EXPERWENTAL RESPIRAFGRY
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ABSTRACT

EXPERIMENTAL RESPIRATORY INFECTIONS IN THE RAT

by Joe D. Burek

A total of 8 experiments were conducted exposing germfree and con-
ventional rats to Mycoplasma sp., Mycoplasma pulmonis, BordeteZZa
bronchiseptica, Pasteurella pneumotrOpica, and a combination of Borde-
teZZa bronchiseptica with Pasteurella pneumotropica. Approximately 200
rats were infected and a similar number of noninfected controls main-
'tained. In each experiment, the acute and chronic aspects of exposure
were studied. Infection was determined by isolation of the organism(s)
and the presenCe of tissue lesions. Pneumonia was more severe in con-
ventional than germfree rats after aerosol exposure to B. bronchiseptioa.
Pasteurella pneumotropica was never isolated from the lungs of conventional
or germfree rats after aerosol exposure. A combined infection of
B. bronchiseptica with P. pneumotropica produced lesions identical to
B. bronchiseptica alone. No enhancement of P. pneumotrOpica occurred as
a result of the combination. Mycoplasma sp. was not capable of producing
respiratory infection by intranasal instillation or aerosol exposure of
broth cultures or pig kidney cell cultures in conventional or germfree
rats. Infection was not produced when the organismeas placed directly
into the trachea after surgical tracheotomy or when the organism.was

injected intravenously with or without agar. Current investigations

Joe D. Burek
demonstrated another mchplas a species of rat lung origin that is capable
of producing an acute to subacute suppurative bronchoPneumonia in germfree

rats. (Supported by NIH grant 5 R01 FR00383.)

EXPERIMENTAL RESPIRATORY INFECTIONS IN THE RAT

By

Joe D? Burek

A THESIS

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

MASTER OF SCIENCE

Department of Pathology

1970

 

”A 7 Q 5

7 /~ 70

Dedicated to

Janet

ii

 

ACKNOWLEDGEMENTS

vaish to express my indebtedness to Dr. C. K. Whitehair, my
major professor, for his generous guidance and counsel.

Also, a special thanks to Dr. G. R. Carter and his associates for
their assistance with the microbiological aspects of this investigation.

I also wish to thank Dr. C. C. Morrill, Chairman, and the entire
Department of Pathology at Michigan State University for providing the
Opportunity to pursue a graduate study.

Special thanks go to Janet, my wife. She has been extremely patient
and understanding and has unceasingly encouraged and assisted me through-

out.my years as a student.

iii

TABLE OF CONTENTS

INTRODUCT ION O O O O O O O O O O O 0 O I O O O O O O
L ITEMTURE REV IEW O O I O O O I O O O O O O O O O O O

The Mchplasma Organisms. . . . . . . . . . .

The BordeteZZa bronchiseptica and PasteureZZa pneumo-

tropica Organisms . . . . . . .‘. . . . . . .
Summary . . . . . . . . . . . . . . . . . . .
MATERIALS AND METHODS. . . . . . . . . . . . . . . .
Source of Animals . . . . . . . . . . . . . .
Source of Organisms . . . . . . . . . . . . .
Exposure. . . . . . . . . . . . . . . . . .
NecrOpsy and Cultures . . . . . . . . . . . .
Experiments . . . . . . . . . . . . . . . . .
RESULTS. . . . . . . . . . . . . . . . . . . . . .
Experiment 1. . . . . . . . . . . . . . . . .
Experiment 2. . . . . . . . . . . . . . . . .
Experiment 3. . . . . . . . . . . . . .
Experiment 4. . . . . . . . . . . . . . . . .
Experiment 5. . . . . . . . . . . . . . . . .
Experiment 6. . . . . . . . . . . . . . .
Experiment 7. . . . . . . . . . . . . . . .
Experiment 8. . . . . . . . . . . . . . . . .
DISCUSSION . . . . . . . . . . . . . . . . . . . . .

Mycoplasma Infection in the Rat . . . . . . .

iv

' 12

14
14
14*
15
15
17
20
20
21
21
21
21
23
30
32
37

37

 

 

Page
Bordetella bronchiseptica Infection in the Rat. . . . . . . 37
PasteureZZa pneumotropica Infection in the Rat. . . . . . . 38

Combined Bordétella bronchiseptica and PasteureZZa
pneumotropica Infections in the Rat . . . . . . . . . . . . 39

SUMY MD CONCLUSIONS 0 O O O O A O O O O 0 O O O O O 0 O O O O I O 40
REFERENCES 0 O O O O O O O O 0 O O O O O O O O O O O O O O O O O O 41

VITA o e e "or o e o o o o o o o e e o e e o o o e e e o o e o o o e 45

 

Table

LIST OF TABLES

Page
Summary of the isolations of Bordetella bronchiseptioa
in conventional rats that were exposed to the organism
by aerosol exposure . . . . . . .'. . . . . . . . . . . . . 24

Summary of PasteureZZa pneumotropica in conventional
rats that were exposed to the organism by aersol
exposure 0 O O O O O O O Q 0 O O O O O O O O O O O l. I O O O 31

Summary of the isolations of Bordetella bronchiseptioa

and Pasteurella pneumotropica in conventional rats that
were exposed to the organisms by aerosol exposure . . . . . 33

vi

 

LIST OF FIGURES

Figure Page
1 Left lobe of the lung from a S-week-old rat, 14 days
after intranasal exposure to Mycoplasma sp.. . . . . . . . 22
2 Focal area of bronchOpneumonia in a 4-week-old rat, 7
days after aerosol infection with B. bronchissptioa. . . . 25
3 Lung from a 4-week-old rat, 7 days after aerosol
exposure to B. bronchiaeptica. . . . . . . . . . . . . . . 25

4 Minimal lymphocytic infiltration along bronchiole in
a 5~week-old rat, 14 days after aerosol exposure to
B. bmnchiseptica O I O O I O 0 O O O O O O O O O O O O O I 27

5 A large focal area of pneumonia in a deeek-old rat,
14 days after aerosol exposure to B. bronchiseptioa. . . . 27

6 A 7-week-old rat lung with regressing pneumonic lesions,
4 weeks after aerosol exposure to B. bronchiseptica. . . . 29

7 Focal pneumonia in a 5-week-old rat, 14 days after
aerosol exposure to B. bronchiseptica and P. pneumo-
tropioa O O Q 0 O O O O O O 0 O O O O O O O O I O O O O O O 35

8 Lung from a 7-week-old rat, 4 weeks after aerosol
eXposure to B. bronchiseptica and P. pneumotropica . . . .‘ 35

vii

 

INTRODUCTION

Respiratory diseases are a serious problem in the laboratory rat
and have complicated experiments for many years. The most persistent
problem is chronic pneumonia with its insidious onset, its prolonged as
course and eventually terminal outcome.
There has been an inability to definitively categorize, that is, to
establish a definite etiology for all forms of the chronic respiratory
disease complex in the rat. As a result, it is quite easy to lump all
respiratory diseases into one broad classification such as "Chronic “I
Respiratory Disease" or "Chronic Murine Pneumonia". However, to erradi-

cate or control pneumonia it is essential to determine the primary agent,

the source, and the mode of transmission of the pneumonia.

