VITAMINS A AND E IN GERMFREE AND
CONVENTIONAL RATS EXPOSED T0
MYCOPLASMA PULMONIS'

Thesis for the Degree of M. S.
MICHW STATE UNIVERSITY
HAROLD WILLIAM TVEDTEN

‘ 1973

 

 

LEF": F?" RY
Mice ‘ mate
Ui‘é‘ - "J‘Ey

 

 

 

ABSTRACT

VITAMINS A AND E IN GERMFREE AND CONVENTIONAL
RATS EXPOSED TO MYCOPLASMA.PULMUNIS

By

Harold William.Tvedten

Ninety—six rats were used in 2 concurrent 2 x 4 factorial experi-
ments under germfree or conventional conditions. One-half of the 48
germfree rats were exposed to Mycoplaama pulmonis, as were one—half
of the conventional rats. These 4 groups of 24 rats were each further
divided into 4 groups of 6 rats fed either a (l) basal, vitamins A and
E deficient, (2) vitamin A deficient, (3) vitamin E deficient, or (4)
vitamins A and E supplemented ration.

Vitamins A and E prevented mycoplasmosis in the exposed rats;
therefore, vitamin supplementation at high levels may be a method of
reducing the disease in rat colonies. Vitamin A also prevented
ascending Pseudbmonas infections of the urinary tract of conventional
rats. Vitamin A supplemented rats had higher levels of glycoproteins
such as gamma globulins and other serum proteins than vitamin A
deficient rats.

Thyroid metaplasia and keratinization were present in vitamin A

deficient, germfree rats. Hepatic necrosis and myocardial degeneration

with calcification, previously attributed to vitamin A.deficiency,

were only present in vitamin E deficient germfree rats. Other vitamin

A or E deficiency lesions were similar to those previously reported.

VITAMINS A AND E IN GERMFREE AND CONVENTIONAL

RATS EXPOSED TO MTCOPLASMA.PULMUNIS

BY

Harold William Tvedten

A THESIS

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

MASTER OF SCIENCE

Department of Pathology

1973

Dedicated to my wife

Gretchen

ii

ACKNOWLEDGEMENTS

I am very grateful for having had the Opportunity to do this
research and to have studied pathology during the year of 1972. The
following groups and individuals made this possible.

The Upjohn Company provided a postdoctoral fellowship during 1972
to provide my income. Professor C. K. Whitehair provided abundant
counseling and help in research procedures and in the writing of this
thesis. Dr. George Jersey was invaluable in the planning and expediting
of this research. Professor R. F. Langham helped in the interpretation
of the histopathology of the tissues and provided much of my advanced
training in pathology. Professor G. R. Carter and his staff unselfishly
helped with the microbiologic aspects of this research. The members
of the Department of Pathology all cheerfully gave assistance whenever

it was needed and made 1972 a productive and enjoyable year.

iii

TABLE OF CONTENTS
Page
INTRODUCTION AND OBJECTIVES. . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW. . . . . . . . . . . . . . . . . . . . . . . . . 2
General . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Vitamin A and Resistance to Disease . . . . . . . . . . . . 2
Vitamin A Deficiency Lesions. . . . . . . . . . . . . . . . 4
Vitamin E . . . . . . . . . . . . . . . . . . . . . . . . . 5
Mycoplasma pulmonis Infection of Rats . . . . . . . . . . . 6
MATERIALS AND METHODS. . . . . . . . . . . . . . . . . . . . . . . 8
Experimental Animals and Design . . . . . . . . . . . . . . 8
Housing . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Diet and Feeding Practices. . . . . . . . . . . . . . . . . 9
Experimental Exposure of the Rats . . . . . . . . . . . . . ll
Termination of the Experiment . . . . . . . . . . . . . . . 12

culturing MethOdS O O O O 0 O O O O O O O O O O O O O O O O 12

Laboratory Tests. . . . . . . . . . . . . . . . . . . . . . 12

PathOIOgy O O O O O O O O O O O 0 O O O O O O O I O O O 0 O 13

RESULTSo . . o . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Clinical Signs. . . . . . . . . . . . . . . . .
Clinical Pathology. . . . . . . . . . . . . .
Microbiology. . . . . . . . . . . . . . . . . . . .

