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University
Microfilms
International
300 N. Zeeb Road
Ann Arbor, Ml 48106
N
1320110
MAPOTHER, MARY ELIZABETH
IN VITRO INTERACTION OF MYCOBACTERIUM AVIUM WITH INTESTINAL EPITHELIAL CELLS
THE UNIVERSITY OF ARIZONA
University
Microfilms
International
300 N. Zeeb Road, Ann Arbor. MI 48106
M.S.
1982
IN VITRO INTERACTION OF MYCOBACTERIUM AVIUM
WITH INTESTINAL EPITHELIAL CELLS
by
Mary Elizabeth Mapother
A Thesis Submitted to the Faculty of the
DEPARTMENT OF ANIMAL PHYSIOLOGY
In Partial Fulfillment of the Requirements
For the Degree of
MASTER OF SCIENCE
In the Graduate College
THE UNIVERSITY OF ARIZONA
1 9 8 2
STATEMENT BY AUTHOR
This thesis has been submitted in partial fulfillment of
requirements for an advanced degree at the University of Arizona
and is deposited in the University Library to be made available
to borrowers under the rules of the Library.
Brief quotations from this thesis are allowable without
special permission, provided that accurate acknowledgment of
source is made. Requests for permission for extended quotation
from or reproduction of this manuscript in whole or in part may
be granted by the head of the major department or the Dean of the
Graduate College when in his judgment the proposed use of the
material is in the interests of scholarship. In all other
instances, however, permission must be obtained from the author.
SIGNED
APPROVAL BY THESIS DIRECTOR
This thesis has been approved on the date shown below:
/
J. G(^So)iger
rofessor of Veterinary Sciences
ACKNOWLEDGMENTS
I would like to extend my appreciation to Dr. J. G. Songer
for his encouragement, guidance, and often necessary sense of
humor.
To the entire staff of the Veterinary Science Department,
my sincere gratitude for their assistance and patience.
For his valuable time and expertise concerning computer
analysis, I wish to extend a very special thanks to Dr. L. M.
Kelley of the Department of Microbiology.
And, to my parents, Richard and Francis Mapother, my
greatest appreciation for their continuous faith and love; without
them, this thesis would not be possible.
TABLE OF CONTENTS
Page
LIST OF ILLUSTRATIONS
v
LIST OF TABLES
vii
ABSTRACT
viii
1.
REVIEW OF LITERATURE
1
2.
MATERIALS AND METHODS
25
Experimental Design
Bacterial Cultures
Cell Cultures
Antiserum Preparation
Method of Assay
Staining Techniques
Microscopy
Statistical Analysis
3.
25
25
26
28
30
35
37
37
RESULTS AND DISCUSSION
40
Kinetics Studies
Host Cell Effects on Uptake
Bacterial Effects on Uptake
4.
40
48
54
CONCLUSIONS
67
LIST OF REFERENCES
68
iv
LIST OF ILLUSTRATIONS
Figure
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Page
Kinetics of the Interaction of M. avium
Serotype 2 with HIE Cells
41
Percent of M. avium Serotype 2 Shown to be
Intracellular when Associated with HIE Cells
42
Percent of M. avium Serotype 8 Shown to be
Intracellular when Associated with HIE Cells
44
Percent of M. avium Serotype 10 Shown to be
Intracellular when Associated with HIE Cells
45
Kinetics of the Interaction of M. phlei
with HIE Cells
46
Kinetics of the Interaction of M. smegmatis
with HIE Cells
47
Difference in Uptake of Autoclaved and Viable
M. avium Serotype 2 by HIE Cells
55
Staining Characteristics of Methanol-Fixed and
Unfixed HIE Cells Infected with Autoclaved
M. avium Serotype 2
56
Difference in Uptake of Formalin-Killed and
Viable M. avium Serotype 2 by HIE Cells
57
Staining Characteristics of Methanol-Fixed
and Unfixed HIE Cells Infected with
Formalin-Kilied M. avium Serotype 2
59
Difference in Uptake of M. avium Serotype 2
Irradiated with Ultraviolet Light for
0 to 60 Seconds
60
Staining Characteristics of Methanol-Fixed and
Unfixed HIE Cells Infected with M. avium
Serotype 2 Irradiated with Ultraviolet
Light for 0 to 60 Seconds
61
v
Figure
13.
14.
15.
Page
Interaction of M. avium Serotype 2 Suspended
in Various Concentrations of Homologous
Antiserum with HIE Cells
63
Interaction of M. avium Serotype 2 Suspended
in Bovine Milk or in MEM with HIE Cells
64
Interaction of M. avium Serotype 2 Suspended
in Bovine Colostrum or in MEM with HIE Cells
65
vi
LIST OF TABLES
Table
1.
2.
3.
4.
Page
Effect of Cytochalasin B on Interaction
of M. avium Serotype 2 with
HIE Cells
49
Effect of Dibutyryl-cAMP on Interaction
of M. avium Serotype 2 with
HIE Cells
51
Effect of lodoacetate on Interaction
of M. avium Serotype 2 with
HIE Cells
52
Effect of 2,4-Dinitrophenol on Interaction
of M. avium Serotype 2 with
HIE Cells
53
vii
ABSTRACT
An in vitro model was developed to study the infection of
cultured Henle intestinal epithelial (HIE) cells with pathogenic
Mycobacterium avium and saprophytic mycobacteria.
Results indicate that uptake of pathogenic M. avium occurred
after 2 to 3 hours of incubation.
Saprophytic mycobacteria did not
attach to or penetrate the host cells of this model.
Conditions
necessary for uptake of M. avium serotype 2 by HIE cells include
viability, expression of surface molecules, and normal metabolic
activity of the tubercle bacilli.
Furthermore, results indicate that
the mechanism of uptake is an endocytic process induced by virulent
mycobacteria.
viii
CHAPTER 1
REVIEW OF THE LITERATURE
Swine tuberculosis is a disease problem that has affected the
pork industry of the United States since the early 1900's.
1 OR
The
prevalence of tuberculosis in swine has been estimated at 0.85% of all
swine slaughtered under federal inspection,
109
representing an almost
5 35
$6 million annual loss to the pork industry. '
The incidence of tuberculosis in swine is related to the
opportunity for direct or indirect contact with tuberculous primates,
cattle, avian species, and numerous environmental sources.
Over the
years most tuberculous lesions in U.S. swine have been found to be of
avian origin.^' ^ U.S.D.A. statistics have shown that chickens in
the north-central region of the United States have the highest per­
centage of condemnations due to lesions of tuberculosis.^' ^
This
coincides with reports that states of the west north-central and east
north-central accounted for more than 83% of swine condemnations and
for more than 63% of carcasses retained for tuberculosis in 1971.^' ^
Nationally, the incidence of swine lymphadenitis has declined. 6
Most
of this decline is largely attributable to an increase in the prac5
tice of maintaining all-pullet farm flocks among poultry producers. '
20
Control of tuberculosis among cattle, through tuberculin testing
and slaughter of reactors, is also thought to have decreased the loss
among hogs.
5
Of the 31,843,375 cattle slaughtered under federal
1
2
inspection in 1972, only 246 carcasses (0.0008%) had tuberculous
lesions.
In contrast, of 83,126,396 swine slaughtered during the same
year, 712,227 carcasses (0.85%) had lesions suggestive of tuberculo5 20
sis, nearly 1000 times more than were found in cattle. '
The
above figures indicate that tuberculosis may no longer be considered
such a serious problem in swine in the U.S., but the decrease of the
disease has not been as pronounced as for bovine and avian tubercu­
losis.
In 1972, the Food Safety and Inspection Service of the
U.S.D.A. instituted new rules and regulations concerning tuberculous
lesions in swine.
57
Before 1972 when these granulomatous lesions were
found at slaughter, they were excised and discarded, and the remainder
5 20
of the carcass was processed as usual. '
Following new regulations,
swine carcasses, and/or parts, are labeled as condemned, passed for
cooking (PFC), or passed without restriction for human food.
57
The
entire carcass is condemned when the character of the lesions is not
indicative of a localized condition.
Portions of carcasses and
whole carcasses are labeled PFC if they reveal lesions indicative of
a localized condition.
Such carcasses or parts must be cooked at
76.7°C for 30 minutes after removal of the infected part to be
considered fit for human consumption.
Portions of carcasses and car­
casses of swine are passed without restriction provided the carcass
is found free of lesions or when lesions are found confined to one
primary site of infection.
demned carcasses is obvious.
The economic devastation due to con­
Cooked carcasses lose about 2/3 of
3
their original value. 5 '
20' 106
This loss is felt by the packing
industry and the consumer, but mostly by the pork producer, since he
not only obtains a lower price for his hogs, but may lose his market
altogether. 5 '
2 ®'
^
Etiology
Historically, the etiologic agents of tuberculosis in swine
have been the same agents which have caused tuberculosis in the
animals with which swine have had contact.
Before the start of
attempts to eradicate both human and bovine tuberculosis, isolations
of tubercle
bacilli from lesions in swine were often shown to be of
either bovine (Mycobacterium bovis) or human (Mycobacterium tuberculosis) type.
20
However, Mycobacterium avium causes most mycobacterial
disease i n swine i n the U.S. t o d a y . - A d d i t i o n a l
evidence implies that M. avium serotypes 1 and 2, which also cause
disease in chickens, are the organisms most frequently associated with
tuberculous lesions in s w i n e , ^ a l t h o u g h many myco­
bacteria in the M. avium complex are also involved.^'
Other
species of mycobacteria less frequently isolated in connection with
swine tuberculosis include M. bovis, M. tuberculosis, M. scrofulaceum,
M. xenopi, M. kansasii, M. microti, M. ague, and M. chelonei.
20
'
36
'
37, 62, 102, 105, 108
Corynebacterium equi has also been isolated from lymph nodes
of swine with tuberculos+s^Tfce—'lesions. 2 ^'
The
organism produces localized lesions and cannot be easily differen­
tiated from tuberculous processes macroscopically or histologically.