Objectives-

 

The objectives of this research were-to compare several types of
infections in laboratory rats and determine microscopically how the rat
responds to each. BordsteZZa bronchiseptiaa and PasteureZZa pneumo-
tropica were chosen to represent bacterial pathogens of the respiratory
tract of rats. Mycoplasma puZmonis and Myooplasma sp. were used to
represent myc0p1asma agents.

The specific aims of this research were:

1. To determine the pathogenicity and clinical signs of infections

due to the above-mentioned organisms.

2
2. To determine and compare histOpathology and microbiology during
various stages of the diseases.
3. With the information obtained under objectives 1 and 2, to formu-

late methods of prevention and control.

 

 

LITERATURE REVIEW

A voluminous amount of literature is available on respiratory
infection in the rat. _Summaries and reviews have been presented by
Nelson (1957), Nelson (1963), Innes at al. (1956), Joshi at al. (1967),
Giddens (1968), Brennan at al. (1969), and Pankevicius et a1. (1957).
Much of the research that has been conducted has dealt with the mouse
and, to a limited extent, the rat. This review is limited primarily
to.the major articles dealing with respiratory infections in the_rat.

The current status of chronic respiratory disease in the rat con-
sists of 2 major forms: (1) chronic murine pneumonia (CMP), which is
a slow, progressive respiratory disease. It is characterized by few
if any clinical signs and is believed to be of viral origin.. Histo-
logic examination of the lungs has demonstrated 1ymphocytic_infiltra—
tion and hyperplasia. Only occasionally are other inflammatory cells
noted. Bronchiectasis is not a consistent lesion but may be present;
(2) infectious catarrh is a disease produced by MyOOpZasma pulmonis.

It is seen as a rhinitis, otitis media or labyrinthitis and occasional
snuffling and sneezing.* In advanced cases, exudates may be found around
the eyes and nose. Histologically, there is inflammation of nasal pas-
sages and middle ear predominantly of lymphocytes and neutrOphils. The
lung lesions are often large areas of suppuration, lymphocytic and
neutrOphilic exudates in the bronchi and occasional bronchiectasis.

The pneumonia is believed to be an extension of the upper respiratory

disease.

4
TherMchplasmaOrganisms'

Myc0plasma is the only genus of organisms belonging to the Order
Myc0plasmatales. They are highly pleomorphic, reproduce by breaking up
of filaments into coccoid elementary bodies, lack a rigid cell wall and
may be either parasitic or saprOphytic.

The morphology of the mchplasma organisms (pleuropneumonia-like
organisms; PPLO) is rather difficult to describe because of the great
amount of pleomorphism. The smallest reproductive units (minimal repro-
ductive units) are the elementary bodies, granules or coccobacilliform
bodies. They are spherical in shape and consist of a single cell that
may range from 110 to 250 millimicrons, depending on the species.' This
is significant because these bedies will pass bacteria-retaining filters
and may therefore be confused with virus particles or lead to some unjus-
tified conclusion when tissue filtrates are used to infect experimental
animals. The elementary bodies or "PPLO-cells" (Kleineberger, 1962),
lack a rigid cell wall and are bounded by a triple-layered unit membrane
as seen by electron microscOpy and described by Tanaka et a1. (1965).
This lack of a rigid cell wall may explain the extreme pleomorphism but
also accounts for the complete resistance of these organisms to penicil—
lin and other cell wall inhibiting antibiotics. It may also aid one in
understanding the reproduction of these organisms, which is accomplished
by the formation of filaments which develop from the granules. The fila-
ments break up into granules of various sizes which are in turn capable
of filament formation. Each PPLO—cell may vary in size, but are identi-
cal insofar as cellular potential is concerned.

To isolate mycoplasma, an enriched media is absolutely essential.
The culture media varies with the species to be isolated but, in general,

all cell-free media contains similar constituents. The broth media is

5
prepared as a beef heart infusion peptone media.that is enriched with
10 to 25% serum to provide the necessary proteins and fresh yeast extract
to supply the needed sterols. It should be pointed out that one of the
ways PPLO differ from bacteria is their requirement for cholesterol or
a similar steroid. There are a few exceptions to the requirement of a
high lipid media, but these are among the saprOphytic species. The agar
media is prepared in a similar fashion but agar is added to produce a
semisolid to solid media.

Culturing is the only positive method for isolation, but several
other techniques are available to aid in identification. Any direct
examination for the organism is useless, but if the tissues or exudates
are fixed in methyl alcohol and stained with Giemsa's stain, the elemen-
tary bodies may occasionally be seen (Kleineberger, 1962). Other useful
aids include complement fixation, which is probably the best serologic
method for identification, the use of fluorescent antibody techniques,
darkfield microscopy, and electron microscOpy.

A useful stain to use for colonies on artificial media is the
Dienes stain* (Dienes, 1945) because it can be used on the colonies
directly without previous fixation. A drOp of the stain is spread over
the surface of a coverslip and allowed to dry. The coverslip is then
placed stain side down, onto the PPLO colonies and, after a 10- to 20-
mdnute wait, to give the colonies time to take up the stain, the colonies
are examined under the light microsc0pe. They stain a blue to violet
color, and the dark center of the colonies, along with the granules,
'are readily identified. Bacterial L forms also take up this stain but

Imay lose the deep staining characteristics after a few minutes. This

‘

*Methylene blue O.5g.; Azure II O.25g.; Maltose 2.0g.; Na2CO3
(3.053.; benzoic acid 0.043.; distilled water 20.0m1.

 

6
fading does not always occur and, to positively differentiate PPLO from
bacterial L forms, one must rely on cultural characteristics and serial
passage in an antibiotic free media.

Mycoplasma pulmonis was first isolated by Klieneberger and Steabben
(l937)-from the lungs of rats with bronchiectatic lesions and from normal
rats. This organism was defined at this time as L3 (L for the Lister
Institute where Klieneberger worked; has no relation to the bacterial L
forms) and was isolated from the lungs of 17 of 19 pneumonic rats. In
later research, the L3 organism was isolated from the lungs of 138 rats
from a group of 251 laboratory rats. Lung lesions were observed in 108
rats. Attempts to reproduce the disease failed. Their conclusion at
this time was that there was no definite evidence for or against L3 being
the-etiologic agent of bronchOpneumonia in rats.