PathOIOgy O O O O O O O I O O O O O O 0

iv

DISCUSSION . . . . . . . . . . . . . . .
Vitamins A and E in Resistance to
Pathology . . . . . . . . . . . .

SUMMARY AND CONCLUSIONS. . . . . . . . .

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

v ITA O O O O O 0 O O O O O I I O O O O 0

Infection

Page
33
33
34
37
38

42

LIST OF TABLES
Table Page
1 Experimental design . . . . . . . . . . . . . . . . . . . . 9
2 Basal ration. . . . . . . . . . . . . . . . . . . . . . . . 10

3 Comparison of mean serum protein electrophoresis values
in m/loo ml. 0 O O O O O O C I O I O O O O O O I O O O O O 19

4 Mycoplasma isolations . . . . . . . . . . . . . . . . . . . 21
5 Bacterial isolations from conventional rats . . . . . . . . 21
6 Urogenital infections of conventional rats. . . . . . . . . 28

vi

Figure

2a

2b

LIST OF FIGURES

Comparison of 2, 59-day—old, conventional rats. The

rat on the left had been fed the ration deficient in
vitamins A and E for 38 days and had xerophthalmia.

The rat on the right had been fed a vitamin A deficient
ration for 38 days and did not have signs of vitamin A
deficiency, although both rats had no detectable hepatic
vitamin A stores. . . . . . . . . . . . . . . . . . . . . .

Growth curves of conventional non-exposed rats. The A

and E deficient rats had died by 3 months of age, while
the other groups continued to grow. The A and E sup-
plemented rats had a lower average weight since they

were mainly females . . . . . . . . . . . . . . . . . . . .

Growth curves of conventional exposed rats. The A
deficient rats grew at a slower rate soon after exposure
at 9 weeks of age and lost weight as the infection pro-
gressed. The E deficient rats behaved similarly a few
weeks later. The A and E supplemented rats grew until
termination of the experiment . . . . . . . . . . . . . . .

Rhinitis in an A and E deficient, conventional rat.

Most inflammation involves the keratinized turbinate,
while the columnar epithelium remaining on the nasal
septum (arrow) is free of exudate . . . . . . . . . . . . .

Tracheitis in an E deficient, conventional rat. Hyper-
plasia and folding of the epithelium was a typical response
of vitamin A supplemented rats to M: pulmonzs . . . . . . .

Tracheitis in an A deficient germfree rat° Metaplasia

and keratinization (arrow) was present rather than the
hyperplasia seen in Figure 4. Keratinization of 2 thy-
roid acini was also present (K) . . . . . . . . . . . . . .

Characteristic purulent bronchitis with bronchiectasis
and metaplasia of M2 pulmonis infection in an A deficient,
conventional rat. The lumen was filled with purulent
debris and a large focus of mononuclear cells (arrow) is

adjacent to the bronchiole. . . . . . . . . . . . . . .

Pneumonic lungs of an E deficient conventional rat. The
apical portions (arrow) are typically the most severely
affected in M. pulmonis infections. . . . . . . . . . .

vii

Page

15

17

17

22

23

23

26

26

Figure

10

ll

12

13

14

15

Multiple renal abscesses (arrow) in an A and E deficient,
conventional rat. White flocculent material was present
in the urinary bladder. . . . . . . . . . . . . . . . . .
Cystitis and keratinization in the urinary bladder of an
A deficient conventional rat. . . . . . . . . . . . . . .

Penis, testes and large hemorrhagic urinary bladder of
an A and E deficient germfree rat . . . . . . . . . . . .

Photomicrograph of a keratinized urethra (large arrow)
in the penis of an A and E deficient, germfree rat. The
outer horny layer (small arrow) of the penis was present.
Keratinization of the urethra contributed to enlarged
bladders such as in Figure 10 . . . . . . . . . . . . . .

Aplastic seminiferous tubules of an A deficient, germ-
free rat. Fusion cells (arrow), characteristic of
degenerate testis, were present . . . . . . . . . . . . .

Active spermatogenesis in the testis of a conventional
rat fed a vitamin E deficient ration. . . . . . . . . . .

Degeneration and calcification of the skeletal muscle
of a germfree vitamin E deficient rat . . . . . . . . . .