4
21
'
43
'
108
It has been reported that Corynebacterium equi is often
found in association with tubercle bacilli in swine J'
^
Other
workers have reported that it occurs with equal frequency in normal
and M. avium-infected lymph nodes.
20
Although the relationship
between £. equi and M. avium and the possible role of
equi in
producing tuberculosis-like lesions in swine needs further investiga­
tion, its effect on occurrence of the disease is probably minimal.
Taxonomy
Koch's report of the tubercle bacillus in 1882 was-followed
in 1889 by Rivolta's description of the avian type bacillus, and in
1898 by Theobold Smith's differentiation of the bovine type
bacillus.
20
However, in the 100 years since, the taxonomy of myco­
bacteria has become more awkward and complex.
This is especially
true for organisms now included in the M. avium complex.
Schaefer is the person considered instrumental in taxonomy of
what is now the M. avium complex.
In 1935, he began his study of the
serological properties of the "atypical" mycobacteria.^
He found
that all avian strains were of 2 serotypes, characterized by their
reactions with type-specific antisera.
He also found that other
"atypical" mycobacteria (Battey type) could also be typed serologically.
117
M. intracellular and M. scrofulaceum were named as
species separate from M. avium, although biochemical differences
among these 3 are so minimal that they cannot be reliably differen­
tiated by this means.
Almost 20 serotypes have been established
among cultures identified as M. intracellular, and several among
5
those identified as M. scrofulaceum.^' ^
In spite of objections by Runyon, who believed M. intraQC
cellulare should remain a distinct species,
now been grouped into the M. avium complex.
these organisms have
The problem has been
resolved somewhat by the new scheme proposed by Wolinsky and
Schaefer in 1973.^
Individual species names are dropped and the 3
are grouped together in the "Mycobacterium avium
complex."
Twenty
numbers are reserved in the scheme after the former M. intracellular
group for possible new types related to this group or to M. avium,
and it is believed this new scheme will be a great aid in the study
of these organisms.
Epidemiology
Because most swine tuberculosis in the United States is of the
avian type, and appears to be most prevalent in those geographic
regions where tuberculosis in chickens is greatest, it might be
assumed that infected chickens are the source o f this d i s e a s e . ^ H o w ­
ever, it should be kept in mind that this data comes from meat inspec­
tion records which may be misleading.
Swine are often transported to
abattoirs at great distance from their origin and, in addition, diagnosis is made on the basis of the macroscopic appearance of lesions.
Some liesions may not be macroscopically visible and, therefore, some
infections are missed.
Also, there are reports of tuberculosis in
hogs in areas where there is no association with chickens.
Although
tuberculous chickens may be the primary source of the disease in
swine, it seems very probable that other aspects of the swine
108
6
environment also have a role in the origin and transmission of the
disease.
The opportunity for transmission of tuberculosis among
tuberculous poultry, cattle, and swine has been documented by many
w
o
r
k
e
r
s
.
^
B o v i n e t u b e r c l e b a c i l l i a r e n o t a
common cause of tuberculosis in swine in areas where the disease in
cattle is controlled.
14
Where tuberculosis does occur in cattle,
the infection may be transmitted to swine by feeding of unpasteurized
milk and dairy by-products.^
Also, feces of tuberculous cattle may
contain viable tubercle bacilli, which makes the maintenance of
swine and cattle in common feedlots impractical.^
Tuberculosis in chickens is found in the pectoral muscles, eggs,
and reproductive organs.^ 5
The feeding of offal of tuberculous
chickens, and the raising of swine in areas previously occupied by
tuberculous chickens have been shown to be of great significance in
the transmission of avian type tuberculosis.
14
'
23
Schalk and co­
workers found viable and pathogenic avian tubercle bacilli in the
soil and litter of a chicken coop after 4 years with no chickens.
56
They concluded that soil contaminated by feces of tuberculosis fowl is
one of the most important sources of infection for swine, and recom­
mended that young birds be reared on clean ground and that all fowl
more than 1 year old be disposed of regularly.
The transmission of tuberculosis from pig-to-pig has been
attributed to ingestion of contaminated feces,
56
intrauterine infec­
tion, and aerosol infection from pigs showing pulmonary lesions.®
7
However, work being done in our laboratory indicates that fecal
shedding of the organism does not occur, and that transmission from
pig-to-pig may be rare.
Human tubercle bacilli have been isolated from lesions in
O
swine.
People known to have active TB should not be permitted to
have contact with domestic animals.
Soil, water, and house-dust have also been found to be possible
sources of mycobacterial infection.
28
'
56
'
84
Sawdust is often used as bedding for swine and has recently
been shown to be a source of mycobacterial infection for swine.
81 , 85, 95, 96
jn
g stuc jy
^ Beerwerth and Popp, M. avium complex
organisms were isolated from more than 3% of sawdust and wood shavings
samples examined.
Reznikov and Leggo found sawdust to be an especially
favorable medium for the multiplication of these organisms due to the
relatively high temperatures that develop in the sawdust piles.
84
Be­
cause swine often ingest sawdust, this reservoir could be an important
source of infection and could account for many of the cases of M. avium
complex disease in swine.
Wildlife is another possible source of the infection for pigs.
6 34 38
Many wild birds have been shown to be infected. '
'
As early as
1940, Hignett and McKenzie reported the occurrence of avian tuberculosis in starlings to be 1.3 - 4.8%.
34
They suggested the possibility
of the starling acting as the vehicle of infection in the spread of
avian tuberculosis.
Tubercle bacilli could have been introduced into
the swine herd by these birds.
Other wild birds found infected with
members of the M. avium complex include a trumpeter swan,
crane,
103
and a white-headed tree duck.
42
a sandhill
This emphasizes the impor­
tance of adequate quarantine and test procedures to detect diseased
birds before importation.
There has been an increased interest in tuberculosis in cap­
tive exotic species following recent reports of the disease in ani­
mals in zoos, primate centers, animal colonies at universities, and
animal and game parks.
The importance of these occurrences of tubercu­
losis is emphasized due to the public health hazard, the economic
losses, and the difficulty of replacing rare and endangered exotic
animals.
Thoen et al. summarize results of mycobacteria ogic examina­
tions conducted on 474 specimens submitted from exotic animals sus­
pected of having tuberculosis.^^
The widespread occurrence of tuber­
culosis in exotic animals maintained in captivity emphasizes the
importance of these infections.
The exact source of tuberculosis for swine is not always known.
The organisms may be present in many areas of the swine environment or
originate from only 1 source common to all animals.
Many workers
agree that the key lies in more accurate and reliable procedures for
detection of M. avium.
Diagnosis
Clinical signs are of limited value in the diagnosis of M.
avium infection in swine.
Animals with progressive disease very sel­
dom show symptoms of an infectious disease.
If symptoms are present,
they are not sufficiently characteristic to establish a diagnosis of
9
tuberculosis.
In cases of rapidly disseminating disease, a circum­
stance which is not characteristic of M. avium infection, there may
be clinical evidence of infection including an elevation in body
temperature, anorexia, and weight loss.^' ^
Tuberculous lesions in swine are usually limited to the lymph
nodes of the pharyngeal and cervical regions and of the mesentery.
108
19
'
However, the lesions may be found to involve such organs as
liver, lung, spleen, and kidneys in widely disseminated or generalized
cases. 14 '
17'
»•
109
As stated previously, tuberculosis in swine may be caused by M.
bovis, M. tuberculosis, or M. avium.
Differentiating between porcine
lymphadenitis caused by M. avium and that caused by M. tuberculosis or
M. bovis on the basis of the gross lesions they produce is difficult.
Thoen and Karl son described some gross features characteristic of
each.
109
In avian tuberculosis, the lymph nodes are enlarged and firm
with no discrete purulent foci; there may be 1 or more soft caseous
areas with indistinct borders.
Calcification is seldom seen.
The cut
surface of a lesion has a neoplastic appearance with a few caseous
foci.
There may be diffuse fibrosis but no encapsulation; the granu­
lomas are not easily enucleated.
Mammalian lesions, on the other hand,
are well encapsulated and easy to separate from surrounding tissue,
calcification is prominent, and individual foci are discrete and
caseous.
Microscopically, avian tubercle lesions demonstrate diffuse
proliferation of epithelioid cells and giant cells.
There may be some
necrosis but calcification is seldom seen and only in older lesions.
There is a proliferation of connective tissue elements, but there is
little tendency to form a well-defined wall of fibrosis.
Mammalian
lesions become encapsulated by a well-defined zone of connective
tissue.
Early caseation and marked calcification are also
non+ 6» 1°6>
prominent.
109
It is generally agreed that it is impossible to identify the
causative agent on the basis of gross or microscopic appearance.
This
is probably due to the fact that there is a great similarity between
localized tuberculous lesions and those associated with C^. equi and
other bacteria.
Furthermore, it may be difficult to differentiate
among granulomatous lesions due to M. avium infection, parasitic nodules, and neoplasms.
109
A definitive diagnosis can only be made on
the basis of bacteridogic procedures designed for isolation, identifi­
cation and typing of mycobacteria.
However, there is 1 drawback.
It
is well known that tubercles can resolve into nodules of fibrous tissue
failing to yield tubercle bacilli.
83
Cultures should be incubated at 41°C in an atmosphere of 5-10%
COg; room temperature and 37°C without COg are also adequate.
Cultures
should be examined daily for the first week and then weekly for at
least 8 weeks.
