using 1M sodium bicarbonate.

Medium for these experiments was made fresh every
7 to 10 days, as preliminary experiments suggested older
medium was unsatisfactory for culturing these cells.

Peritoneal cells were harvested in the following
manner. Mice were killed by cervical dislocation. Five

ml of cold tissue culture media were injected into the

20

peritoneum. Gentle massage was applied before attempting
withdrawal of the fluid. After making a mid-ventral
incision, the skin was stripped from the peritoneum. The
peritoneal fluid was withdrawn with an 18 gauge needle and
syringe. A volume of 4 ml was collected. Cell suspensions
were placed immediately into culture flasks, after removing
the needle from the syringe. Culture flasks were 25 ml
Pyrex flasks that had been repeatedly siliconized with
Siliclad (Clay Adams, Parsippany, New Jersey) to minimize
adherence of cells to the glass. To further minimize the
adherence of cells to the flask walls, the flasks were kept
chilled just prior to inoculation. Immediately after plac-
ing the cells in the culture flasks, they were placed on a
Gyrotary Water Bath Shaker (New Brunswick Scientific, New
Brunswick, New Jersey). Cultures were grown in a humidi-
fied atmosphere containing 5% carbon dioxide and 95% air.
The cultures were continually shaken to minimize adherence
to glass as well as to promote greater contact between
bacteria and cells during the infection period of later

experiments. Incubation temperature was 37C.

Determination of Cell Kinetics in Culture

Peritoneal cell cultures were harvested and placed
in the culture flasks. Cells were incubated as above and
samples were taken at 0 hr., 6 hr., and 14 hr. intervals
thereafter over a period of 48 hr. Samples of 0.2 ml were

removed and cell counts were made for macrophages,

21

lymphocytes and polymorphonuclear leukocytes. The cells
were stained with a crystal violet stain. Cells were
counted on an Improved Neubauer Haemocytometer (Spencer AO).
Cells were counted from normal, E. EEEE immune, and E. gggig
immune cultures.

Testing showed that both bacteria used in the pres-
ent study were sensitive to ampicillin. As a control upon
the system for the later growth experiments, the cell
kinetics were studied to determine the effects of parasiti-
zation and control of extracellular bacteria with ampicil-
lin. Therefore, cultures were collected as above. After
6 hr. incubation to achieve a constant cell population, the
cultures were incubated with 5x105 bacteria per m1 of
either E. 33213 or E. EEEE- After a period of one hour for
phagocytosis, llug per ml of Ampicillin (Omnipen-Weyth) was
added to the culture flask. This concentration represented
the minimal inhibitory dose for both bacteria. Samples

were removed as above at 0 hr., 6 hr., 18 hr., 30 hr., and

48 hr. and differential counts were made.

Accumulation of Ampicillin within Immune Peritoneal Cells

 

Immune cells have increased metabolism and phago-
cytic capabilities (32). Therefore it was possible that
they might accumulate the antibiotic used to control extra-
cellular bacterial growth. B. ggig and E. gggig immune
cells were tested for accumulation of ampicillin. Peri-

toneal cell cultures were harvested as above and incubated

22

for 6 hr. to achieve a constant cell population. The cells
were then incubated with llug of ampicillin per ml. After
24 hr. or 48 hr. of incubation an entire culture was
removed to a siliconized conical centrifuge type and cen-
trifuged at 1400 revolutions per minute on an International
Clinical Centrifuge (International Equipment, Needham Hts.,
Massachusetts) for 10 minutes. The supernatant was removed
and tested for killing ability by incubation with either

E. EEEE or E. EEEiE- Samples of bacterial growth were
taken at various times over 48 hr., plated on Trypticase
Soy agar and counted 4 days later. The growth of these
bacteria in ampicillin diluted in tissue culture medium
that had not been incubated with cells served as the con-

trol.