Centrolobular hepatic necrosis (C) in a vitamin E

deficient germfree rat. The other hepatocytes had some
postmortem degeneration . . . . . . . . . . . . . . . . .

viii

Page

27

27

29

29

31

31

32

32

INTRODUCTION AND OBJECTIVES

Massive amounts of certain vitamins, such as vitamins E, B complex
and C, are commonly being ingested for the prevention of disease. This
practice is suggested by veterinarians and physicians alike, who are
of the opinion the published minimum daily requirements of these
vitamins are adequate to prevent deficiency signs but not adequate to
prevent infectious disease. Although previous research (Scrimshaw,
1966) demonstrates the importance of nutrition in the resistance to
disease, neither the optimum dosage, nor specific mechanisms of action
of the specific nutrients, is sufficiently understood.

One objective of this research was to clarify the action of 2
specific nutrients, vitamins A and E, in the most important disease of
rats, chronic respiratory disease (CRD) due to Mycoplasma'pulmonis.
Various theories of the action of vitamins A and E were discussed, as
was the possible use of these vitamins to reduce the incidence of
mycoplasmosis in rat colonies.

The second objective was to describe the pathologic lesions of
vitamin A or E deficient rats with emphasis on lesions of controversial

origin or those not previously reported in the germfree rat.

LITERATURE REVIEW

General

The role of specific nutrients, such as vitamins A and E, in the
body has usually been determined by creating a deficiency of the par-
ticular nutrient and determining what functions are lost. The functions
which are lost are then attributed to the missing nutrient. Scrimshaw
(1966) reported some of the functions lost during malnutrition as (1)
formation of specific antibodies, (2) activity and response of phago-
cytes, (3) membrane integrity, (4) maintenance of a normal intestinal
flora, (5) lysozyme activity in tears, sweat and peritoneal fluid,
(6) interferon production, (7) endocrine balance, and (8) resistance

to bacterial toxins.

Vitamin A and Resistance to Disease

 

Vitamin A was labeled as an anti-infective agent in 1928 (Green
and Mellanby). Vitamin A deficiency is still the most important
pediatric nutrition problem in India (Sirakumar and Raddy, 1971), where
it has been incriminated as a predisposing cause of respiratory and
gastrointestinal infections. Squamous metaplasia of epithelia in the
vitamin A deficient animal has been established since 1925 as an
impairment of the body's protective coating (wolbach and Howe), but
only recently has another role in the body's defense mechanism been

established.

3

DeLuca and WOlf (1970) demonstrated the need for vitamin A in
secretion of intestinal mucus. In vitamin A.deficiency the number of
goblet cells is reduced in half. They were of the opinion that vitamin
A acts as a sugar carrier lipid intermediate in the synthesis of
glycoproteins, such as mucus.

Rogers at al. (1971) similarly speculated that vitamin A was
required in the production of other protective glycoproteins such as
antibodies, interferon and lysozyme, as well as being required for the
maintenance of epithelia and the maturation of phagocytic leukocytes.

The requirement of vitamin A in the cellular differentiation of
specialized cells such as mucus—secreting goblet cells, osteoclasts,
and seminiferous tubules has been postulated by Hayes (1970) in a
review article. He stated that bipotential cells such as in the
respiratory tract can synthesize either mucous glycoproteins or fibrous
keratinizing proteins. In a vitamin A deficiency these cells only dif-
ferentiate into keratinizing epithelium. In unidirectional differentiat-
ing cells such as spermatogonia, no differentiation is possible, which
results in testicular degeneration.

The process of cellular differentiation consumes vitamin A and,
in a growing animal, cells are rapidly developing, thus requiring maximum
amounts of vitamin A (Hayes, 1970). Similarly, during infections such
as bronchitis and enteritis, the turnover of mucous epithelia is
increased, thus increasing the need for vitamin A as well as increasing
its depletion.

Sirakumar and Raddy (1971) also reported the increased depletion of
vitamin A during infection, and that isotope-labeled vitamin A was

poorly absorbed by the intestines during infection of children in India.

4

Thus, this anti-infective vitamin is not only depleted more rapidly
during infection, but an animal is less able to supply its needs.

Vitamin A has been used in the prevention and therapy of epithelial
tumors (Bollag, 1970). The mode of action was to either prevent
squamous metaplasia to a precancerous state or to revert already meta-
plastic epithelia back to normal.: Hitchcock and Bell (1952) have
reported that squamous metaplasia can lead to carcinomas. A specific
example is the prevention of squamous cell carcinomas in hamster cheek

pouches after administration of a carcinogen (benzpyrene) (Bollag,

1970).