Nelson (l940a,b) reported otitis media and pneumonia in the albino
rat and reported coccobacilliform.bodies in the cytoplasm of leukocytes.
It was at this time that he coined the term "infectious catarrh" of the
albino rat to refer to this particular infection. His attempts to repro-
duce the pneumonia with pure cultures of L3 were fruitless, but he was~
able.to reproduce the pneumonia with lung suspensions or with minced
chick embryo tissue suspensions with the organism.

Further studies by Nelson (1946a,b) revealed "endemic pneumonia"
to be present in the absence of PPLO. Clinically the signs were that
of rhinitis with a mucopurulent exudate, and a low grade nasal irritation
with little or no snuffling. Since PPLO were not isolated and this
disease was in a rat colony free of infectious catarrh, a virus-like
agent was thought to be the etiologic agent. Attempts to culture an
organism in eggs or cell cultures were unsuccessful. This virus-like
agent was transmissible by direct contact and was probably passed from

the mother to the young.

7

In order to eliminate the disease, rats were derived by cesarean
and artificially reared in barrier isolation. It was only by this.method‘
that Nelson (1951) was able to remove the virus—like agent and the PPLO
from his colonies.

In a summary, Nelson (1958) concluded CRD in rats could be divided
into 2 separable diseases. The first was infectious catarrh produced
by PPLO. The lesions were a persistent exudative inflammatory response
of the nasal cavity, middle ear, and lungs. The second disease was
endemic pneumonia, or rodent bronchiectasis. The agent is probably a
virus and produces a chronic bronchopneumonia with bronchiectasis. These
2 syndromes may exist independently or may be superimposed upon each
other. Both are reproducible in SPF rats by lung suspensions, middle
ear exudates and nasal washings (1963).

Klieneberger was-not convinced by Nelson‘s conclusions. Klieneberger-
Noble and Chang (1955) found PPLO (L3) to be associated with bronchiectasis
in lobes of lungs that were ligated at the bronchi. Lungs were negative
before surgery, but after ligation PPLO were found in high numbers in
the stagnated exudate in the ligated, bronchiectatic lobes. This pro—
cedure was repeated by Ventura and Domardzki (1967) with similar results.

It was.also noted by Klieneberger and Steabben (1940) that L3
increased directly with lesion severity until the lungs became purulent.
They were reduced in numbers or absent when the whole lobe became dry
and caseous. Also observed at this time were rats between 1 to 3 months
of age that had been subjected to the stress of castration or ovario-
hysterectomy. The L3 organism increased in frequency compared to stock
rats of the same age. Eighteen of 23 developed lung lesions and 22 of

23 had the mchplasma.

 

8

Although she was not completely swayed by Nelson, Klieneberger
stated that , "All kinds of injections of L3 cultures into rats, with or
without agar, have so far not resulted in producing acute pulmonary
disease." She does feel at this time that if the L3 is the causative
agent, an additional factor may be needed to render it pathogenic.
However, the possibility of the L3 agent being only a secondary invader
is still plausible.

Other attempts to produce lesions with pure cultures of PPLO have
also proven to be unsuccessful (Joshi at al., 1965; Pankevicius at al.,
1957).

PPLO infections in rats and mice are apparently not passed from
mother to fetus in utero. Research by Lemcke (1961) suggests that
although the mothers had PPLO in the lung and nasaopharynx, the young
did not acquire the infection until later in life. As the age of the
rat increased, the percentage of total rats that had a titer for PPLO
increased. Within a few weeks of life, PPLO could occasionally be iso-i
lated from the nasopharynx, but not the lung.

In 1956, Innes et a2. established the term chronic murine pneumonia
(CMP) and suggested it as a better term than Nelson‘s.enzootic bronchi-
ectasis. The term CMP is now used to indicate the disease in rats
described by Passey at al. (1936) and Innes at al. (1956), in which
mchplasma are not isolated and the lesions are characterized by a peri-
bronchial lymphocytic infiltration with bronchiectasis occasionally

noted in advanced cases.

The BordsteZZa bronchiseptica and Pasteurelga pneumotropica Organisms
The etiology of GNP has long been a question that has remained

unanswered. The primary role or even the secondary role of bacteria in

9
the etiology and pathogenesis of the classical chronic murine pneumonia
is believed to be relatively insignificant (Giddens, 1968; Innes st aZ..
1956; Joshi et aZ.. 1961; Nelson, 1957; Newberne et aZ., 1961;
Pankevicius at al., 1957; Vrolijk, 1957). Even though the role of bac-
teria in CMP is dubious, mention should be made of bacteria that are
found to be agents of distinct respiratory problems in the rat. Of
these, Bordstella bronchieeptica and Pasteurella pneumotropioa are
frequently associated with the rat respiratory tract and will therefore
be discussed and their pathological features alluded to.

BordsteZZa bronchiseptica is a motile, noncapsule producing Gram
negative rod that is a common respiratory inhabitant of many animals.

It may remain in the nasOpharynx or more commonly in the trachea and be
innocuous or it may progress to an acute infection and result in death
within a few days.

In Bergey's Manual (1925 and 1957), Galli-Vallerio in 1896 is
described as the first to isolate this organism from dogs with distemper
and gave it the name of Bacillus oanioulae. Lignieres (1901), in the
same sources, described this organism as a Phateurella, but Ferry (1911)
again.placed the organism in the genus Bacillus. The organism has been
further classified as.AZanigenss bronchiseptious (Bergey's Manual, 1925),
BruceZZa bronchiseptica and finally BordsteZZa bronchiseptioa (Bergey's
Manual, 1957).

Winsser (1960) has described B. bronchiseptioa infections as being
either latent, present as carriers, or in overt illness. He-has described
a catarrh in the nasal passage of laboratory animals related to this
organism and has isolated it from nasal discharge and from the soft
palate of animal caretakers. BordsteZZa bronchiseptica was isolated from

a child that developed a mild whooping cough 12 days after receiving a

 

 

10
rabbit with snuffles. BordsteZZa bronchiseptica was also isolated from
the rabbit after it accidentally died. He described the white rat as
being quite resistant but that carrier states do occur since he was able
to isolate the organism from the lungs of rats with no gross lesions.r

Griffin (1955) found hemolytic Streptococcus and B. bronchiseptica
(Bacterium bronchisepticum) to be the 2 most common isolates from guinea
pigs with respiratory disease. The lesions produced were that of focal
pneumonia with areas of congestion or consolidation. Ganaway et a1.
(1965) also described acute B. bronchiseptica pneumonia in a guinea pig
colony where bronchOpneumonia and death occurred overnight in several
breeders.