Assay of Brucellar Growth in Normal and Immune Cell Cultures

Peritoneal cells were harvested from 4 to 5 mice
and cultured according to the procedures outlined above.
After an incubation period of 6 hr. to allow stabilization
of cell populations, the cultures were infected with 5x105
bacteria per ml. This concentration represented an infec-
tive dose of approximately 1:1. This low dose was chosen
to limit a large extracellular population of bacteria.
IPhagocytosis was allowed to continue for one hour before
adding llug per m1 of ampicillin to the culture. At 0 hr.,
6 hr., 18 hr., 30 hr., and 48 hr., 0.3ml samples were

removed from the culture flasks. The sample was diluted

23

in 1.7 ml of saline-peptone and centrifuged at 1400 rpm for
5 minutes. The supernatant was placed in a sterile tube
and chilled while the packed cells were washed in one
additional ml of saline-peptone. The washed cells were
again centrifuged and the supernatant of the second was
combined with that of the first. The packed cells were
then resuspended in 0.5 ml of Staphylococcal delta-
hemolysin. This is an exotoxin produced by the Staphylo-
coccus which lyses animal erythrocytes and leukocytes.

The delta-hemolysin used in these experiments was prepared
by Dr. F. A. Kapral (Ohio State University) and supplied by
Mrs. D. Muirhead (Michigan State). Testing indicated that
better than 99% of the peritoneal cells in the samples were
lysed after 20 minutes incubation at ambient temperature,

with lOOHD5 units per m1, without damage to the brucellae.

0
Intracellular and extracellular bacterial numbers
were determined by the plate count method using Trypticase

Soy agar and reading plates after 4 days incubation at 37C.

RESULTS

Cell Kinetics of Peritoneal
Cells—in Culture

 

 

The first control experiments upon the 12.21EEQ
system for the measurement of cellular immunity were to
determine the cell kinetics in the system. It was found
that after 6 hrs. incubation the cell populations reached a
relatively constant number of macrophages and lymphocytes.

The harvested macrophage populations in each group,
normal, E. 521$ immune, and E. EEEi§_immune, numbered about
1.5x106 per ml. After 6 hrs incubation the normal macro-
phage numbers drOpped to 6 to 7x105 per ml, while the
E. §23§_and E. EEEEE immune macrophages were 3 to 5x105 per
ml. These levels were maintained over the period of experi-
mentation as shown in Figures 1, 2, and 3. Therefore, all
following experiments were incubated 6 hrs. prior to begin-
ning the experiment to allow maintenance of a constant
macrophage population.

Lymphocyte populations also remained relatively

constant over the period of experimentation. The normal

6 5

lymphocyte numbers dropped slightly from 1.5x10 to 8x10

per m1 over 48 hrs. The E. suis immune lymphocytes

5 6

remained from 9x10 to 1x10 per m1, and the E. canis

24

25

.1 ‘ V cflflofism

+ chm0 .m + mmumoonmaq : . v

 

CHHHHOHQEM + mflsm .m + mmumooam8>q
: . v omuommcfics mmumoogmfimq I 4 v

CHHHHOHmEm + mflcmo .m + mommnmouomz

 

“A nu v CHHHAOHQEM + mash .m.+ mmmmnm
nouomz I O v pmuommcflcs mommnmouomz "maongm
.Hmpmq usom

mco popp< GHHHHOHmad paw waamosum sufl3 pmuoomsH
mmHSDHso Hamo Hmmcouwnmm Hmfiuoz mo mowuocwx Hamo

 

.H musmflm

Ml

lDl Gills PER

10

26

 

 

30

0
TIME NI:

 

27

.1 ‘V cflfioflfi

+ mflcmo .m + mmeoonmemq : . V

 

CHHHAUHQEM + mflsm .m + mmpmoonmfiwq
: .V Umuommcflcs mmuwoonméflq : G V

cHHHflonEm + mflcmo .m + mommnmouomz

 

“A nu V cflaafloflmfim + mflsm .m + mommnm
nouomz I O V omuommcfics mommnmouomz "maongm
.Hmumq noon mco coupe

caaaaoamE< use waaoosum nuazl pmuommcH mmusuaso
Hamu Hmmcoufiumm mGSEEH mwcmo .m mo mowuwcflm Hamo

.N musmfim

ll. Dill! PEI ll

 

  
 

28

 

A __ ~A
{" ...._._--I‘l’ II
\n/

\

fl\§i >O-A _QA ‘O—A

I. 20 40 50
III! IDS

 

29

.1 4V 538315..

+ mflcmo .m + mmuwoogmfiwq 11 . V

 

GAHHHOHQEM + mHSm .m + mmuwoosmemq

I . V omuommcflcs mmuwoonmgq 2 4V

GHHHAOHQEM + mwcmo .m + mommzmouomz

 

“A flu V CHHHHonEm + mflsm .m + mommnm
nouomz : O V pouomwcflcs mommnmonomz "maonim
.Hmnmq usom oco coped

GAHHHOHmfid can maamosum muflsrpmuommcH mousuasu
Hamu HomGODAHmm masEEH mflsm .m mo moaumcwx Hamu

.m wusmflm

Dill: PEI ll

30

 

" 2' nut In: “

 

31

immune lymphocytes remained at 6 to 7x105 per ml as shown
in Figures 1, 2, and 3.