Usually growth occurs in 14-21 days on solid media, but
it may take up to 5 weeks.
Colonies are round, smooth and glistening
with a light buff-yellow color.
white and translucent.
On Middlebrook 7H10 colonies are flat,
Morphologically, M. avium is a short, acid-fast
rod in lesions, but in cultures it may develop as acid-fast branching
filaments. 20,
41'
53
11
Routine biochemical tests on Mycobacterium spp. include niacin
production, hydrolysis of Tween 80, nitrate reduction, and tellurite
reduction.
M. avium is negative for all the above.
20
In the outer layer of their cell walls mycobacteria contain
a substance known as Wax D that is responsible for the relative
difficulty of staining of these organisms by conventional methods.
This problem is overcome by using the classical acid-fast stain.
41
With
this stain, mycobacteria stain cranberry red.
Two fluorescence .techniques are currently employed in myco­
bacterial staining: auramine O-acridine orange (AOAO) and the indirect
fluorescent antibody technique (IFAT).
29
'
39
'
72
The former is more
simple and requires fewer and less costly reagents, but the latter
may aid in speciating an isolate.
A special technique has been developed for detection of mycobacteria by electron microscopy.
32
The tuberculin test, used on a herd basis, seems to be a use­
ful tool in the diagnosis of tuberculosis in swine.
Tuberculins are
mixtures of soluble components of mycobacteria grown in liquid media.
Robert Koch, in an effort to find a treatment for tuberculosis
developed old tuberculin, a concentrate of the broth culture filtrate.
It produced a marked reaction when injected intradermally into people
with tuberculosis.
Old tuberculin was later shown to be unsuitable
for treatment, but its usefulness as a diagnostic tool was quickly
realized.
Currently, a purified protein derivative (PPD) of myco­
bacteria is used.
These reagents are fractions of the culture fil-
trate prepared by various methods of precipitation.
3
PPD reagents
12
have been prepared from M. tuberculosis, M. avium, M. fortuitum, M.
scrofulaceum, M. marinum, and M. kansasii.
PPD is now produced from
mycobacteria grown in synthetic liquid medium rather than in broth.
3
This procedure yields a very pure product, free from nonmycobacterial
protein.
The test is usually performed by injecting 0.1 cc of tuberculin
intradermally into either the ear or vulva, and results are read 48 and
72 hours after administration.
65
The size, shape, and consistency of
the tissue response do not reflect the degree of infection.
Unfortu­
nately, reports indicate that results from tuberculin skin tests in
pigs may be unreliable.
As early as 1952, Luke reported that there was
a large percentage of error due to non-specific sensitivity or to
residual sensitivity from healed lesions.^
Animals with negative skin
test reactions had lesions at slaughter which Luke ascribed to the
ability of the pig to overcome and apparently sterilize existing
lesions.
There still exists some controversy over the reliability and
reproducibility of the tuberculin skin test.
A peripheral blood lymphocyte stimulation test has been
studied and compared with the tuberculin skin test. 5 ^'
^ 06
Results indicate that in vitro lymphocyte immunostimulation may be of
considerable value in detecting tuberculosis in animals in which skin
testing has been used previously.^®' ^
More work needs to be done on
this test, especially a comparison between the 2 tests using
naturally infected pigs.
Lastly, the enzyme-linked immunosorbent assay (ELISA) has
recently been described for detecting antibodies in swine infected with
13
M. avium.^
Positive ELISA reactions were seen in experimentally and
naturally infected pigs.
It is a rapid, automated test, which may be
of value on a herd basis.
Pathogenesis
The mechanism by which mycobacteria produce disease in man and
animals is relatively unknown.
It is not known which properties of
the bacteria are responsible for virulence, and the fact that the
pathogenicity varies within different species of animals makes this
matter even more complex.
Different hosts seem to be susceptible to different types of
mycobacteria.
Rabbits, cattle, and cats are susceptible to M. bovis,
but M. tuberculosis causes little problem in these animals.
Man and
guinea pig are very susceptible to M. tuberculosis, however, the rat
is resistant to this species.^' ^
In man, differences in age have
been reported to affect the incidence of M. tuberculosis infection.
The disease is found to be more frequent in the very young and in the
elderly, perhaps due to the occasional inadequate immune status of
these age groups.
139
Pregnant women seem to be more susceptible to
tuberculosis for the same reason.
Other studies have shown that the body temperature of the host
may also play a role in susceptibility.
Chickens, whose body temper­
ature is 42°C, are susceptible to infection with M. avium which grows
well in vitro at 43°C.
On the other hand, chickens are resistant to M.
bovis and M. tuberculosis which require growth temperatures of 37°C.
It has been known for some time that immunity to infection by
facultative intracellular parasites, such as mycobacteria, is not
mediated by an antibody.
118
This fact is supported by the fact that
in either the natural or experimental disease, there is no relation­
ship between the amount of antibody formed and immunity to tuberculosis.
82
Experimentally, workers have been unable to transfer
immunity from immunized animals to nonimmunized animals by injecting
serum,
82
but have now shown that immunity can be transferred by
using lymphoid cells taken from infected animals.
98
'
99
About 2-4
weeks following infection with tubercle bacilli, the host's cellmediated immune response attempts to destroy the organism.
The macro­
phage seems to be the most important cell type involved in this
destruction process.
is engulfed.
15
The bacillus attaches to the macrophage, and
This results in the formation of a phagosome.
These
phagosomes then fuse with lysosomes containing hydrolytic enzymes.
This fusion results in the formation of phagolysosomes in which
hydrolytic enzymes destroy the microorganism.
are resistant to intracellular destruction.
However, mycobacteria
In fact, the tubercle
bacilli multiplies in and will usually kill the macrophage.
It is not
certain how mycobacteria accomplish this feat, but different theories
exist.
The tubercle bacilli may use the enzymes present in the
phagolysosome, or it may block fusion of the phagosome and lysosome.
26
It is generally agreed that the resistance of mycobacteria to lysozyme is due to the high lipid content of the cell wall.
One of the outstanding features of the host-tubercle relation­
ship is the formation of granulomas.
This granulomatous inflammation
first becomes evident in 2-4 weeks, after cellular immunity develops.
116
Macrophages accumulate at the site of infection, and enlarge following
ingestion of bacilli.
Macrophages are modified, lose their phago­
cytic ability, and are termed "epithelioid" cells because of their
resemblance to epithelial cells.
Epithelioid-cell formation is
accompanied by a tendency for the cells to be arranged in groups
rather than in diffuse sheets.
Frequently, when macrophages encounter
insoluble material or organisms causing chronic inflammation, they
coalesce to form giant cells.
Therefore, in a tuberculoid reaction,
in addition to epithelioid cells, a considerable number of giant
cells are also present.
Surrounding the mass of epithelioid cells,
there is a diffuse zone of lymphocytes with a few plasma cells and
fibrocytes.
Within 2 weeks, caseation necrosis takes place, and the
tissue forms a dry, firm, coagulated mass.
This is thought to be
caused by the development of hypersensitivity to the bacilli.
The end
result is a fully formed tubercle follicle, consisting of a central
mass of caseation surrounded by epithelioid cells and giant cells.
This, in turn, is surrounded by a zone of round cells.
With swine
tuberculosis, these tubercles can be found most commonly in cervical
or mesenteric lymph nodes.
As previously stated, the properties which cause mycobacteria
to be pathogenic are poorly understood.
A factor noted early which
may relate to pathogenesis of tubercle bacilli is the formation of
cords by the organisms in liquid medium.
It is thought that the cell
wall and its lipid components, in particular a lipid identified as
trehalose-6, 6'-dimycolate,
55
'
73
" are responsible for this phenomenon.
Cord factor can be extracted, and is known to inhibit the migration of
lymphocytes.^
Experimentally, it was noted that some animals, inocu­
lated with extracted bacilli, developed disease more slowly than
those inoculated with unextracted bacilli.
Further studies showed
that the level of succinic dehydrogenase in the animal decreased with
an increase in the severity of tuberculosis.^
Cord factor apparently
inhibits host production and/or secretion of succinic dehydrogenase
and, therefore, the host is less able to prevent, or more likely
control, advancement of the infection.
45
Cord factor has also been shown to cause swelling and lysis
of mouse liver mitochondria.
47
The effect of cord factor on other
organelles has also been studied.
It has been found to induce sepa­
ration of the ribosomes from the rough endoplasmic reticulum of liver
cells in mice.
24
The nature of these actions is not well understood.
Other compounds isolated from virulent mycobacteria are
sulfolipids.
sulfolipids.
27
In fact, many species are known to have common
Sulfatides have been shown to prevent fusion of phago-
somes and lysosomes.
47
This would allow the bacilli another means,
aside from cord factor, of escaping and/or prolonging digestion by
lysosomal enzymes.
affect mitochondria.
These sulfolipids, like cord factor, are known to
In fact, it appears the 2 compounds may act
synergistically, as sulfolipid was found to drastically increase the
toxicity of cord factor.^
During phagocytosis, there is an increased production of
superoxide by the host tissue, another attempt at stopping infection.
17
An iron-containing superoxide dismutase has been isolated from M.
tuberculosis, and has been found to protect the organism from the
toxic effect of superoxide.
52
' With M. avium, colony morphology is correlated with
virulence.
113
Those colonies which are flat, transparent, and
colorless are usually more virulent than those that are dome-shaped,
smooth, and opaque.
These "virulent" colonies can also be identified
by their growth requirement for palmitic acid; nonpathogenic M. avium
has no such requirement.
64
M. avium infection in swine is primarily a disease of the
lymph nodes associated with the gastrointestinal tract, more specifi­
cally the small intestine.
The following is a brief discussion of
the anatomy and physiology of the small intestine with an emphasis on
those structures involved in the uptake and transport of macromolecules from the gut.