BordeteZZa bronchiseptica, Aerobacter cZoacae and Staphylococcus
aZbus were isolated from pneumonic lungs of albino rats by Smith at al.
(1930). The gross lesions consisted of abscess formation, single or.
multiple, and in one or several lobes. Bronchiectasis was occasionally
noted along with adhesions. Nelson (l940a,b) isolated B. bronchiseptica
along with Actinobacillus muris from rats. Pankevicius et al. (1957)
found BordsteZZa bronchiseptica to be the most common bacteria isolated
from pneumonic and grossly normal lungs in a stock colony of rats.

Six of 21 wild rats that were trapped at a small town dump con-
tained B. bronchiseptica (Switzer et al., 1966). Switzer also noted
that skunks, foxes, raccoons, and cats were carriers of this organism.

Filion et al. (1967) reported a respiratory condition in young
turkeys characterized by rhinitis, bronchitis and bronchOpneumonia with
B. bronchiseptica as the possible agent. Attempts to experimentally
infect turkeys and chickens were successful, but mice were apparently

resistant.

”(AAIKK I." “A: -‘ ‘. '5‘ 9 .‘ . LII-"IVE

 

ll

BordeteZZa has been noted as a pathogen of swine for several years
(Phillips, 1943; Switzer, 1956; Duncan et aZ., 1966; Ross et al., 1967;
Arskog, 1967). Switzer, in his attempts to reproduce atrophic rhinitis,
has at times incriminated this organism as a possible agent (Harris and
Switzer, 1968).

The trachea is the best location to attempt isolation and culture
in most species (Ross et'aZ., 1967; Switzer et aZ., 1966; Ganaway, 1965).

PasteureZZa pneumotropica is a Gram negative, nonhemolytic rod that g
is differentiated from Pasteurella multocida in that it is both indol
and urease positive and does not produce acid from mannitol or sorbitol
(Wheater, 1967; Brennan et aZ., 1965).

PasteureZZa pneumotropica is a common isolate from the respiratory

 

I L‘” .7L417-nglll - - '

tract of rats and mice. Jawetz (1950) and Jawetz and Baker (1950) first
isolated and characterized this organism and alluded to its pathogenicity.
They described the infection as latent in the respiratory tract and
felt that the organism generally would not produce clinical illness
except under artificial conditions. In one study, Jawetz (1950) found
the incidence in infected animals to be: white mice, 30 to 1002; white
rat NIH, 87.5%; cotton rat, 25%. Generally, the natural spread of the
organism from parent to offspring was not serious enough to result in
spontaneous illness. However, his research indicated that latent
P. pneumotropica infections increased susceptibility of experimental
infection via intranasal instillation. PasteureZZa free mice were lees
susceptible to intranasal infection.

The lesions in mice, described by Jawetz and Baker (1950), began
as peribronchial alveolar pneumonitis with the alveoli filled with poly-
morphonuclear cells and some fibrin. There were areas of focal alveolar

coalescence with some polymorphonuclear cells in the bronchi 48 hours

12‘

after experimental exposure. After 72 hours, gray hepatization began to
occur with consolidation of upper lobes of lungs. Bronchi were filled
with exudate. He described the lesion in Swiss mice as "...a picture
of peribronchial consolidation with spreading and coalescence of involved
areas." Their conclusion of resistance of young mice to infection was
not related to subclinical infection but to "nonspecific hereditary
factors."

Flynn et a1. (1965) isolated P. pneumotropica from all but 1 of 11
colonies. The organism was isolated from nasal passages of 90% of the t
rats he examined and also isolated the organism from a "high incidence"

of the lungs and from the uterus of several breeders. é

 

PasteureZZa pneumotropica was cultured on several occasions from
mice by Hoag et a2. (1962). Most isolations were from uteri and brain.
The mouse colonies appeared normal.

Giddens (1968) isolated this organism from several rats with chronic
murine pneumonia. Isolations from the nasal passage of one rat from one
colony and from the middle ear of 2 rats, trachea of one and lung of one
of another colony.

Spontaneous outbreaks of P. pneumotropica in rodents have been
reported by Heyl (1963) and Wheater (1967).

This organism is isolated almost entirely from rats and mice, but
there is one report of the isolation of P. pneumotropica from a normal

human respiratory tract (Henricksen and Jyssum, 1961; Henricksen, 1962).

Summary.

While respiratory disease in the rat has received considerable
investigative attention, still much needs to be examined regarding the

disease. The pathogenesis of specific microorganisms in a defined animal

13
requires additional study. In addition, the interrelationship of dif-
ferent pathogens needs to be established. In this literature review,

references to experimental work with germfree rats were not found.

 

MATERIALS AND METHODS

The germfree and other facilities at.Barn 5, at the Veterinary
Research Farm, were used to conduct these experiments. This research
was part of a larger project on murine pneumonia and was carried out

over a 2-year period.

Source of Animals

 

Two sources of experimental animals were used: (1) Germfree rats

‘-

 

were procured from a Sprague-Dawley derived colony from Notre Dame Uni- L
versity. The colony was originally cesarean-derived and maintained

under germfree conditions for several generations. These rats were‘

reared to maturity and bred to establish a germfree "subcolony" at

Michigan State University. It is from this subcolony that rats were

obtained for germfree studies; (2) Conventional rats used in the follow-

ing experiments were obtained from a local breeder. They were originally
cesarean—derived and were barrier-maintained.

Conventional rats were maintained while on experiment in clear
plastic cages with autoclaved wood shavings. The plastic cages were
disinfected by cleaning with a strong iodine preparation. Filter-taps
made of compressed fiberglass were used to cover the cages and thereby

reduce contamination via aerosol.

Source of Organisms
Both BordsteZZa bronchiseptica and PasteureZZa pneumotropica.were
originally isolated from rat lung tissue. The source of these 2 organisms

l4

15
was the Michigan State University microbiology collection.

Mycoplasma puZmonis. kon strain, was originally isolated from rat
lungs by KlienebergervNoble and maintained by the American Type Culture
Collection. The A.T.C.C. #19612 was the source of this organism.

Mycoplasma 3p,, also of rat lung origin, was isolated from rats
with chronic murine pneumonia. These rats were from the GMT-3 group

used by Giddens (1968).

Exposure

Two methods of exposure were used to infect the rats with the dif-
ferent microorganisms.