The polymorphonuclear leukocyte populations in each
type of culture were in the 104 per ml range and generally
tended to drop in numbers with continued culture.

Since it was possible that the infection of the
cell cultures with brucellae might cause a disruption of
the cell kinetics, which could alter interpretation of the
growth curves, it was decided to determine the cell kinetics

under infection conditions. Ampicillin was shown by experi-

,1

W
. 3141.13

 

ment to be an effective means of controlling the growth of
brucellae. Therefore it was chosen to control the extra-
cellular brucellar growth in the infected cell cultures.
Thus determining the cell kinetics under the combined con-
ditions of infection and antibiotic control would serve a
dual purpose. It would show any toxic effect of the anti-
biotic on the cells in culture, as well as any toxic effect
of brucellae products or brucellae growth on the cell popu-
lations.

Figures 1, 2, and 3 show that neither brucellae
infection nor ampicillin treatment caused any change in the
macrophage kinetics in cell culture for any of the three
types of cells. The lymphocytes appear to follow a similar
pattern of behavior, remaining relatively constant over the
period of experimentation, although the E. §3i§_immune
lymphocytes appear to decrease slightly over 0.4 log per

ml in 48 hrs. in cultures infected with E. suis.

32

Accumulation of Ampicillin within
Immune Peritoneal Cells

 

 

Ampicillin was chosen to control the growth of
extracellular brucellae in the cell culture system. The
usage of antibiotics made necessary a series of controls
to assure that the antibiotic was not taken up by the immune
cells to any great degree. Should the antibiotic be taken
up by the immune system it would confuse any killing
results seen later. The method of attacking this problem

was to incubate the ampicillin with the cell cultures for I g

 

various times, spin down the cells, and test the supernatant
of the incubated cultures for killing ability. This was
compared to the control curves of unincubated ampicillin.

Figure 4 shows the growth curves of E. BREE in
ampicillin incubated for 24 and 48 hrs. with E. Eggig
immune cells. The ampicillin incubated with peritoneal
cells for 24 hrs. showed a killing rate which was not sta-
tistically different from the control as determined by the
Students t test comparing the difference between two means
at a 95% confidence interval. However analysis of the 48
hr. incubated ampicillin indicated that at some points
there may be accumulation. Therefore, it was decided to
use the growth of E. gggig as a test system on the accumu-
lation of ampicillin by E. EEEEE immune cells. The results
are shown in Figure 5. Statistical analysis using the

Students t test showed there was no evidence of significant

.1 AC‘V .muc

we MOM maamo QDHB poumnsocfloum cHH

33

 

Igoflmfim + mflsm .m 11 4V .mu: em How

mHHmo LDH3 omumnsocwwum CHHHAOHQEM

 

+ meow am 11 nu V maamo suflz cowumn

 

usocflmum usonufl3 cwaafloflmfim + mfism .m "maonemm

 

.Emummm umma on» mm mflsm .m mo suzonw mam
mcflmn mousuaso Hamo Hmwcouflnmm mcsEEH mflsmo .m
we sfiaafloflmad mo coaumasedood umHsHHoomuucH

.v musmflm

3S

.1 ‘V .93 we now maamo Sufi; pmumn
Isosflmnm GAHHAOAQEM +.MHmmm .m 11 AN V
.mug em How maamo QDAB pmumnsocflmud
323% . Iml .m .1 IV .92 o
How maamo QDHB pmumnsucflmum CHHHHUHQEM
+ mmmmm am 11 mu V mHHmo £DH3 cofiumn

Isocflmum usonuflz CAHHHUHQEM + macho .m

1maonfimm

.Emummm puma on“ mm mflcmo .m mo £u3onw map

mchD mousuasm Hamo Amocouwumm mcsEEH macho .m

ma cflHHHOHmE¢ mo coaumHDEsoom HMHDHHmUMHHGH

.m musmwm

   

37

accumulation of ampicillin over the 48 hrs. of incubation
with E. canis immune cells.