The small intestine has 4 main functions: secretion, motility,
digestion, and absorption.
87
'
94
Here, we are mostly concerned with
absorption or the mechanisms by which molecules transverse the intes­
tinal wall.
Through an understanding of these mechanisms involved in
absorption, a better understanding of the potential mechanisms by
which M. avium may travel from the intestinal lumen to the regional
lymph nodes may be obtained.
The wall of the small intestine is composed of 4 layers: the
mucosa, submucosa, muscularis externa, and serosa.
The mucosa is
composed of an epithelial cell layer with its filamentous basement
membrane.
The basement membrane is a lamina propria containing blood
vessels, lymphatics, smooth muscle cells, nerve fibers, plasma cells,
lymphocytes, fibroblasts, eosinophils, macrophages, reticular cells,
mast cells, collagen, and reticular fibrils and is separated from the
submucosa by the muscularis mucosa.
The submucosa contains larger
blood vessels, lymphatics, more connective tissue, nerves and ganglia,
and more lymphoid elements.
The muscularis externa is divided into
an inner circular layer and an outer longitudinal layer of smooth
muscle, with the myenteric plexus between the two.®''' ^
The small intestine is characterized by 3 structures which
increase its surface area and aid in absorption.
circumferential folds of mucosa and submucosa.
First, there are
They are responsible
for the "feathery" appearance of the small intestine.
Second, the
villi are projections of mucosa which are usually fingerlike.
The
individual cells of the villi possess filamentous microvilli, which in
turn are coated with a more fine filamentous structure.
Lastly, the
mucosal crypts lie between the villi and extend to the muscularis
mucosa.
The surfaces of the villi and the linings of the crypts are
covered by a layer of columnar cells.
87
'
94
The microvillus plasma membrane is covered with an adherent,
filamentous structure called the glycocalyx.
It is composed of
sulfated, weakly acidic mucopolysaccharide which appears to be contin­
uously synthesized by the Golgi apparatus of the epithelial cell,
especially at the villous tips.
The plasma membrane and its asso-
ciated glycocalyx constitute a digestive-absorptive unit.
87
'
94
The process of absorption is initiated in the lumen of the
small intestine and is completed by the specialized absorptive cells,
the plasma membrane of which maintains composition and electrical
charge between the lumen and inside the cell by regulating the rate
at which molecules enter the cell.
The difference between the
function of this membrane at the cell's apical and basal surfaces
results in a net movement of molecules through the cell or
absorption.
77
'
94
Because a main function of the small intestine is
the absorption of lipids, and M. avium has a high lipid content, a
special interest was taken in the mechanism by which lipids are
absorbed.
Fats are continuously broken down and emulsified into
very small complexes called chylomicrons.
These enter the lacteals
by reverse pinocytosis, and electron microscopy has demonstrated
that pinocytic vesicles appear to occupy approximately 15% of the
endothelial cell cytoplasm.
87
'
94
The GI tract of mammals is complete with immunologically
competent tissue.
It is capable not only of experiencing but also
of mounting an immune response to antigenic stimulation.
Two of the
most important components of this system are the Peyer's patches and
specialized cells, the microfold or M cells.
The Peyer's patches lie in the lamina propria and submucosa
throughout the small intestine, usually on the antimesenteric wall.
They occur more frequently in more distal areas of the intestinal
tract, but some are always found in the duodenum.
Light microscopy
has shown that regardless of how many follicles a patch contains, each
20
patch is only one follicle thick, thus preserving the close relationship between the follicle and the overlying epithelium.
77
'
79
This
tissue is also commonly called gut-associated lymphoid tissue or
GALT. 77
The surfaces of Peyer's patches are characterized by
specialized epithelium which differs from that of adjacent areas and
possesses features which enhance trapping and uptake of antigenic and
8 77-79
particulate material. '
M cells appear responsible for antigen transport within the
8 75 79 88
epithelium. '
'
'
These variably surfaced cells with their
scattered, short protuberances are interspersed among the ordinary
epithelial cells.
They form cytoplasmic bridges between ordinary
epithelial cells, and separate the lumen from underlying nests of
lymphoid cells.
The reticulum formed by M cells allows cells from
the lymphoid series (above) to approach the gut lumen very closely
without being lost in the lumen.
At the same time, the M cells main­
tain tight junctions with adjacent epithelial cells so that the
integrity of the gut epithelium is preserved. 77
M cells transport
antigens to the lymphocytes through a tubulovesicular system, repre­
sented by pits in the luminal cell surface.
The underlying lymphoid
cells then migrate into mucosal nodules, or are carried away by
afferent lymphatics to mesenteric lymph nodes.
In a study by Owens
and Jones, they concluded that 1) the lymphoid cells contained within
M cell pockets could be responding there to antigenic material trans­
ported from the intestinal lumen via the M cells, or 2) M cells may
couple IgA to secretory components and transport the immunoglobulin
complex into the lumen. 77
All of these lymphoid "structures" maintain a lymphoepithelial
relationship which is similar to that of the thymus and the avian
bursa of Fabricius.
The former is responsible for T cell immunity and
the latter, in avian species at least, regulates the development of
B cell immunity.
Most immunoglobulin-containing cells in the small
intestine are mature plasma cells, although some immunoglobulins are
found within immature plasma cells and lymphocytes.
It is well
established that IgA is the predominant immunoglobulin in the lymphoid
cells and secretions of the small intestine.
IgM, IgE, and IgD are
1 114
also present. '
Secretory IgA has been demonstrated to possess antiviral and
antibacterial activity as well as the properties of isohemagglutinms.
114
Aside from these immunospecific mechanisms, any bacteria
colonizing the intestinal tract would have to contend with the non­
specific resistance mechanisms functioning in the tract.
This would
require bypassing the low pH of the stomach, and the peristalsis and
villous action in the small intestine.
They would have to resist the
toxicity of conjugated or unconjugated bile acids.
However, those
organisms sensitive to bile acids may avoid this by taking up
residence in the upper small intestine, where the concentration of
bile is low. 77
Mucin is another barrier against microbial invasion in the
small intestine.
Mucin traps particles, such as bacteria, and then
clears them away through the villous action and peristalsis in the
stomach and intestines.
overcome this.
However, certain bacteria are known to
In fact, they may colonize mucins and use them as
sources of carbon and energy.^
Indigenous microorganisms also may block, or limit, coloni­
zation and growth of pathogenic bacteria in the GI tract.
However,
this normal flora changes under conditions such as antibiotic treat­
ment, stress, diet, etc.
Pathogens may take advantage of these
circumstances, and infect only "altered" hosts.^
Although the neonatal small intestine has long been known to
have the ability to absorb macromolecules, until recently it was
thought that the adult mammalian gut maintained a complete barrier
against such absorption.
12
'
113
Today, there is increasing evidence that the normal adult
intestine is permeable to macromolecules, not in sufficient quantities
to be of nutritional importance, but in quantities that may be anti­
genic or biologically active.
mechanism.
12
'
113
The process employs an endocytic
Initially, there is an interaction between these
large molecules within the intestinal lumen and components of the
microvillus membrane of intestinal absorptive cells.
When a
sufficient quantity of molecules comes in contact with the cell mem­
brane, invagination occurs, and small vesicles are formed.
This
process of uptake is energy-dependent and it has been shown to be
inhibited by metabolic inhibitors of both glycolysis and oxidative
phosphorylation.
12
After invagination, macromolecules travel within
23
membrane-bound vesicles (phagosomes) to the supranuclear region of the
cell where the vesicles fuse with lysosomes to form phagolysosomes.
In these structures intracellular digestion occurs.
However, small
quantities of macromolecules escape breakdown and may be deposited in
the intercellular space by reverse endocytosis or exocytosis.
53
These molecules or antigens may come in contact with local lymphoid
tissue or may pass into the systemic circulation.
Numerous studies
using macromolecules such as HRP (horseradish peroxidase), PVP,
insulin, and vitamin
have confirmed this finding.^'
70, 89, 93, 113
The pathogenic mechanism involved in the uptake and processing
of M. avium from the small intestine is not known.
Through studies
involving other intracellular parasites, it is believed that this
process may be similar to the endocytic-type uptake discussed in
relation to macromolecular uptake from the gut.
7 f\
However, after the
formation of phagolysosomes, intracellular digestion does not occur.
Instead, tubercle bacilli reside and multiply within the involved
macrophages.
By escaping breakdown, bacilli may be deposited in the
intercellular space by exocytosis, and come in contact with local
lymphoid tissue.
At this time, the cellular immune response of the
host, and the corresponding granulomatogenesis, would be initiated to
form the typical lesions found upon postmortem examination and
slaughter.
If M. avium does penetrate into intestinal epithelial cells
by a phagocytic process, the various events in this process may be
elucidated by studying them in vitro using cultured intestinal
epithelial cells.
1)
The objectives of this investigation are:
To determine the maximum rate of infection of
HIE cells by M. avium serotype 2.
2)
To evaluate any differences in the interaction
of pathogenic versus saprophytic mycobacteria
with HIE cells.
3)
To assess the properties of M. avium that may
be responsible for its invasiveness.
4)
To determine what role, if any, the host
epithelial cell plays in uptake of M. avium.
CHAPTER 2
MATERIALS AND METHODS
Experimental Design
Each assay consisted of Henle intestinal epithelial (HIE) cells
grown to confluency in cluster 6 well tissue culture dishes. 3
Each
well was inoculated with 0.25 mg (wet weight) of the appropriate
organism suspended in minimum essential medium (MEM).' 3
Following incu­
bation, plates were washed in PBS, stained, and examined microscopi­
cally under oil immersion (100X).