Vitamin A Deficiency Lesions

 

The earliest complete description of vitamin A deficiency was by
Wolbach and Howe in 1925. They summarized the lesions as widespread
epithelial keratinization and atrophy of glands. However, they also
reported humped postures of the rats, rough hair coats, puriform infil-
tration of turbinates, bronchitis, and bronchiectasis, which are all
characteristic of mycoplasmosis in rats (Lindsey et al., 1971).

Not until 10 vitamin A deficient rats were maintained germfree by
Beaver (1961) could concurrent infection be avoided. Beaver's paper
serves as a classic breakthrough in the study of vitamin A but some
discrepancies have been observed. Hepatic necrosis and focal myocardial
necrosis with infiltration of mononuclear cells and calcification
attributed to vitamin A deficiency by Beaver, have not been reported
by later workers (Rogers, Bieri and McDaniel, 1971; Raica at al.,

1969). Similar lesions have instead been associated with vitamin E

deficiency (Michel et aZ., 1969; Trapp, 1970).

5

Although no difference in longevity between germfree and conven-
tional vitamin A deficient rats was noted by Beaver (1961), much
greater survival times have been reported in germfree, vitamin A
deficient rats (Raica at al., 1969; Bieri at aZ., 1969; Rogers et aZ.,
1971). These later researchers reported that, although growth stopped
as vitamin A stores were depleted, the rats could survive up to 272
days in contrast to the approximate lOO-day survival time in conven-
tional vitamin A deficient rats°

Raica et a1. (1969) studied the pathology of 2 male and 2 female
germfree, vitamin A deficient rats and also reported widespread
keratinization, flocculent material in the urinary bladder, and calci-
fication of kidney tubules, as did Beaver (1961). However, neither
liver necrosis, myocardial necrosis, nor xerophthalmia was present
in Raica's rats°

Rogers at al. (1971) attempted to infect germfree vitamin A
deficient rats with Eacherichia coli (strain 0127—B9), but failed to
obtain clinical signs. Thus, to date, no infection of germfree,

vitamin A deficient rats has been conducted under controlled conditions.

Vitamin E

Vitamin E was first described in 1922 as a dietary requirement for
normal reproduction in the rat (Evans and Bish0p). In 1965 Tappel
proposed that vitamin E functions as an antioxidant in preventing the
accumulation of free radicals and lipid peroxides which cause tissue
damage and membrane fragility. The peroxidation products that accumu- .
late are the ceroid and other lipofuscin pigments present in steatitis
of cats and mink (Smith, Jones and Hunt, 1972) or brown bowel disease

of dogs (Hayes, Nielson and Rousseau, 1969). Selenium is believed to

6
have a similar function along with vitamin E in acting as an antioxidant
in preventing injury to membranes of mitochondria, endoplasmic reticulum,
erythrocytes and microsomes (Tappel, 1965).

Only limited information on a role of vitamin E in specific infec—
tions has been reported (Scrimshaw, 1966). Laboratory animals deficient
in vitamin E are more susceptible to human leprosy infection (Mason and
Bengel, 1955) and vitamin E deficient ducklings are more susceptible and
more severely affected by avian malaria than are ducklings fed adequate
amounts of vitamin E (Yarrington, 1971). In a report on naturally
occurring vitamin E and selenium deficiency in swine, Trapp et a1.
(1970) reported that the deficiency was accompanied by a high incidence
of pathogens in various tissues and organs. Vitamin E may have 2 roles
in the prevention of infection: (1) serving indirectly as an antioxi-
dant for vitamin A which otherwise might be readily oxidized, or (2)
more specifically in preventing membrane damage by free radicals and

lipid peroxides.

@gggglasmagpulmcnis Infection of_§ats

 

Chronic respiratory disease (CRD) is the most common and important
health problem of albino research rats (Coleman, Zbijewska and Smith,
1971), and the pathology of this disease has been described (Giddens,
Whitehair and Carter, 1971; Giddens and Whitehair, 1969). Lindsey
et al. (1971) and Whittlestone, Lemcke and Olds (1972) have adquately
proven that Mycoplasma pulmonis causes all forms of CRD. However,

Adams et al. (1972) still regard mycoplasmas isolated from a colony of
rats with an epizootic of respiratory disease as an incidental finding.