1. Intranasal instillation. The rats were anesthetized with ether

 

and a pipette was used to place 1 dr0p (0.05 ml.) of culture medium -
into each nostril. The rat was held in a vertical position to permit
maximum use of the force of gravity to gravitate the broth medium down
toward the lungs.
2. Aerosol exposure. All rats to be exposed were placed in a clear
plastic cage that was.covered with a fiberglass filter-tOp. A hole was
cut in-the filter-top to permit insertion of a nebulizer.* Five milli-
liters of broth were placed in the nebulizer and, with 15 pounds of pres-
sure, an aerosol was created in the cage. Rats were left in the closed
cage for 30 minutes to assure maximum exposure and then transferred to

other cages.

Necgopsy and Cultures

 

All animals were necropsied and cultured as follows: Rats were

euthanatized with ether and placed in dorsal recumbency with all 4 legs

 

*DeVilbiss No. 640 nebulizer, The DeVilbiss Company, Somerset,
Pa., U.S.A.

16

held out in extension. The skin was removed from the ventral aspect
of the carcass as well as the entire cranium. The abdominal cavity was
Opened and the viscera examined for gross lesions. Next, the ventral
rib cage was removed, exposing the thoracic viscera. .A section of the
left lobe was removed for culture. The trachea was then dissected from
the surrounding cervical tissue and was transected at the pharynx. A
sterile platinum loop was inserted into the trachea to obtain material
for culture. This loop was then used to streak a blood plate and/or
PPLO plate and to inoculate BHI broth. The trachea, lungs, and heart
were then removed for fixation.

The tip of the nostrils were cut off with scissors. A platinum

loop was inserted, the mucus collected, and the 100p used to streak a

 

blood plate and a PPLO plate and inoculate broth. The head was then
placed in formalin.

Bacterial cultures from the lungs were obtained by taking a sterile
piece of lung and streaking the cut surface over a blood agar plate. A
portion of this lung was then macerated with the aid of scissors and
placed in BHI (brain heart infusion) medium for 24 hours.

Mycoplasma cultures were obtained by first streaking a PPLO plate
with the cut surface similar to the technique used for bacteria. This
portion of lung was then ground in a tissue grinder and placed in PPLO
broth medium and incubated for 3 to 4 days. At this time, 1 ml. of this
inoculated broth was transferred to new PPLO media as well as streaking
another PPLO plate. A total of 3 transfers in broth and PPLO agar plates
were made on all PPLO samples. If no growth was recorded after the
third transfer, the samples were considered negative for mchplasma.

All tissues were fixed in 10% buffered formalin. The lungs were

perfursed with formalin by slowly inflating the lobes with a syringe

l7
filled with formalin. The trachea was then tied off and the lungs placed
in formalin.

Three sections were made through the lungs for histopathologic
examination. The first section was taken through the apical and portions
of the cardiac lobes as well as the apical portion of the left lobe. The
second section included the remainder of the cardiac, a portion of the
azygous lobe, and the midportion of the left lobe. The third section
included the diaphragmatic lobe and the distal portion of the left lobe. .m
Two cross sections were made of the trachea. The head was decalcified
for 24 hours and 2 cross sections of the nostrils were made. The first
section was 1/2 inch from the tip of the nose. The second section was
1/4 inch anterior to the medial canthus of the eyes.

The mchplas a broth medium was prepared from the dehydrated Bacto
PPLO Enrichment Broth from Difco Laboratories. It was enriched with 20%
horse serum and 10% yeast extract. To this, thallium acetate and peni-
cillin were added to retard bacterial growth.

The agar medium was prepared from.the dehydrated Bacto PPLO Agar
from Difco Laboratories. The constituents are identical to the broth

medium plus the addition of 1.5% agar.

Experiments

 

The following experiments were conducted. In all the trials, con-
trol rats were given the sterile broth or tissue culture medium and a
similar number of nontreated controls were maintained separate from
the experimental animals, but under similar conditions. The eXperiments

may be summarized as follows:

l8
1. Sixteen conventional weanling rats were exposed to broth cul-
tures of Mycoplasma sp- Eight were exposed by aerosol and 8 by intra-
nasal instillation. The broth cultures were incubated for 3 days before

exposing the rats. Rats were examined daily and necropsied after 14 days.

2. Eleven germfree 21-day-old rats were exposed to an intranasal
instillation of Mycoplasma sp. growing for 5 days in pig kidney cell (PKC)

cultures. Rats were necropsied at 2 weeks, 4 weeks, and 5 weeks.

3. Four conventional weanling rats were exposed to Mycoplasma sp.
by surgically entering the trachea and placing l drOp of_broth culture

directly into the trachea. 'Rats were euthanatized and necropsied at 4

 

weeks past infection.

4. Six conventional weanling rats were exposed to a broth culture
of Mycoplasma sp. intravenously. The rats were observed for 4 weeks and

then necropsied.

5. Twenty conventional rats were exposed to ML pulmonis, 10 by
intranasal instillation of the organism growing 5 days in PRC cultures
and 10 by aerosol exposure of the same culture. Rats were necropsied

after 4 weeks.

6. Eleven conventional rats were exposed to a 24-hour broth culture
of B. bronchiseptica. Necropsy was performed at selected intervals up

to 2 months after exposure.

7. Same as experiment 6, using P. pneumotropica.

l9
8. Eight conventional rats were exposed to a combination of
B. bronchiseptica and P. pneumotropica. NecrOpsy was performed as in

experiments 6 and 7.

 

RESULTS

Experiment 1

 

On daily examination clinical signs were not observed. Therefore,
after 14 days, all rats were euthanatized. NecrOpsies were-performed
on each rat and cultures were taken from the rats as described previously.

Cultures from the nasal passage were negative for Mycoplasma. How-
ever, nasal contaminants were noted in nearly all rats, both experimental
and controls. These contaminants were mainly a combination of Micro-
coccus and Staphylococcus sp.

Bacteria were not isolated from the trachea, but Mycoplasma sp.
was isolated from one rat. All other rats were negative for Mycoplasma.

Cultures from the lungs of all rats were negative for bacteria and
Mycoplasma.

On gross and microscOpic examination, the nasal passage, trachea,
and lungs were normal. There were no changes that were even suggestive
of a response to the organism in any of the rats with one exception.