The same approach was used to test E. BREE immune
cells for accumulation of ampicillin. In this instance
E. E213 growth was again used as the test system. The
results in Figure 6 show there is no evidence of signifi-
cant accumulation of ampicillin by B. EEEE immune cells

over 48 hrs.

Growth of Brucellae in Normal and
Immune Cell Cultures

 

 

Both E. canis and E. suis grow within normal mouse
peritoneal cells in culture as shown in Figure 7. In the
£3 vitro system, the extracellular brucellae, under ampicil-

3 to 104 per m1

lin control, drop in numbers into the 10
range within 6 hrs. and remain constant throughout the
experiment. Both E. EEEEE and E. ggig behave in a similar
manner extracellularly.

Intracellularly, there is an initial drop within
the first 6 hrs. of the experiment. After the initial
drop, both E. gggig and E. ggig steadily increase intra-
cellularly throughout the experiment. Statistical analysis
using the Students t test for comparison of two means indi-
cate that although the curves appear similar they are sta-
tistically different. The curve of intracellular E. EEEE
drops to a somewhat lower level at 6 hrs. than that of

E. canis, however from there increases at a greater rate

than intracellular E. canis.

 

38

.14V .31

em MOM mHHmo zuflz Umumnsocflmum QHHHHU

 

IHQEM + mflsm .m 11 AV V .mun we “Om

maamo QDHB pmumnsocflmnm cHHHHOHQEM

 

+ mHSm .m 11 mu V maaoo suHB coflumn

 

Isocflmum unonuflz :HHHHOHmEm + mHSm .m

 

"maonfimm

.Emumwm umoa ecu mm meow .m mo nuzouo mam
mcflmm mmusuHsO Hawo HomQODfiumm ocSEEH mean .m
an cHHHH0flmE¢ mo coHumHseooo¢ ansHHmomuucH

 

.m mHsmHm

39

AA”.

1: an; ¢1¢m_u- :91

20 4| 50
IIME IRS

10

4O

.1 ‘V .13

Idaamomuuxm mflcmo .m 11 AN V HMHSHHmo

Immune mflcmo .m 11 . V Hmasaamomuuxm

 

 

mash .m 11 mu V HMHSHHmomuucH mHSm .m 1maonewm

I .mmusuaow
HHmU Hmmsouflumm HmEHoz CH mwcmo .m one mash .m
mo npzouo Hmasaamomupxm ocm HmHsHHmomuucH

 

.h mnsmflm

42

Figure 8 shows the results of E. gggig and E. EEiE
growth in E. EEEE§_immune cells. Again the extracellular
behavior of both bacteria is similar, dropping into the 103
per ml range between 6 and 18 hrs. and remaining constant
over 48 hrs. The intracellular results between the two
brucellae are markedly different. E. EEEE decreases intra-
cellularly about 2 logs per ml in the first 6 hrs. and
slowly decreases over 48 hrs. E. EEEEE drops in number
about 1.2 logs per ml in 6 hrs. and remains constant over
48 hrs. These curves are obviously different and have been
shown statistically different.

The results of E. gggig and E. §2i§_growth in
B. Egi§_immune cells are shown in Figure 9. The E. EEEEE

extracellular curve behaved as before, decreasing to the

103 per ml range in 6 to 18 hrs. and remained constant
thereafter. However, the E. E212 extracellular curve drops
further than the E. 92215 by about 0.5 log per ml and main-
tains a lower level over 48 hrs. The E. EEEEE intracellular
curve in E. EEEE immune cells appears similar to that of

E. canis in E. canis immune cells. It drops 1.2 logs per

ml in 6 hrs. and remains constant over 48 hrs. E. suis

 

growth in E. suis immune cells drops 2.1 logs per ml in 18
hrs. and generally decreases over 48 hrs. to a level

similar to that in E. canis immune cells.