In the research of Lindsey at al. (1971) germfree rats were inocu-

lated with various strains of Mycoplasma pulmcnis received from other

7
researchers in the field. All forms of CRD were consistently repro—
duced with the most severe lesions caused by strain M/J69 isolated and
provided by Dr. George Jersey at Michigan State University. The
disease caused by this organism is a model which can be used in germ-
free or conventional rats to provide further information to control
or eliminate CRD from rat colonies or provide basic information on

infectious processes and resistance to themo

MATERIALS AND METHODS

Experimental Animals and Design

Ninety-six weanling Sprague-Dawley strain* rats were used in 2
concurrent 2 x 4 factorial experiments. The experiments were performed
under germfree or conventional environments utilizing 48 rats in each
experiment. All rats were the progeny of 18 female germfree rats which
were fed a ration deficient in vitamins A and E after parturition to
minimize liver stores of vitamins A and E in the experimental young rats.

One-half (24 rats) of each experimental group was exposed to.M.
pulmonis. The 2 exposed and 2 non-exposed groups were subdivided into
4 groups of 6 rats fed a (l) vitamins A and E deficient, (2) vitamin A
deficient, (3) vitamin E deficient, or (4) vitamins A and E supplemented

ration (hereafter known as A and E deficient, A deficient, E deficient,

or A and E supplemented).

Housing

The 48 rats in the germfree experiment were maintained in 3 flexible

plastic isolators (Trexler, 1959) under standard germfree technique

(Coates, 1968). The 48 ex-germfree rats in the conventional experiment

were transferred out of the germfree isolators and maintained in filter

topped "shoe-box" cages initially containing 6 rats each. Metal dividers

were used in the "shoe-box" cages to separate male and female rats to

prevent cannibalism or pregnancy.

 

fA. R. Schmidt Co., Inc., 2826 Latham.Dr., Madison, Wise.

8

Table 1. Experimental design

 

Type of environment and number of rats

 

 

germfree ex- ex—germfree**
Diet germfree infected* germfree** infected*
(l) Basal ration 6 6 6 6
(Table 2)
(2) + vit. E 6 6 6 6
(l8 IU/lOO
gm. diet)
(3) + vit. A 6 6 6 6
(600 IU/100
gm. diet)
(4) + vit. A & E 6 6 6 6

 

*
Infected at 9 weeks of age with strain M/J69 Mycoplasma pulmonis.

**
Reared under germfree conditions, then transferred to conven-

tional surroundings.
Diet and Feeding Practices
The basal ration deficient in vitamins A and E was a modification of

the ration used by Rogers et a1. (1971) (Table 2). The vitamin mix was
based on that used by Raica at al. (1969) (Table 2). At weaning the
21-day-old rats were marked by ear notches, and the appropriate rats
were supplemented by subcutaneous injection of 710 International Units

of vitamin A* and/or 25 International Units of vitamin E.** The rats

were fed the basal diet free choice and the appropriate groups were

subsequently supplemented with vitamins in the feed. The ration was

*
8Upplemented with 600 1.0. of vitamin A per 100 gm. of diet and/or 18

m8. of vitamin E** per 100 gm. of diet. Vitamin levels were tripled,

 

*Rocavit, Roche Chemical Div., Hoffmann-LaRoche, Inc., Nutley,
N.J. 07110.

**Vitamin E Injectable (aqueous), An. Health Res., Hoffmann—

LaRoche, Inc., Nutley, N.J. 07110.

 

10

since some loss was expected when the ration for the germfree animals

was autoclaved.