For all studies, 5 wells on each plate were used as treatment
groups and the sixth as the control.
randomly assigned to treatment groups.
Individual plates and wells were
59
Thirty cells per well were
counted at random, and the number of bacteria associated with each
cell recorded.
Each assay was performed 3 times and the results were averaged.
Bacterial Cultures
Cultures of M. avium serotype 2 were obtained from Dr. C. 0.
Thoen at Iowa State University.
Those of serotype 8 and serotype 10
originated from pigs with mycobacterial lymphadenitis in Arizona, and
cultures of M. phlei and M. smegmatis from the mycobacterial collection
a.
Bellco.
b.
Gibco.
Vineland, New Jersey.
Grand Island, N.Y.
25
at the Trudeau Institute, Saranac Lake, NY.
Stock solutions of each
organism were prepared and stored at 0°C for future use to avoid an
excessive number of passages from the original slants.
Throughout
this entire study, no bacterial culture exceeded 4 passages from the
original slant.
c
d
Tubes of Dubos broth containing 1.0% horse serum
were inoculated with the appropriate culture, and incubated at 37°C
until a density equivalent to a McFarland Tube 1 was reached.
The
suspensions were aseptically transferred to sterile vials containing
2.0 ml final volume and frozen at 0°C.
Seven days prior to assay, the vials were thawed in a 37°C
water bath.
One ml of the stock culture was inoculated into 8.0 ml
of Dubos broth containing 1.0% horse serum.
Following 7 days incubation at 37°C, each bacterial culture
was centrifuged at 2000 rpm for 20 minutes, and resuspended in 40.0 ml
of MEM.
Two mis of this suspension were overlayed on each well.
This
resulted in approximately 0.25 mg (wet weight) of bacteria per well.
Cell Cultures
Henle 407 human intestinal epithelial (HIE) cells e were
cultured in MEM containing 10.0% fetal calf serun/ (FCS), 200 IU of
c.
Difco.
Detroit, Michigan.
d.
Gibco.
Grand Island, N.Y.
e.
Maryland.
f.
American Type Culture Collection (CCL 2).
Gibco.
Grand Island, N.Y.
Rockville.
penicillin per ml, 200 ug of streptomycin per ml, and 5 ug of
9
n
fungizone per ml in 25.0 cm tissue culture flasks.
All media were
filter-sterilized using a 0.20 y cellulose-acetate filter.
Once a
monolayer was established, cultures were either divided 1:3 into
new flasks to facilitate new growth, or transferred 1:1 into cluster
6 well tissue culture plates for assay.
The procedure for passage
of cells is outlined below.
1.
Flasks containing monolayers were washed twice with
MEM without FCS.
2.
1.0 ml of 0.25 percent trypsin -0.10 percent EDTA
solution was added to each flask.
3.
Flasks were incubated at 37°C with 5% CO2 until gentle
tapping detached the monolayer from the vessel (about
2 minutes).
4.
A 2.0 ml solution of MEM containing 10% FCS was
added to each flask to neutralize the action of
trypsin and to wash the cells from the surface of
the flask.
5.
The contents of each flask were dispensed into 15.0
ml centrifuge tubes, and centrifuged at 800 rpm for
10 minutes.
6.
The supernatant fluid was discarded, and the pellet
was resuspended in MEM containing 10% FCS and 2.0%
antibiotic.
g.
Bellco.
It was at this point that a bifurcation
Vineland, New Jersey.
28
in the procedure occurred dependent upon whether the
passage was into another flask or into a 6-well plate
for assay.
FLASK
7a.
8a.
The pellet was resus-
PLATE
7b
The pellet was resus-
pended in 1.5 ml of MEM
pended in 12.0 ml MEM
containing 10% FCS and
containing 10% FCS and
2% antibiotic.
2% antibiotic.
0.5 ml of this solution
8b.
A sterile coverslip
was dispensed into each
was placed in each well
2
of three 25.0 cm flasks.
of the tissue culture
plate for assays using
the I FAT.
9a.
The total volume was
9b
2.0 ml of the bacterial
adjusted in each flask to
suspension in (7b) were
5.0 ml with MEM contain­
added to each well.
ing 10% FCS and 2% anti­
biotic.
10a.
Flasks were incubated at
10b.
Plates were incubated
37 C with 5% COg until
at 37 C until conflu­
confluent.
ent.
(4 days)
(3-4 days)
Antiserum Preparation
Antiserum against M. avi um serotype 2 was prepared for use with
the indirect fluorescent antibody stain.
Antisera against serotypes 8
and 10 were previously prepared by Dr. J. G. Songer, and were available
for testing.
Antisera against M. phlei and M. smegmatis were not
available, and alternative staining techniques were employed for
assays involving these bacteria.
The preparation of hyperimmune serum against serotype 2, the
immunization of rabbits, and the collection of serum was as follows:
L
1.
Tubes of Dubos broth containing OADC
J
and Tween 80
were
inoculated with M. avium serotype 2, and incubated at
37°C for 14 days.
2.
One ml of this suspension was transferred to each of
L
several plates of Middlebrook 7H10 Agar
containing
OADC enrichment and 4.1 mg of sodium pyruvate per ml
of medium.
Plates were incubated at 37°C for 14
days.
3.
Cultures were checked daily for contamination.
If
pure, phenolized phosphate buffered saline (PPBS) was
added to each plate, and the cells harvested.
4.
Cells remained in PPBS, at a density equivalent to a
McFarland III Standard, for 5 days.
5.
Phenolized cell suspensions were centrifuged and the
pellet was resuspended in 2.0 ml of PBS.
This was
used to prepare a cell suspension of 0.4 optical
density at 525 nm in PBS.
h.
Difco.
Detroit, Michigan.
i.
Sigma.
St. Louis, Missouri.
30
6.
The marginal ear vein of New Zealand White rabbits was
injected with 1.0 ml of the final suspension on days
0, 4, 8, and 12.
7.
Two mis of the same suspension were injected into the ear
vein on days 16, 20, 24, and 28.
8.
Rabbits were exsanguinated on day 35.
The blood was
allowed to clot and then centrifuged.
9.
The serum was collected and stored at -20°C until needed.
Method of Assay
Kinetics Studies
Kinetic studies were performed on M. aviurn serotypes 2, 8, and
10, M. phlei, and M. smegmatis.
Each assay required 6 tissue culture
plates and 60 ml of the appropriate bacterial culture.
Each well was
washed twice with MEM and 2.0 ml of the bacterial suspension were
overlaid onto the monolayer of each of 5 wells.
tained MEM only, and served as a control.
The sixth well con­
Plates were incubated at
37°C for 30, 60, 120, 180, 240, or 300 minutes, after which the
bacterial suspension was aspirated, and each well was washed 4 times
with PBS.
Cells were then fixed for 30 minutes, or processed without
fixation, depending upon the staining technique to be used.
Assays Involving Phagocytic Inhibiting Reagents
These studies involved M. avium serotype 2 only.
The proce­
dures for preparing HIE cell monolayers and bacterial cultures were the
same as previously described, except covers!ips were not used.
The
reagents used included cytochalasin B 1 at concentrations of 0.05,
0.10, 0.50, 1.0, 2.0, and 4.0 mg/ml, dibutyryl cyclic AMP 1 ' at concen­
trations of 1.0, 2.0, 4.0, and 10.0 mM, iodoacetic acid 1 at concentra­
tions of 0.01, 0.03, 0.05, .10, .20, and ,30 mM and 2,4-dinitrophenol 1
at concentrations of 0.001, 0.01, 0.1, 1.0, and 2.0 mM.
All reagents
were diluted in MEM except cytochalasin B which was prepared as a
1.0 mg/ml stock solution in dimethyl sulfoxide 1 and then diluted in
MEM.
Two different infection protocols were used in these assays.
In
Procedure A, 2.0 ml of the appropriate concentration of reagent were
applied to each well, and the plates were incubated at 37°C for 3
hours.
Following incubation, monolayers were washed 4 times 'in MEM
and infected with 2.0 ml of the appropriate bacterial suspension.
After an additional incubation period of 3 hours at 37°C, plates were
washed 4 times in PBS and fixed with formalin for 30 minutes.
In
Procedure B, equal volumes of the bacterial suspension and reagents
were incubated together at 37°C for 3 hours.
This suspension was used
to infect monolayers by overlaying each well with 2.0 ml of the appro­
priate solution and incubating an additional 3 hours at 37°C.
Each
well was then washed 4 times in PBS and fixed with formalin for 30
minutes.
All plates were stained with the acid-fast stain.
One plate
was run for each reagent per replication except in those instances
where there were more dilutions than wells on each plate, and then it
was necessary to run 2 plates for that reagent.
Each assay incorpor­
ated 1 monolayer not subject to chemical treatment to serve as a
j.
Sigma.
St. Louis, Missouri.
32
control.
Studies Involving "Altered" Bacteria
These studies involved M. avium serotype 2 only.
The procedure
for preparing HIE cell monolayers and bacterial suspensions were the
same as previously described.
A 7 day culture of M. avium, grown in Dubos broth, was autoclaved and centrifuged at 2000 rpm for 20 minutes.
The supernatant
fluid was discarded and the pellet was resuspended in 40 ml of MEM.
Prior to infection of monolayers with this suspension, Middlebrook 7H10
Agar plates were inoculated to determine the viability of mycobacteria
after autoclaving.
Two confluent, 6-well plates of HIE cells were
prepared per replication.
After washing each well twice with MEM, the
cells from randomly selected wells were infected with 2.0 ml of the
above bacterial suspension.
The sixth well was overlaid with 2.0 ml
of viable M. avium serotype 2 suspended in MEM, and served as the
control.
Following 3 hours of incubation at 37°C, the coverslips were
removed from each well, labeled, and washed 4 times in PBS.