In one rat, from the group exposed by internasal instillation,
MchpZasma sp. was isolated from the trachea. The nasal passage and
trachea were normal and histologic examination did not reveal microscopic
changes. However, one portion of the lungs contained an area of bron-
chiectasis, lymphocytic proliferation and infiltration, peribronchial
and perivascular edema, lymphangiectasis, lymphatics filled with lympho-
cytes, and hypertrOphy of sections of bronchiolar epithelium. There were

numerous alveolar macrOphages within the lumen of the bronchi, with an

20

21
increased production of mucus. The alveolar walls were thickened in
this area with edema and lymphocytes. Emphysema and atelectasis were
also present in this localized area but did not involve much of the lung
area. Because this reaction involved most of the left lobe of the lung
and could be followed through several histologic sections at different
levels of this lobe, this reaction is considered to be an early response

of the lung to this particular microorganism (Figure 1).

Experiment 2 !

 

The 11 germfree rats that were eXposed to intranasal instillation
of MycopZasma sp. growing for 5 days in pig kidney cell tissue cultures

were euthanatized at 2 weeks, 4 weeks, and 5 weeks. No gross or micro-

 

scOpic lesions were observed in the nasal passage, trachea, or lung. -

Experiment3'

 

Attempts to infect rats by surgical entrance into the trachea and
thus admitting l drOp of mchplasma broth culture directly into the

trachea were all negative both culturally and histOpathologically.

Experiment 4
The results of intravenous inoculations with mchplasma broth cul-

tures, with and without agar, were also negative.

Experiment 5
The 10 conventional rats that were exposed to M. puZmonis by intra—
nasal instillation of the organism growing for 5 days in PRC cultures were
negative on culture and no tissue lesions were seen.
The 10 rats exposed by aerosol to the same culture were also

negative.

22

   

 

 

 

Figure l. Distal portion of the left lobe of the lung
from a 5-week-old rat, 14 days after intranasal exposure to
Mycoplasma sp. Note the peribronchiolar edema, dilation
of subepithelial lymph vessels, atelectasis and focal peri-
vascular lymphocytic cuffing. H & E stain. x 40.

23
Clinical signs were not apparent in any of the rats. Gross and
microscOpic examination did not localize-any abnormalities. Lymphocytic

hyperplasia or infiltration around the bronchi was not marked.

Experiment 6

1. One week after aerosol exposure. All rats had rough hair costs,
were anorectic and had respiratory distress. Food consumption was mar-
kedly reduced when compared to controls. Average weight was 75 grams,
which compared to an average of 92 grams for control rats.

Two rats were killed at this time and were found to have B. bronchi-
septica in their nasal passage, trachea and lungs (Table 1).

An increased amount of mucus or exudate was in the nasal passage
and trachea upon gross examination. The lungs had numerous focal pale
areas .1 to .3 mm. in diameter, suggestive of focal necrosis. Distribu-
tion of these areas was uniform throughout all lobes without predilection
for any area of the lungs.

The nasal submucosa of both rats contained a few lymphocytes and
an occasional neutr0phil but did not appear significantly different from
controls and was therefore considered normal. There was, however, an
increased quantity of exudate in the nasal passage. This exudate con—
sisted of lymphocytes, neutrOphils and an occasional macrophage all in
a mucous matrix.

The trachea of both rats did not contain microsc0pic lesions.

Microscopic examination of the lungs revealed numerous focal areas
of bronchOpneumonia. The pneumonic areas contained a core of lymphocytes
and a few neutrOphils. Many of these cells were necrotic. Around the
core were numerous alveolar macrOphages and lymphocytes and a few fibro—
blasts and neutrophils (Figure 2). In several areas the focal pneumonic

lesions appeared to coalesce (Figure 3).

 

24

Table 1. Summary of the isolations of BordsteZZa bronchiseptica in
conventional rats that-were exposed to the organism by
aerosol exposure

 

 

 

No. of
No. of weeks
animals in after Nasal
group exposure Passage Trachea Lung
2 1 2 2 2 *7.
5 2 5 5 4
2 4 2 O 2

 

 

 

 

25

 

 

 

 

Figure 2. Focal area of bronchOpneumonia with lympho-
cytes, neutrOphils and macrophages in a 4-week-old rat, 7 days
after aerosol infection with B. bronchiseptica. Note the
areas of necrosis within the pneumonic lesion and the lack of
cellular reaction next to the focal lesion. H & E stain. x 40.

1,

 

 

 

Figure 3. Lung from a 4-week-old rat, 7 days after
aerosol exposure to B. bronchiseptica. Focal pneumonic
lesion with coalescence and necrosis. H & E stain. x 40.

 

26
Bronchiolar epithelium in the region of the inflammatory reaction
was hyperplastic. An exudate similar to that found in the nasal passage

was in the lumen of several bronchioles.

2. Two weeks after aerosol exposure. Occasional sneezing was the
only clinical sign observed. Food consumption was comparable to controls
and average weights were lower than controls, but they were not sig-
nificantly different.

Exudates were not observed around the nostrils, nor was there any
sign of respiratory distress.

Five rats were euthanatized. All 5 contained B. bronchiseptica in

the nasal passage and trachea. All but 1 had the organism in the lung

 

(Table l). ._.

No gross lesions were observed in any of the 5 rats.

The nasal passage of.3 rats had a mild rhinitis characterized by
lymphocytes and a few neutrOphils in the submucosa and infiltrating
between the nasal epithelial cells. An increase in nasal exudate was
also in these rats, but not as extensive as was observed 1 week earlier.

Two rats had a mild lymphocytic infiltration along with a few neu-
trOphils in the trachea. Generally, the trachea was considered normal.

All 5 rats had a moderate hyperplasia of lymphoid tissue along the
bronchi and bronchioles. Three of the 5 had lymphoid infiltration along
the bronchi and occasionally into the epithelial lining (Figure 4). The
same 3 rats also had areas of bronchOpneumonia with focal inflammatory
infiltration and coalescence. There was a mdnimal amount of necrosis
(Figure 5). Increased fibroblasts and evidence of collagen were noted
by the use of trichrome stains. Bacterial colonies, some emphysema and
extensive subepithelial lymphoid cuffing of bronchioles were noted in

2 of the pneumonic rats.

27

 

 

Figure 4. Minimal lymphocytic infiltration along bron-
chiole in a Seweek-old rat, 14 days after aerosol exposure to
B. bronchiseptica. H & E stain. x 100.

 

 

 

Figure 5. A large focal area of pneumonia with coalescence
and hyperplasia of adjoining bronchiolar epithelium in a 5-
week-old rat, 14 days after aerosol exposure to B. bronchi-
septica. H & E stain. x 40.

 

28

3. Four weeks after aerosol exposure. By the end of 4 weeks, no
clinical signs were observed and gross lesions were not noted after
euthanasia of 2 rats.

Both animals contained B. bronchiseptica in their nasal passages
and lungs. Both tracheas were negative upon cultural examinations
(Table l).