 

 

 

Table 2. Basal ration
Ingredient Percentage
Casein, vitamin free 22.0
*

Vitamin mix

3X customary level 6.0
Salt mix (Briggs and Fox) 6.0
Corn oil (plus antioxidant) 4.0
Corn starch 58.0
Cellulose 4.0 A

 

*
Vitamin mix

 

 

Ingredient mg. added per kg. of diet
Vitamin D3 0.15
Menadione 150
Thiamine HCl 80
Riboflavin 66
Nicotinic acid 200
Calcium pantothenate 300
Inositol 300
Choline 4000
Pyrodine HCl 60
Biotin 1.5
Folic acid 10.0
PABA 50
Cyanocobalamine 100
Vitamin C 3000

 

11
Experimental Exposure of the Rats

The rats were exposed to M. pulmonis by aerosol inhalation when the
basal and E supplemented rats depleted their hepatic stores of vitamin
A. This was estimated to be at 7 to 11 weeks of age from growth curves
of similarly treated rats in previous work (Rogers et al., 1971). To
determine the exact point when vitamin A was depleted, 4 ex—genmfree
rats (1 from each nutritional category) were necropsied at weekly
intervals starting at 7-1/2 weeks and hepatic vitamin A levels were
measured. The rats were deficient at 8-1/2 weeks of age and were then
exposed to approximately one—third milliliter per rat of a 72-hour
broth culture of Mycoplasma pulmonis strain M/J69* through a glass
nebulizer.**

To expose the germfree rats, flame-sealed glass ampules containing
the mycoplasma in broth culture were first washed in 70% ethyl alcohol
and then passed into the germfree isolators through a peracetic acid
entry lock. The peracetic acid fumes were allowed to dissipate from
the isolator for 30 minutes, after which the isolator's air inlet and
outlet were sealed. The mist was directed at the nostrils of each rat
for at least 1 minute per rat and allowed to remain in the isolator an
additional 30 minutes before the circulation was reopened.

The conventional rats were similarly exposed to 2 ml. of the 72~
hour, second passage broth culture per cage of 6 rats. The aerosol
was produced by the same type nebulizer using a low pressure stream of

compressed nitrogen gas. The spray was directed through a hole cut in

*
Ph.D. Thesis, Dr. George Jersey.

**
DeVilbiss No. 640 Nebulizer, The DeVilbiss Co., Somerset, Pa.

 

12
the filter-top cages for 10 minutes per group of 6 rats. The hole was

immediately taped shut following nebulization to trap the mist inside

the cages.

Termination of the Experiment
When one-half of the exposed rats in each nutritional group had
died or were moribund, the rest of that group were euthanatized. All
rats were euthanatized by 150 days of age. Corresponding non-exposed
rats were euthanatized for comparison when exposed rats died. After

ether anesthesia, euthanasia was performed by exsanguination.

CulturingfiMethods
The middle ear and lung of all rats were cultured with PPLO
enriched broth* and agar.** Thirty percent of the agar and broth was
composed of a commercial supplement.*** The middle ears and lungs of
all rats, the kidneys of conventional rats, and the cecal contents of
germfree rats were cultured on blood agar plates. The germfree iso-
lators were extensively cultured for bacteria and fungi before, during,

and after the experiment.

Laboratory Tests
Livers from all rats were frozen after necropsy for later analysis
of vitamins A and E. Hepatic vitamin A levels were determined by the

trifluoroacetic acid method described by Neeld and Pearson (1963).

 

*
Bacto—PPLO enrichment broth, Difco Laboratories, Detroit,
Mich.

**
Bacto-PPLO Agar, Difco Laboratories, Detroit, Mich.

***
Bacto-Mycoplasma Supplement 8, Difco Laboratories, Detroit,

Mich.

 

13

Hepatic vitamin E levels were determined by the micromethod described

by Fabianek et al. (1968).

Serum protein electrophoresis was conducted by the cellulose
acetate strip method described by Klainer et al. (1968) on frozen serum
collected at necropsy. A refractometer was used to determine total
serum protein levels (Medway, Prier and Wilkinson, 1969). Whole blood
from 14 rats was used for packed cell volume determinations and white

blood cell counts. The microhematocrit and unopette methods were used

(Medway, Prier and Wilkinson, 1969).

Pathology

After euthanasia or death, all rats were necropsied and their
tissues were fixed in 10% neutral buffered formalin. One eye from each

rat with gross ophthalmic lesions was fixed in Zenker's solution and

stored in 70% ethyl alcohol until sectioning. Tissues were sectioned

at 6 microns thickness and stained with hematoxylin and eosin or other
selected special stains as described in the Armed Forces Institute of

Pathology Manual of Histologic and Special Stainfing Technics (1960).