 

EMA-1‘1

41.,

 

43

 

14:3

loaamomuuxm mflcmo .m 11 AN V umHSHHmo

Imnucfl mficmo .m 11 - V Headaamomuuxm

 

mHSm .m 11 mu V Hmasaamomuucfl mash .m "maonfiwm

IIIII I IMMWMDDHDU Hamu Hmmm
noeflnmm ocsEEH mflcmo .m CH mflcmo .m can meow .m

mo £u3ouo Haasaamomuuxm paw HmasaamomuucH

 

.m onsmflm

 

 

 

 

 

 

 

 

45

 

.1 ‘V .13
Ioaamomuuxm mflcmo am 11 AN.V Hmasaamo

IMHDGH mflcmo .m 11 n- V udeHHmomupxm

 

mflsm .m 11 mu V amasaamomuucfl mesh .m "maonfimm

 

I @3950 :60 Hmmm
uouflumm masEEH mHSm .m 2H mflcmo .m paw mflsm .m
mo nusouw HmHDHHmomupxm cam HmHSHHmomHucH

 

.m onsmflm

[DD IICIEIIA PEI Ml

‘

III

 

 

46

20

 

3D
IIME HIS

 

40

DISCUSSION

As indicated in the results section, the experimental
work in this paper consisted of three groups of experiments.
The experiments on cell kinetics and antibiotic accumulation
were controls on an 13 31359 system devised to measure cell
mediated immunity. The last set of experiments measured
cellular immunity using restriction of bacterial growth as
a parameter.

Most other 12.2iggg systems for measuring cellular
immunity utilize the Leighton tube as a culture vessel and
the adherence of cells to glass for purification. The sys-
tem used in the present study utilizes a suspension tech—
nique which more closely resembles an ig_yéyg_situation.
This technique allows easy sampling without using numerous
tubes. The cell population can be easily monitored in the
suspension system. In this case one suspension culture
represents one mouse, and it is easier to keep cultural
conditions constant. Using the Leighton tube method, it is
possible to obtain relatively pure populations of macro-
phages which would allow somewhat more defined experiments
in areas concerning the role of the macrophage in cellular
immunity. The suspension type system uses a mixed popu-
lation of cells which is what is seen EE 2329. Attempts in

47

48

this laboratory to purify macrophages by adherence to glass
and then culture in suspension have failed, as we were
unable to detach the macrophages. Since there are indi-
cations that glass adherence may stimulate macrophage acti—
vation, precautions were taken to minimize adherence in the
suspension system.

Once it was decided to use this system, the first
necessary control was to monitor the cell kinetics in the
suspension culture. As shown in the results a constant
pOpulation of macrophages and lymphocytes was achieved
after 6 hrs. incubation. The populations remained constant
over 48 hrs. The polymorphonuclear leukocytes represented
a small proportion of the cells present in culture, and
decreased in number with increased incubation.

Even under infection conditions the macrophage and
lymphocyte populations remained at a constant level, the
only exception being E. EEEE immune lymphocytes in the
presence of E. EEEE' This drop is unexplained, however it
possibly may be due to a hypersensitivity reaction.

It could be argued that the decline of the poly-
morphonuclear population could be causing some of the bac-
terial killing seen in the immune system, by release of
enzymes such as myeloperoxidase. This however is doubtful,
since polymorphonuclear leukocytes seem to play little if

any role in immunity to Brucella (34). Although readily

 

phagocytized by polymorphonuclear leukocytes, brucellae are

49

not killed, and even multiply within. Therefore, it is
doubtful that polymorphonuclears play any significant role
in the present experiments.

The use of antibiotics to control the extracellular
population of bacteria is a controversial technique. Many
early workers studying cellular immunity ig_yi£52 used
streptomycin as a means of control (24, 46). However
recent work by Bonventre and Imhoff (5) and Patterson and
Youmans (42) suggests that streptomycin enters macrophages
in culture. Methods used to avoid the use of antibiotics
include extensive washing of cells to eliminate extracellu-
lar bacteria, and using extremely low infection doses (19).
These techniques are inapplicable to the system used in the
present experiments. The suspension culture does not allow
extensive washing, as this requires repeated centrifugation,
after which resuspension becomes very difficult. The low
dose infection technique is impractical with mouse peri-
toneal cell cultures as mice are relatively resistant to
infection by brucellae and it takes a relatively high num-
ber to establish infection. The percentage of phagocytosis
of Brucella is low in these cells, which necessitates use
of larger numbers to give assayable results. Therefore,
antibiotic treatment was a necessity. It was decided to
use a member of the penicillin family of drugs, as this
antibiotic is thought not to enter cells.

Eagle (12) had shown that epitheloid cells treated

with low doses of penicillin did not accumulate penicillin.