The nasal passage of 1 rat had a mild rhinitis with a moderate
increase in lymphocytes diffusely below the epithelial surface. A few
neutrophils were noted. The other rat was normal.

The trachea of both rats was histologically normal.

The lung of 1 rat was characterized by diffuse lymphoid infiltra-
tion along the major bronchi and arterioles. The lung parenchyma and
alveoli were normal and did not differ from controls in their overall
appearance.

The lung of the second rat contained numerous areas of regressing
bronchOpneumonic lesions. These areas were surrounded by alveolar macro-

phages and some connective tissue, fibroblasts and lymphocytes (Figure 6).

4. Eight weeks after aerosol exposure. Clinical signs had not
been observed and the last 2 rats were examined. No gross lesions were
noted.

One rat had B. bronchiseptica in the nasal passage, trachea and
lung. The other rat was negative from all locations.

5 Both rats appeared to have a mild rhinitis characterized by infil-
trations of lymphocytes and a few neutrophils. However, both controls
also had a similar response in their nasal passage, leading one to the
conclusion that this is a function of age rather than irritation from

BordeteZZa.

29

 

   

Figure 6. A 7-week-old rat lung, 4 weeks after aerosol
exposure to B. bronchiseptica. Note the apparent regression
of pneumonic lesions with lymphocytes and macrOphages as the
predominant cell types. H & E stain. x 100.

30

A few lymphocytes were noted in the trachea, but this was considered
normal.

The lesions in the lungs of both rats were of focal areas of lymphoid
hyperplasia or nodules with a reduction in the diffuse lymphocytic infil-
tration around most bronchi. A few small areas remained that appeared
to be regressing pneumonic lesions. Multifocal histiocytosis were noted-
in these areas as well as in areas that appeared nonmal. The only diffuse

lymphoid infiltration was around the large bronchioles and bronchi.

Experiment«7

 

1. One week after aerosol exposure. All rats were observed and no
clinical signs were noted. Feed consumption was comparable to controls
as were the average weights.

Two rats were euthanatized. There were no gross lesions. PasteureZZa
pneumotropica was isolated from the trachea of.l rat (Table 2).

Nasal passage and trachea did not have microscOpic lesions. The

lung of 1 rat had focal lymphoid hyperplasia, but no evidence of.pneumonia.

2. Two weeks after aerosol exposure. Five rats were observed and
no clinical signs were noted. Feed consumption and average weights were
comparable to controls. Of the 5 rats, only 1 was found to harbor Pas-
teureZZa. It was isolated from the nasal passage and trachea, but not
the lung (Table 2).

Nasal passage and trachea of all 5 rats were normal.

On microscoPic examination 2 rats had a distinct increase in lymphoid.
follicles as well as a mild increase in lymphoid elements between the

basement membrane and epithelial cells. The other 3~rats were normal.

 

31

Table 2. Summary of PasteureZZa pneumotropica in conventional rats
that were exposed to the organism by aerosol exposure

 

 

 

No. of
No. of weeks
animals in after Nasal-
group exposure Passage Trachea Lung
2 l O l O
5 2 1 l O
2 4 1 l O

 

 

32

3. Four weeks after aerosol exposure. No clinical signs were
observed. Weights and feed consumption were similar to controls.

Of the 2 animals examined, 1 had the organism in the nasal passage
and trachea (Table 2).

MicroscOpic examination of the upper respiratory tract did not reveal
any significant changes. Both rats had an increased amount of lymphoid
elements around the bronchi, but this did not differ from controls and

was considered normal.

4. Eight weeks after aerosol exposure. The last 2 rats had no
clinical signs after 8 weeks.

Both rats had areas of multifocal histiocytosis, that is, focal
areas of alveolar macrophages in the alveoli but no evidence of pneumonia.

Nasal passage and trachea when compared to controls were normal.

Experiment 8

 

1. One week after aerosol exposure. Hair costs were rough, but
none of the rats appeared anorectic, nor were any clinical signs appar-
ent. Feed consumption was comparable to controls. Average weights of
these rats were similar to controls.

Both rats were found to have B. bronchiseptica in the nasal pas-
sage, trachea and lungs (Table 3). However, P. pneumotropica was iso-
lated from the trachea of both rats.

On gross examination, the nostrils had increased amounts of mucus.
Microscopic examination demonstrated a mild rhinitis with the predomi-
nant cell the lymphocyte, with an occasional neutrophil and macrOphage.
There was evidence of inflammatory cells infiltrating into the nasal

epithelium.

33

 

 

 

 

 

 

Table 3. Summary of the isolations of Bordetella bronchiseptica and
PasteureZZa pneumotropica in conventional rats that were
exposed to the organisms by aerosol exposure

No. of
No. of weeks Nasaerassage Trachea Lung
animals in after Bords- Pasteur- Borde- Pasteur- Borde- Pasteurb
group exposure teZZa eZZa teZZa eZZa teZZa eZZa
2 1 2 0 2 2 2 O
2 2 2 0 2 l 2 O
2 4 2 2 2 0 2 0
2 8 2 0 2 0 l 0

 

 

34
The trachea appeared normal on gross and microsc0pic examination.
Lungs were characterized by a diffuse lymphoid reaction around
bronchi and a.vasculitis. Focal pneumonitis was noted with lymphocytes
and macrophages predominating and occasional neutrOphils and fibroblasts
observed. Coalescence was noted in several areas. This response was

nearly identical to the lungs in the BordeteZZa group of rats.

2. Two weeks after aerosol eXposure. Two rats were euthanatized.
They did not differ from controls in feed consumption, average weights
or clinical appearance.

Both rats harbored B. bronchiseptica in the nasal passage, trachea
and lungs. Only 1 rat had P. pneumotropica present in the trachea
(Table 3).

Some exudate was noted in the nostrils but a rhinitis was not pres-
ent. The trachea had no gross lesions but a mild lymphoid infiltration
was observed.

Lungs were characterized by focal lymphoid hyperplasia and infiltra—
tion around bronchi as well as a few focal lesions of lymphocytes and

macrOphages, and a few foci of coalescence (Figure 7).

3. Four weeks after aerosol eXposure. Two rats were euthanatized.
Both rats were clinically normal. .

Cultures were positive for B. bronchiseptica in both rats from the
nasal passage, trachea and lungs. PasteureZZa pneumotropica was iso-
lated from the nasal passage of both rats (Table 3).

The upper respiratory tract had no gross or microsc0pic lesions.

The lung of l rat had a diffuse as well as focal lymphocytic pro-
liferation around bronchi (Figure 8). A few small foci of pneumonia

were observed in both rats.