 

RESULTS

Clinical Signs
The earliest lesion was xerophthalmia in 55-day-old rats fed the
basal, A and E deficient ration. This began as a slight periorbital

crusting and progressed until the entire cornea was dry and the eyelids

 

crusted shut (Figure l). The xerophthalmia began in the germfree and
conventional A and E deficient rats at the same time but did not appear

in the rats deficient in only vitamin A until 1 to 2 weeks later, although
both had depleted their liver stores of vitamin A (Figure l).

Retarded growth occurred first in the A and E deficient rats, and
only a few of these rats lived slightly over 3 months (Figure 2). The
exposed, A deficient, conventional rats lost weight next and had clinical
signs of mycoplasmosis. At 4 months of age the exposed, E deficient,
conventional rats also began to lose weight and had signs of respira-
tory infection. Non-exposed, A deficient, conventional rats did not
lose weight but several died suddenly of Pseudomcnas infections. The
growth rate of rats supplemented with vitamins A and E was slower at
4-1/2 months, but the rats did not have clinical signs of Mycoplasma
pulmonis infection.

As the ML pulmonis infection progressed in all afflicted rats,
they became more emaciated, arched their back to aid in breathing,
developed an unkempt haircoat and were cachectic. Terminally, breathing
became difficult and exaggerated, and the respiratory sounds were moist
and loud. Ataxia was present in those animals with severe weight loss.

14

15

 

Figure 1. Comparison of 2, 59-day—old, conven-
tional rats. The rat on the left had been fed the
ration deficient in vitamins A and E for 38 days and
had xerophthalmia. The rat on the right had been fed
a vitamin A deficient ration for 38 days and did not
have signs of vitamin A deficiency, although both rats
had no detectable hepatic vitamin A stores.

16

Figure 2a. Growth curves of conventional non—exposed rats.
The A and E deficient rats had died by 3 months of age, while the
other groups continued to grow. The A and E supplemented rats had
a lower average weight since they were mainly females.

Figure 2b. Growth curves of conventional exposed rats. The
A deficient rats grew at a slower rate soon after exposure at 9
weeks of age and lost weight as the infection progressed The
E deficient rats behaved similarly a few weeks later. .

The A and E
supplemented rats grew until termination of the experiment.

 

Weight (‘qm )

Weight (gm)

 

 

17

 

 

 

 

 

Vitamin AoE Deficient ............
400 - Vitamin A Deficient ---...
Vitamin E Deficient - -----
Vitamin Ac E Supplemented —
300 -
200 '-
l 00 .—
1 1 1 1 J
0 2 3 4 5
Months of Age
Figure 2a
Vitamin A e E ouicigm .........
400 - Wamin A Deficient .--..-
Vitamin E Deficient - -----
Wamin A e E Supplemented -—
300 -
200 -
IOO -
1 1 1 4
O 2 3 4 5

Months of Age

Figure 2b

18

The A and E deficient rats, whether germfree, conventional,
exposed, or non-exposed, died of infection or inanition before any
rats of the other 3 nutritional categories became noticeably ill
(Figure 2). The A deficient, exposed, conventional rats grew slower
and developed clinical signs of mycoplasmosis before the E deficient,
exposed, conventional rats. The conventional A and E supplemented rats
continued to grow throughout the experiment and had no clinical or
histopathologic lesions of mycoplasmosis at 150 days of age.

The same ranking of A and E deficient, A deficient, E deficient
and A and E supplemented rats (worst to best, respectively, in regard
to growth and resistance to infection) was present in the germfree
experiment, but it was not as pronounced. The germfree rats fed the

autoclaved diet did not grow as well as their conventional counterparts.

Clinical Pathology

 

Absolute values of total serum protein, albumin and gamma globulin
in grams per 100 milliliters were lowest in the A deficient or A and
E deficient rats (Table 3). Of these 2 groups, lower total protein,
albumin and gamma globulin levels were present in the A and E deficient
conventional rats (Table 3), reflecting the vitamin A sparing action of
vitamin E supplementation. The gamma globulin levels were essentially
the same in the A deficient, conventional, exposed rats as in the A
and E supplemented rats. The conventional, exposed rats had higher
gamma globulin levels than the conventional, non-exposed rats.

Packed cell volume, white blood cell count and total protein
levels were evaluated on 14 rats. These few results indicate that
lower white blood cell counts and total protein levels occur in germfree

than in conventional rats.