50

Unpublished experiments in our laboratory also showed no
evidence that macrophages incubated with 25 units of peni-
cillin per ml for 48 hrs. accumulate significant penicil-
lin. Therefore, ampicillin was chosen as an effective
penicillin to control the extracellular growth of brucellae.
A series of controls were run to determine if any ampicillin
were taken up by the immune macrophages in the suspension
culture system. The results showed no evidence that immune
macrophages take up significant amounts of ampicillin over
48 hrs. incubation. Therefore, any killing of bacteria by
immune macrophages was not due to antibiotic treatment.

As shown in the results E. E212 and E. Eggig both
multiply within normal peritoneal cells. The behavior of
E. Egig intracellularly was expected, as early i§_yi§£9_
workers (23, 24) showed that smooth strains of brucellae
multiplied rather rapidly in both chick embryo fibroblasts
and guinea pig and mouse peritoneal monocytes. These
authors showed that non—smooth strains multiplied poorly
or not at all in these cell cultures. E. gggi§_is not a
smooth strain brucellae, yet multiplies within normal cells
as shown in Figure 7. The intracellular growth rate of
E. gggig is slightly slower than that of E. ggig, however
the results show the patterns to be similar. E. gggig also
differs from most other non-smooth brucellae in that it
causes disease.

The behavior of E. EEEE and E. Eéflifi in immune

cells in culture adds some knowledge to the role of cellular

51

immunity in Brucella infections. The results show that
both bacteria are killed intracellularly in immune cell
cultures. In both E. EEEE and E. g§§i§_immune cell cul-
tures E. ggig decreases in number steadily over 48 hrs.
E. 233i§_drops within the first 6 to 18 hrs., then remains
constant over 48 hrs. Since the intracellular patterns are
similar in both E. BREE and E. EEEEE immune cells, it would
suggest that both immunizing agents produced a similar
degree of cellular immunity. The results also support the
concept of the nonspecific nature of cellular immunity, for
E. 9321§_immune cells are equally efficient in limiting the
growth of E. ggig as the E. §21§_immune cells.

The necessity for immune serum in antibacterial
cellular immunity, has been questioned by some and con-
sidered necessary by others. In working on acquired resis-

tance to Listeria monocytogenes, Miki and Mackaness (37)

 

were unable to detect any effect of immune serum on cellu-
lar immunity. Mackaness (31) suggested that the nonspecific
nature of cellular immunity made it unlikely that an
antigen-antibody reaction on the bacterial cell surface was
an important factor. He suggested that antibody bound to
the macrophage cell surface would activate it nonspecifi-
cally and enhance antibacterial activity.

The situation in immunity to Salmonella is also

 

controversial. Turner et a1. (56) reported that immunity

in mouse typhoid is due primarily to humoral antibodies.

52

Mackaness (32) and Blanden (4) reported that immune serum

increases clearance of Salmonella from blood, but does not

 

prevent growth in the tissues, which they feel is dependent
on enhanced antibacterial ability of macrophages.

The role of immune serum in anti-Brucella immunity

 

is also confused. The $2 vitro work of Holland and Pickett

(24) indicated that anti-Brucella antisera had no effect on

 

the growth or survival of brucellae within normal or immune
monocytes. More recently, Ralston and Elberg (48, 49)
reported the appearance of two serum factors following

immunization with E. melitensis Rev I. The activity of

 

both could be absorbed out with the organism. These fac-
tors enhanced the ability of the monocyte to attach and
ingest the organism, restrict growth intracellularly, and
enhanced bactericidal activity. In the present study, the
nonspecificity seen in the results and the better elimi-
nation of E. EEEE rather than E. EEEEE in the E. gggig
immune cells lends support to the concept of a minimal role

if any for immune serum in anti-Brucella immunity.

 

It is interesting to note that E. Eéflii is not
eliminated well by either E. EEEE or E. EEEEE immune cells.
One possible explanation for these results resides in the
antigenic nature of E. Eéflifi' This organism is not a

typical rough Brucella. It is mucoid in nature and with

 

continued growth produces much mucoid material. This
material may be protecting the organism from lytic enzymes

and intracellular death.

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Badakhsh, F.F., and Foster, J.W. 1970. Endotoxin-
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Blanden, R.V., Mackaness, G.B., and Collins, F.M.
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Bonventre, F.F., and Imhoff, J.G. 1970. Uptake of
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Deyoe, B.L. 1970. Studies on the Pathogenesis of a
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Diaz, R., Jones, L.M., and Wilson, J.B. 1968. Anti-
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Ellwood, D.C., Keppie, J., and Smith, H. 1967. The
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