35

I .r :L’f“: '

D
'0

 

- 3'
‘. NH,”

 

Figure 7. Extensive focal inflammatory exudate extend—
ing through alveoli with disruption of the normal alveolar
pattern in a 5-week-old rat, 14 days after aerosol exposure
to B. bronchiseptica and P. pneumotropica. H & E stain. x 40.

 

 

 

 

Figure 8. Lung from a 7~week-old rat, 4 weeks after
aerosol exposure to B. bronchiseptica and P. pneumotropica.

Note the extensive peribronchiolar lymphocytic infiltration
and hyperplasia. H & E stain. x 40.

 

36

4. Eight weeks after aerosol exposure. The last 2 rats were
clinically normal.

BordeteZZa was isolated from the nasal passage and trachea of both
rats and the lung of one. PasteursZZa pneumotropica was not isolated
from either rat (Table 3).

Gross and microscOpic lesions of the upper respiratory tract were
not found.

Lung reaction was minimal. Lymphoid tissue was reduced around
bronchi, and little evidence of pneumonia was present. Several areas

of multifocal hyistiocytosis were observed.

 

DISCUSSION

Mycoplasma Infection in the Rat

Only 1 rat was found to contain Mycoplasma in its trachea. This
rat was also the only animal to have distinct lesions in‘its lungs.
However, since this is only one of many rats that were exposed to
MycopZasma sp. and Mycoplasma pulmonis, it is difficult to determine
the significance of this finding.

There are several possible explanations for the failure of Myc0plasma
to localize in the rat respiratory tract when pure cultures of the
organisms are used: (1) the.rats used may have had a resistance to
infection by this organism; (2) the organism loses its virulence when
passed in artificial media; (3) the Mchplasma may be secondary invaders
and are not capable of primary infections in the rat; (4) there are
several strains of Mchplasma that may be found in the rat respiratory
tract and only certain strains of the same species are capable of pro-
ducing respiratory infections; (5) certain age factors of the host may
play a role; and (6) Mchplas a may need a virus togbecpme'pathogenic.
Any one or a combination of these may result in failure of infection

to occur 0

BordsteZZa bronchiseptica Infection in the Rat
BordsteZZa bronchiseptica was consistently isolated from the nasal
passage, trachea and lungs after experimental exposure. The response

was characterized by an acute pneumonic reaction which slowly regressed

37

 

38
over a period of 2 months.r Although an increase in peribronchiolar
lymphocytes was noted between 1 and 4 weeks after exposure, by 8 weeks
the peribronchiolar tissue was similar to controls. Therefore, peri-
bronchiolar lymphocytes can regress if the antigenic stimulus is removed.
This would lead one to conclude that in CMP, where there is a chronic
insidious increase in lymphoid tissue, there is apparently a constant

antigenic stimulation preventing regression of the inflammatory cells.

PasteureZZa,pneumotropica Infection in the Rat

 

PasteureZZa pneumotropica has been reported to produce experimental
pneumonia. However, no isolations were made from the lungs of 11 rats
exposed to P. pneumotropica by itself or from 8 rats exposed to both.

B. bronchiseptica and P. pneumotropica in combination. The_organism
could be isolated from the nasal passage and more commonly the trachea,
thus indicating that the organism is capable of surviving in the rat
respiratory tract. Even though the lung tissue was damaged by Borde-
teZZa, the PasteureZZa was still not capable of lung invasion.

One might speculate that P. pneumotropica needs some added factor
besides lung damage to become pathogenic. This factor could conceivably
be a Mycoplasma, rather than a bacterium. It would be interesting to
see if the 2 organisms together might be capable of lung invasion.

Another point to consider is the age at which the rats were exposed;
to PasteureZZa. There may very well be an age predilection or a time
when the rat is more sensitive to infection by these organisms. Data
should be: collected concerning uterine infections, early embryonal

infections and susceptibility to repeated infections of the same organism.

 

39

Combined BordeteZZa bronchiseptica and PasteuroZZa
pneumotrgpica Infections in the Rat

The results from a combined infection of Bordetella and PasteureZZa
parallel the reaction of Bordetella alone. There was no increase in
pasteurella isolations compared to infection with PasteureZZa alone.
There was no significant difference in lesions in the nasal passage,

trachea or lung as compared to that with BordéteZZa alone.

SUMMARY AND CONCLUSIONS

A total of 8 experiments was conducted over a 2-year period, expos-
ing germfree and conventional rats to MycopZasma sp., Mycoplasma pulmonis,
BordsteZZa bronchiseptica, PasteureZZa pneumotropica, and a combination
of BordsteZZa bronchiseptica with PasteureZZa pneumotropica. In each
experiment, the acute and chronic aspects of the exposure were determined.
Infection was determined by isolation of the organism(s) and the presence
of tissue lesions. Where possible, the infections were compared to
determine host response to each agent.

From these experiments, the following conclusions were made:

1. Mycoplasma sp. was not capable of producing respiratory infec-
tion in the rat with intranasal instillation or aerosol exposure of a
broth culture. Even when the organism was placed directly into the
trachea or when given intravenously, no lesions were produced.

2. Mycoplasma puZmonis did not produce respiratory infection when
a 5-day growth of this organism in PRC cultures was inoculated intranasally.

3. After aerosol exposure to BordsteZZa bronchiseptica from a 24-
hour growth in BHI, an acute to subacute multifocal bronchOpneumonia
was produced that regressed over a period of 2 months.

4. PasteureZZa pneumotrOpica did not invade the lung after aerosol
exposure to a 24-hour growth of the organism in BHI culture media.

5. A combination of B. bronchiseptica and P. pneumotropica infec-
tion produced lesions identical to B. bronchiseptica alone. No enhance-
ment of P. pneumotropica occurred as a result of the combination.

40

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VITA

Joe Dale Burek was born in Joplin, Missouri, on May 7, 1945. He‘
graduated from Edsel Ford High School, Dearborn, Michigan, in June 1963.
He was awarded the Bachelor of Science degree in 1968 and the

Doctor of Veterinary Medicine degree in 1969 from Michigan State Uni-

versity. The author was chairman of the Public Relations Committee,
chairman of the Program Committee and president of the Student Chapter
of the American Veterinary Medical Association while in veterinary
school. He was also the recipient of the-Pfizer Award for scholarship
and leadership.

From December 1969 until June 1970, the author was employed in a
private practice in Ovid-Elsie, Michigan.

The author was appointed to the Upjohn Fellowship, in the Department
of Pathology at Michigan State University, in December 1969.

The author was married September 4, 1965, and is a member of the
American Veterinary Medical Association and the Michigan Veterinary

Medical Association.

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