The mono­
layers, grown on coverslips from 1 plate were fixed in methanol for 30
minutes, while those from the second plate were processed without
fixing.
Those experiments involving formalinized M. avium used the same
protocol as above except the 7 day culture of M. avium was centrifuged
and resuspended in 5% formalin for 15 minutes.
Cells were washed twice
in PBS before resuspension in 40 mis of MEM.
To determine the effect of UV irradiation on M. avium in this
system, a 7-day culture of M. avium was centrifuged at 2000 rpm for
20 minutes and resuspended in 40 ml of MEM.
Four ml of the above
suspension were placed in each of 6 100 x 15 mm petri dishes
a shallow layer.
k
to form
The petri dishes were exposed to UV light for 10,
20, 30, 40, 50, or 60 seconds from a distance of 60 cm.
After irra­
diation, the contents of each petri dish were cultured on Middlebrook
7H10 Agar to check viability.
Using two 6-well tissue culture plates
as treatment groups, each treated bacterial solution was randomly
assigned to 1 of the 6 wells per plate.
were overlaid onto each monolayer.
Two ml of this suspension
The third plate was used as the
control, and cells from each well were infected with 2.0 ml of M.
avium in MEM that had not been exposed to UV irradiation.
were incubated for 3 hours at 37°C.
All plates
Coverslips, upon which monolayers
had been grown, were removed from wells, labeled, and washed 4 times
in MEM.
The coverslips from 1 of the treated plates were fixed in
methanol for 30 minutes, while coverslips from the other treated plates
were processed without fixing.
Three randomly selected coverslips from
the control plate were also fixed in methanol for 30 minutes while the
remaining 3 were not fixed.
M. avium was suspended in MEM containing various dilutions of
antisera to observe the possible effect of masking surface molecules
functioning as receptor sights in attachment.
Serum was filtered
through a 0.20 y cellulose-acetate filter before being diluted 1:16,
1:64, and 1:256 in MEM.
k.
Falcon.
Each replication of this assay required
Oxnard, California.
34
monolayers in three 6-well tissue culture plates, and three 7-day
cultures of M. avium.
The acid-fast stain was employed in this assay
so it was not necessary to incorporate coverslips in these tissue
culture plates.
Bacterial cultures were centrifuged at 2000 rpm for
20 minutes before suspension in 40 ml of 1 of 3 antibody-containing
solutions of MEM.
infection.
All monolayers were washed twice in MEM before
Two ml of the appropriate bacterial suspension were
added to each of 5 wells.
The monolayer in the sixth well was inocu­
lated with M. avium suspended in MEM not containing antisera.
Plates
were incubated for 3 hours at 37°C, washed 4 times in MEM, and fixed
with formalin for 30 minutes.
The format followed in studies involving M. avium cultures
suspended in milk or colostrum was essentially the same as that
described above, except the FA staining technique was used.
Two con­
fluent 6-well tissue culture plates with coverslips, and one 7-day
culture of M. avium were needed per replication for each of the 2
treatments (milk or colostrum).
The unpasteurized milk and colostrum
were obtained from the University of Arizona Dairy from cattle not
receiving antibiotic therapy, and the pH was adjusted to 7.0.
A
culture of M. avium was resuspended in 40 ml of milk or colostrum, and
2.0 ml of this suspension were overlaid onto each of 5 monolayers.
The sixth monolayer, the control, was infected with M. avium suspended
in MEM.
Plates were incubated 3 hours at 37°C, after which the cover-
slips, containing monolayers, were removed, labeled, and washed 4
times in MEM.
The coverslips from 1 plate were fixed in methanol for
30 minutes, while those from the second plate were processed without
fixing.
Staining Techniques
Kinyoun's Acid-Fast Stain
This procedure was employed initially with cultures infected
with M. avium serotype 2 to determine if there was any attachment
occurring, in all assays involving M. phlei and M. smegmatis, and in
those incorporating M. avium suspended in antiserum.
The procedure
described below begins after fixation of cultures with formalin:
1.
Carbol fuchsin^
1 Hour
2.
Rinse 70 percent alcohol
2 Times
3.
Rinse running tap water
5 Minutes
4.
Rinse acid alcohol
3 Times
5.
Rinse running tap water
8 Minutes
6.
Methylene blue^
30 Seconds
7.
Rinse running tap water
10 Minutes
Auramine O-Acridine Orange Stain
This stain was used on all cultures involving the phagocytosis
inhibiting reagents.
After fixation in formalin, infected monolayers
were treated as follows:
1.
Auramine 0^ (0.30% solution)
2.
Rinse running tap water
5 Minutes
3.
Rinse acid alcohol
2 Times
1.
Fisher.
Fair!awn, New Jersey.
15 Minutes
36
4.
Acridine orange m
5 'Minutes
5.
Rinse running tap water
5 Minutes
Indirect Fluorescent-Antibody Stain
Immunofluorescent techniques similar to those described by
49
Kihistrom
and modified to incorporate a counterstain were used to
differentiate intracellular and cell surface adherent bacteria in
kinetic studies involving M. avium serotype 2, serotype 8, and sero­
type 10.
Assays incorporating autoclaved, ultraviolet exposed,
formalin treated, and milk or colostrum coated M. avium serotype 2
were also evaluated using this procedure.
Monolayers, grown on cover-
slips, were either fixed in methanol or processed without fixation
according to the following protocol:
1.
Monolayers were incubated with a 1:64 dilution of rabbit
anti-M. avium serotype 2 antibody for 20 minutes at 37°C.
A dilution of 1:160 was used for serotype 8 and a dilution
of 1:320 was used for serotype 10.
2.
Cells were washed 4 times in PBS.
3.
Monolayers were incubated with 1:32 dilution of fluorescein
isothiocyanate (FITC)-labeled goat anti-rabbit 75 immuno­
globulin 11 for 20 minutes at 37°C.
4.
Cells were washed 4 times in PBS.
5.
A counterstain, consisting of 0.125 percent Evans Blue m
m.
Sigma.
St. Louis, Missouri.
n.
Mi 1es.
Elkhard, Indiana.
37
diluted in PBS, was applied to each coverslip for 10
minutes.
6.
Cells were washed twice in PBS and mounted in 60%
glycerol.
Microscopy
Light
Those cultures stained with Kinyoun's acid-fast stain were
examined under oil immersion using the 100 x objective.
Fluorescence
Cultures stained with the Auramine 0-acridine orange stain and
those involving the indirect fluorescent antibody stain were examined
by incident light fluorescence microscopy at 100X (oil immersion) using
a Leitz photomicroscope with exciter filter II and a 400 nm barrier
filter.
Statistical Analysis
The data were distributed according to a Poisson distribution,
with numerous small integer and zero counts.
because data were found to be highly skewed, a
For this reason, and
1/ X + .5 transformation
was used on most bacterial counts.
Two statistical computer programs, compiled by Kelley,
used for data analysis.
48
were
The first included an analysis of variance
and tests of significance between treatment groups (a = .05) were per­
formed with the F-test.
A second program was devised to find the mean
38
count for each treatment group per assay.
Therefore, each data point
consisted of the average of 3 means corresponding to the 3 performances
of each treatment.
When the percent of intracellular bacteria was needed, the
means for fixed and unfixed monolayers were employed in the following
formula:
Mean Number of Bacteria/Fixed HIE Cell Mean Number of Bacteria/Unfixed HIE Cell
Mean Number of Bacteria/Fixed HIE Cell
x
100
All data from those assays involving reagents to inhibit phago­
cytosis were evaluated without transformation.
into 2 groups: infected and noninfected.
ciated bacteria were defined as infected.
The cells were arranged
Cells with 1 or more asso­
This was done for each con­
centration of each reagent, and because each assay was performed 3
times, an average of the 3 was used in the following formula to give
the percentage of HIE cells infected per monolayer:
Mean Number of Cells Infected
Mean Number of Cells Infected + Mean Number of
Cells Noninfected
x 100
Tests of significance were conducted using the Newman-Keuls mean separa­
tion test and a 95% confidence level.
In some cases, it was necessary to look individually at fixed
and unfixed HIE cell monolayers.
Bacteria could be considered intra­
cellular if the 2 preparations were found to stain differently, and
extracellular if preparations stained alike.
Using raw data, each
HIE cell counted was placed in 1 of 7 groups depending upon the number
of bacteria which were found in association with it.
Groups included
0, 1-10, 11-20, 21-30, 31-40, 41-50, arid 51-60 associated bacteria.
The distributions for fixed and unfixed monolayers were then graphed
for comparison.
CHAPTER 3
RESULTS AND DISCUSSION
Kinetics Studies
Primary assays involving M. avium serotype 2 were conducted
with the intention of establishing the amount of time required for
maximum infection of cultured epithelial cells.
Data indicate that
maximum cellular uptake occurred after 3 hours incubation time.
(Figure 1.)
Because mycobacteria are intracellular parasites, the
validity of the data in Figure 1 is dependent upon the assumption that
cell-associated bacteria seen in acid-fast stained monolayers were in
fact intracellular and not merely adhering to host cell surfaces.
A
49
technique described by Kihlstrom
was used to aid in distinguishing
surface-associated from internal bacteria.
This indirect fluorescent-
antibody test is based upon the fact that immunoglobulin proteins do
not cross the intact plasma membrane^ but diffuse freely into
methanol-fixed cells.
92
This principle was first applied to monolayers
infected with M. avium serotype 2 for time intervals of 30 minutes to
5 hours.
time.
Figure 2 shows the percent intracellular bacteria at each
Maximum infection of epithelial cells occurred after 3 hours of
incubation, and at this time, over 60% of the bacteria seen in associ­
ation with each epithelial cell were, in fact, intracellular.
The fact that the number of intracellular bacteria per HIE
cell decreased as incubation time exceeded 3 hours, may be due to
40
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4.5
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<
v»—
o 1.5
w.
a>
.a
E
z 1.0
0.5
30
60
120
180
240
Time (minutes)
Figure 1.
Kinetics of the Interaction of M. avium
Serotype 2 with HIE Cells.
300
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20.0
10.0
J
30
I
60
I
120
I
180
I
240
I
300
Time (minutes)
Figure 2.
Percent of M. avium Serotype 2 Shown to be Intra­
cellular when Associated with HIE Cells.
43
exocytosis of tubercle bacilli.
53
The kinetics of the interactions of serotypes 8 and 10 with
HIE cells were studied.to determine if they differed from the
supposedly more pathogenic serotypes 1 and 2.
Serotype 8 exhibited
maximum uptake of 42.94% after 3 hours incubation (Figure 3).
13% less than serotype 2 uptake.
is even less with serotype 10.
This was
Figure 4 shows that cellular uptake
Furthermore, this maximum uptake of
only 28.60% occurred after only 2 hours incubation.
The differences
in kinetics of the M. avium:HIE cell interaction may partly explain
the differences in rates of infection with various serotypes.
Other
factors, such as the relative prevalence in the environment of the
different serotypes, may also play a role.
Saprophytic mycobacteria, which are not causally associated
with a disease process, are widespread in the environment.
Two of
these, M. phlei and M. smegmatis, are occasionally isolated from
tuberculous lesions of swine, often with M. avium.
The interaction of
these 2 organisms with HIE cells were studied to compare them with
pathogenic M. avium.
Results are shown in Figures 5 and 6, as the mean
number of bacteria associated with each HIE cell.
The lack of antisera
against these species of bacteria did not allow use of IFAT.
Neither
organism displayed a significant (P = 0.05) ability to attach to or
penetrate, HIE cells.
If an equivalent response occurs in vivo, it
may explain the rare occurrence of these, as compared to M. avium, in
porcine lymph nodes.
tOO.Oi—
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30.0
3
(3!
20.0
10.0
30
J
60
I
120
L
180
240
300
Time (minutes)
Figure 3.
Percent of M. avium Serotype 8 Shown to be
Intracellular when Associated with HIE Cells.
100.0
90.0
8Q0
STO.0
•H
« 60.0
a>
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CD
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o
30.0
8
i2!
20.0
10.0
I
30
1
60
I
120
I
180
I
240
Time (minutes)
Figure 4.
Percent of M. avium Serotype 10 Shown to be
Intracellular when Associated with HIE Cells.
L
300
5.0
4.5
—. 4.0
SU
3.0
25
'§ 20
8
(O
<
»•—
o 1.5
1.0
Q5
30
1
1
60
120
180
240
300
Time (minutes)
Figure 5.
Kinetics of the Interaction of M. phlei with
HIE Cells.
5.0
4.5
4.0
O
in „
<X> 3.5
<330
0>
o 2.5
o
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k.
a>
-O
E
1.0
0.5
J30
I
60
I
120
L
180
240
300
Time (minutes)
Figure 6.
Kinetics of the Interaction of M. smegmatis wi
HIE Cells.
48
The interaction of M. aviurn serotype 2 with epithelial cells
was examined in greater detail by studying separately the contributions
of the HIE cells and the bacteria to the uptake process.
Host Cell Effects On Uptake
HIE cell monolayers were treated with 4 compounds known to
inhibit the ability of phagocytic cells to engulf particles.
Pre­
incubation of host cells with the appropriate reagent (Procedure A)
versus treatment of bacterial cultures with each reagent before
infecting monolayers (Procedure B) was designed to assure that the
effect of each reagent was upon the host cells and not upon the
bacteria.
Cytochalasin B, a fungal metabolite, inhibits phagocytosis by
.
.
2 4
polymorphonuclear leukocytes (PMN) and macrophages. '
Many investi­
gators have reported the disruption of actin polymers of microfilaments
in both phagocytic and nonphagocytic cells treated with cytochalasin
B.2'
^ According to these authors, concentrations of
1-3 mg cytochalasin B/ml disrupt microfilaments and suppress cell
phagocytosis.
Results in Table 1 indicate significant inhibition of
phagocytosis at a concentration of 1.0 mg cytochalasin B/ml, regardless
of which procedure was applied.
This suggests that infection of
epithelial cells in vitro is dependent upon functional host cell micro­
filaments.
Dibutyryl cAMP inhibits particle ingestion by PMN and mouse
Table 1.
Effect of Cytochalasin B on Interaction of
M. avium Serotype 2 with HIE Cells.
PROCEDURE
Concentration
(Uq/ml)
.AA
x
%
4.0
16
SO
2.0
1.0
0.5
0.1
0.05
0
22.33 25.33 39.33 45.33 56.33 59.66
60
60
60
60
60
So~
4.0
2.0
1.0
0.5
0.1
0.05
0
6.66 16.66 21.66 3'».66 40.33 50.66 57.66
~W
60
So 60
60
60
60
26*66 37*22 k2*22 65.55 75-55 93.88 99.43 11*10 27?77 36?10 57.77 67.22 84.43 96.10
* significant (P
t
= .05)
bacteria applied after removal of reagent
reagent and bacteria applied simultaneously
» mean number cells infected
• v ,
r
n
7—r
total number cells counted
.
\
(3 observations)
io
peritoneal macrophages at a concentration of 2.0 mM.
13
'
97
'
115
Experiments were conducted to test the effect of this compound upon
the susceptibility of HIE cells to mycobacterial infection.
Results
indicated that dibutyryl cAMP significantly inhibited the infection
of host cell monolayers at different concentrations dependent upon the
infection procedure used (Table 2).
It was found that 1.0 mM solutions
caused significant inhibition with Procedure A.
However, under Proce­
dure B, significant inhibition did not occur at concentrations less
than 4.0 mM.
Dibutyryl cAMP is soluble in the lipids of the host cell
membrane, thereby allowing it to cross the intact cell membrane and
inhibit phagocytosis.
31
It may be readily absorbed by M. avium due to
the high-lipid content of its cell membrane, thereby impairing the
ability of this compound to affect the HIE cells at lower concentra­
tions.
Because phagocytosis is an energy-dependent process, experi­
ments were done to determine if disruption of carbohydrate metabolism
affected the interaction between M. avium serotype 2 and HIE cells.
Iodoacetate inhibits phagocytosis in PMN,^ blood monocytes,^ and
peritoneal macrophages
74
; in these cells, the energy required for
phagocytosis has been linked to glycolysis which is known to be inhib­
ited by iodoacetate.
2,4-dinitrophenol inhibits Krebs' cycle activity
and, thus phagocytosis, by alveolar macrophages.
74
These 2 compounds
were tested for effects on mycobacterial infection of^H-IE cells.
Tables 3 and 4 show results when iodoacetate and 2,4-dinitrophenol were
employed, respectively.
In both cases it was found that infection was
Table 2.
Effect of Dibutyryl cAMP on Interaction
of M. avium Serotype 2 with HIE Cells.
PROCEDURE
At
Bt
Concentration
mM
X "*
%
10.0
k.O
2.0
1.0
10
So
14.66
60
1*4.33
60
21.33
60
16V67
2V. ; i»3
23*88
35*56
0
10.0
4.0
2.0
1.0
57.66
60
11.66
60
26.66
60
33-33
60
46.33
60
59.33
60
96.10
19 V* 3
kb'lk 3
55.55
77.22
98.88
- significant (P = .05).
t bacteria applied after removal of reagent
T reagent and bacteria applied simultaneously
** mean number cells infected
total number cells counted
(3 observations)
0
Table 3.
Effect of Iodoacetate on Interaction
of M. avium Serotype 2 with HIE Cells.
PRODECURE
Af
Concentration
n*t
X «'»
0.3
0.2
15.33
60
19.66
60
25*55
yi.ll
0.1
0.05
0.03
0.01
0
0.03
0.2
0.1
0.05
0.03
0.01
0
26
35.66
60
55
8.66
So
57.66
60
59.33
So
So
18
27
SO
36.66
60
<14
So
So
52.66
60
So
43*33
53M
91.67
96.10
98.83
1O13
30?00
45*00
16.10
73-33
87.77
98.33
59
^significant (P » .05)
t
bacteria applied after removal of reagent
^ reagent and bacteria applied simultaneously
mean number cells infected
total number cells counted
(3 observations)
cn
ro
Table 4.
Effect of 2,4-Dinitrophenol on Interaction
of M. avium Serotype 2 with HIE Cells.
PROCEDURE
A+
Concentration
2.0
1.0
0.1
0.01
11.66
So"
27.66
60
29.33
60
35
19*43
46*10
48788
0.001
0
2.0
1.0
0.1
0.01
0.001
25
^
29-66
60
32.33
Sir
45.66
So
59-33
60
49743
53.88
76.10
98.88
mM
58.33
43-66
60
59
Zo
20.66
So"
72.77
98.33
34743
41767
* significant (P = .05).
t bacteria applied after removal of reagent
:[: reagent and bacteria applied simultaneously
** mean number cells infected
total number cells counted
(3 observations)
cn
CO
54
significantly reduced at a concentration of 0.10 mM or greater.
Furthermore, there was no difference based upon the infection proce­
dure used.
These data support other studies using cytochalasin B and
dibutyryl cAMP by showing that the uptake of M. avium into intestinal
epithelial cells is at least partially dependent upon the endocytic
activity of the host cell.
Bacterial Effects On Uptake
Uptake of M. aviurn into HIE cells may be partially explained by
factors related to the bacterium, such as its cell surface character­
istics (proteins or lipids) or metabolic products.
This was studied
using cultures of M. avium serotype 2 manipulated in various ways to
alter these characteristics, and their subsequent interactions with
HIE cells.
Initial studies used bacteria killed by autoclaving or by
suspension in formalin.
Killed suspensions were incubated with HIE
cells for 3 hours and the interaction was assessed quantitatively using
the indirect fluorescent antibody stain.
Figure 7 shows the results of incubation of autoclaved M. avium
serotype 2 with HIE cell monolayers versus results from monolayers
inoculated with viable M. avium cultures.
A remarkable decrease in
uptake was evident compared to the control, but a small amount of
endocytosis (3.08%) did occur.
Comparison of the results of methanol
fixed and unfixed preparations (Figure 8), indicates that minimal
engulfment probably reflects extracellularly adhered bacilli.
Figure 9 shows the results of interactions of HIE cells with
55
100.0
90.0
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30.0
8
£ 20.0
10.0
AUTOCLAVED
Figure 7.
VIABLE
Difference in Uptake of Autoclaved and Viable
M. avium Serotype 2 by HIE Cells.
Number of Associated
Bacteria
51-60
UNFIXED
FIXED
41-50
31-40
11-20
I-IO
100.0
80.0
60.0
40.0
20.0
0
20.0
40.0
60.0
BOO
Percent HIE Cells/Monolayer
Figure 8.
Staining Characteristics of Methanol-Fixed and Unfixed
HIE Cells Infected with Autoclaved M. avium Serotype 2.
100.0
100.0
90.0
^ 800
|70.0
•M
« 60.0
0>
o 500
m
= 40.0
d)
o
o
30.0
8
10.0
FORMALIN
TREATED
Figure 9.
VIABLE
Difference in Uptake of Formal in-Killed and Viable
M. avium Serotype 2 by HIE Cells.
formal in-killed and with viable M. avium.
A significant inhibition
(P = 0.05) in uptake of killed bacteria was found when compared to
the control.
Examination of methanol-fixed and unfixed preparations
(Figure 10) indicated that the minimal level of endocytosis (3.79%)
was more likely due to extracellular adhered bacteria than intra­
cellular bacteria, since the 2 preparations stain alike.
Suspensions of M. avium serotype 2 were exposed to ultraviolet
irradiation for 0 to 60 seconds and their interaction with HIE cells
was compared to that of nonirradiated bacteria.
An exposure of 40
seconds or more significantly reduced (P = 0.05) the infectivity of
the organism (Figure 11).
Results of methanol-fixed and unfixed prep­
arations were examined separately (Figure 12).
After 40 seconds of
irradiation preparations stained similarly indicating that the bacilli
were not being taken into the host cell but were only adhered to the
outside.
After 50 to 60 seconds exposure the staining characteristics
of methanol-fixed and unfixed preparations were identical.
Viability tests on the M. avium suspensions paralleled those
shown for infectivity in Figure 11.
After 8 weeks of incubation at
37°C Middlebrook 7H10 Agar plates inoculated with suspensions exposed
for 10, 20, or 30 seconds showed similar growth to plates inoculated
with nonirradiated M. avium.
Plates inoculated with bacterial cultures
exposed for 40 seconds demonstrated a 65% reduction in growth compared
to cultures containing nonirradiated bacilli.
Plates inoculated with
cultures exposed to 50 or 60 seconds of UV light exhibited no growth.
The effects of short-term irradiation (less than 60 seconds)
on mycobacteria are thought to be confined to the nucleic acids.
30
Number of Associated
Bacteria
51-60
UNFIXED
FIXED
41-50
31-40
21-30
11-20
1- 0
)
1
1
I
100.0
8Q0
1-
60.0
I
—1—
40.0
20.0
0
1
1
20.0
40.0
...
1
1
1
60.0
80.0
loao
Percent HIE Cells/Monolayer
Figure 10.
Staining Characteristics of Methanol-Fixed and Unfixed
HIE Cells Infected with Formalin-Killed M. avium
Serotype 2.
60
100.0
90.0
8Q0
S?70.0
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® 60.0
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m
= 40.0
(V
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30.0
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10.0
10
20
30
40
50
Time (seconds)
Figure 11.
Difference in Uptake of M. avium Serotype 2
Irradiated with Ultraviolet Light (260 nm)
for 0 to 60 Seconds.
60
1
NumberofAooctafed
1
20 sec.
Number of Associated
Bocterio
10 sec.
Bocterio
•«0
UNFIXED
FIXED
1
|,
1
SMO
21-30
JJO
|
r
)
-i.
CO
L—Ji
i
i
*
40
20
0
i
•
20
t
i
»
•0(040200
PNcent HIE Cdls/Mooohiyer
20
40
CO
«>
Percent HIE Cells/Monolayer
1
80 10
I
40 sec.
Number of Associated
Bacteria
50 sec.
Number of Associated
Bocterio
91*60
UNFIXED
40
20
0
20
40
Percent HIE Grih/Monolayer
UNFIXED
41-50
I
3M0
1
60 sec.
Number of Associated
Bocterio
I1
51*60
4hS0
1
1
3MO
FIXED
»
«-p
L
1
•
to
•
40
20
*
0
•
20
40
Percent HIE Cells/Monoloyer
Figure 12.
i
BO
.i
CO
.i
40
.i
2D
II
0
20
«0
Percent HIE Cells/Monolayer
60
90
. i
SO
1
I.
60
1
i
i
40
.i
20
0
i. —i
20
40
Percent HIE Cells/Monolayer
Staining Characteristics of Methanol-Fixed and Unfixed HIE
Cells Infected with M. avium Serotype 2 Irradiated with
Ultraviolet Light for 0 to 60 Seconds.
i .
GO
i
80
Therefore, it is likely that surface molecules would be unaltered in
the suspensions irradiated in the present study.
It may be inferred,
from data obtained in these studies, that viability of the bacterium
is an obligate requirement for endocytosis by HIE cells.
The role of
surface molecules in this process must be evaluated further.
Although immunity to mycobacterial infections is cell-mediated
rather than antibody-mediated, the effect of local antibody on the
infection process in swine tuberculosis is unknown.
Porcine anti-M.
avium IgA is not available and, therefore, anti-M. avium serotype 2
serum (rabbit origin) was employed to evaluate the interaction of this
organism with HIE cells.
Results are shown in Figure 13.
Inhibition
of infection/attachment was inversely proportional to the concentration
of antiserum.
This may have resulted from the masking of surface
structures which are required for attachment.
Cultures of the anti-
sera treated suspensions revealed no reduction of growth, implying that
viability is not the sole requirement for M. avium uptake.
Preliminary studies were conducted on the effect of milk and
colostrum on uptake of M. avium by HIE cells.
This was done to simu­
late conditions occurring in the gut of the neonate pig.
Due to the
extreme difficulty in obtaining milk and colostrum from sows, bovine
milk and colostrum were substituted.
Figure 14 indicates an increase
in the number of internalized tubercle bacilli when M. avium was
suspended in milk as compared to M. avium suspended in MEM.
However,
this increase was not statistically significant (P = 0.05).
Figure 15
shows the results when epithelial cells were infected with M. avium
5Q0t—
„— 400
O 15.0
I:256
164
l : 16
Concentration
Figure 13.
Interaction of M. avium Serotype 2 Suspended
in Various Concentrations of Homologous
Antiserum with HIE Cells.
64
100.0
90.0
^ 8Q0
SB™
•«
60.0
\
o
'C
Q>
g 50.0
m
= 40.0
a>
u
o
V.
"c
30.0
8
£
20.0
10.0
MILK
Figure 14.
NO MILK
Interaction of M. avium Serotype 2 Suspended in
Bovine Milk or in MEM with HIE Cells.
65
100.0
90.0
8Q0
|70.0
•M
® 60.0
0>
8 50.0
m
w
= 40.0
0)
o
o
30.0
C
8V.
<D
CL
20.0
10.0
COLOSTRUM
Figure 15.
NO
COLOSTRUM
Interaction of M. avium Serotype 2 Suspended
in Bovine Colostrum or in MEM with HIE Cells.
66
suspended in colostrum.
A statistically significant (P = 0.05) increase
in the percent of bacterial uptake was found when bacteria were
suspended in colostrum.
The difference in bacterial uptake between the 2 suspensions was
possible due to their differences in lipid or immunoglobulin co'ntent. 22 -
54-
70
If pigs are exposed to M. avium during the neonatal period,
not only are they physiologically incapable of preventing bacterial
invasion of the gut, but their diet at this time may also enhance
susceptibility to infection.
This may indicate that sanitation during
the first few days after birth could be of some importance in pre­
venting mycobacterial infection.
CHAPTER 4
CONCLUSIONS
An in vitro model using cultured Henle intestinal epithelial
(HIE) cells was designed to study the early interaction between M.
avium and the host cell in an attempt to better understand the patho- .
genie mechanism involved in swine tuberculosis.
The results of this
study indicate that:
1.
Maximum infection of cultured HIE cells was achieved after
3 hours of incubation with M. avium serotype 2.
2.
Differences in characteristics among different serotypes of
M. avium and saprophytic or nonpathogenic mycobacteria may
account for the observed differences in infection rate with
these organisms.
3.
Because the rate of infection of HIE cells was inhibited by
reagents shown to inhibit particle uptake in known phago­
cytic cells, it was suggested that uptake of M. avium by
cultured epithelial cells was endocytic in nature.
4.
Studies involving infection of HIE cells with dead M. avium
showed that viability of the tubercle bacilli is necessary
for endocytosis to occur.
5.
Surface molecules may play a role in attachment of the
organism to the host cell, but metabolic activity on the
part of the tubercle bacilli is also required for endocy­
tosis.
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