 

Table 3. Comparison of mean serum protein electrophoresis values in

gin/100 ml

 

 

 

 

 

 

Total Gamma
Protein Albumin Globulin

Conventional rats *

A and E deficient (l) 5.3 3.39 0.19

A deficient (3) 5.63 3.74 0.60

E deficient (5) 5.73 4.93 0.84

A and E supplemented (5) 6.02 4.89 0.81
Conventional exposed rats

A and E deficient (l) 4.4 3.15 0.33

A deficient (2) 5.6 2.92 .94

E deficient (4) 6.85 4.35 1.31

A and E supplemented (5) 6.08 4.76 0.99
Germfree rats

A and E deficient (2) 4.7 3.82 0.60

A deficient (2) 4.5 3.49 0.33

E deficient (2) 6.4 4.95 1.07

A and E supplemented (3) 5.8 4.45 0.72
Germfree exposed rats

A and E deficient (0) -- ~~ —-

A deficient (1) 3.7 3.25 0.11

E deficient (0) -— -- -—

A and E supplemented (4) 4.1 3.5 0.38

 

*Number of euthanatized rats per group with enough serum to run

serum protein electrophoresis determinations.

The hepatic vitamin A content of the rats closely reflected their
diet. Those rats fed the basal, A and E deficient ration alone or with
E supplementation were devoid of vitamin A. Those supplemented with
just vitamin A.had an average of 232 micrograms of vitamin A per gram
of liver. The rats supplemented with both vitamins A and E had an
average of 922 micrograms of vitamin A per gram of liver.

The vitamin E determination method was inadequate for use on these
livers. No rats were determined to be deficient in vitamin E, and some

of the highest levels of vitamin E were detected in the rats on the

20
A and E deficient ration. A yellow pigment, possibly ceroid, was present
in the extract of the liver during the determination procedure and was

believed to cause the inconsistent results.

Microbiology

 

The lungs and middle ears were cultured for mycoplasma in order
to fulfill Koch's postulates. All of the exposed, A or A and E
deficient rats in 1 germfree isolator were positive for mycoplasma
(Table 4). Only 1 E deficient rat was positive and no isolations were
made from the A and E supplemented rats. The other isolator of exposed
rats had only one isolation from the middle ear of an A deficient rat.
The conventional rats had a high rate of isolations of M. pulmonis
except in those rats supplemented with vitamins A and E (Table 4).

Bacterial cultures from the lung, middle ear and cecal contents
of 38 germfree rats were all negative, confirming germfree conditions.
The lung, middle ear and kidney of 33 conventional rats were cultured
on blood agar plates. The A deficient, conventional rats, fed either
the A or A and E deficient rations, had a high incidence of Pseudamonas
isolations (Table 5). The Pseudomonas was always isolated from the

kidney but was often found in all 3 organs.

ranges

The lesions of Mycoplasma pulmonis infection (otitis media,
rhinitis, tracheitis and bronchopneumonia) were of the acute form and
were similar to previous descriptions (Lindsey at al., 1971; Giddens
et aZ., 1971). Differences were related to the sqamous metaplasia
and keratinization of the respiratory tract in A deficient rats.
Earliest inflammation was found in the keratinized scrolls of turbinates

(Figure 3) and in the tympanic cavities.

21

Table 4. Mycoplasma isolations

 

 

 

 

 

 

 

Dietary Inoculated Rats Inoculated
Vitamin Germfree Isolates R5 Conventional_ga£§___
Status Lung Middle Ear Lung Middle Bar
A and E deficient 2/2* 2/2 2/4 3/5
A deficient 3/3 3/3 2/4 4f4
E deficient 1/3 1/3 4/5 4/5
A and E supple- 0/3 0/3 0/5 0/5
mented

*

Number of positive cultures/number of rats.
Table 5. Bacterial isolations from conventional rats
Dietary
Vitamin
Status Pseudomonas Coliform Microcoocus
A and E deficient 5/7* 3/7 Of?
A deficient 7/8 2/8 2i8
E deficient 1/9 lf9 2f9
A and E supplemented 0/9 1/9 3f9

 

*
Number of positive cultures/number of rats.

The typical hyperplasia and mucus secretion of the infected tracheas

of rats supplemented with vitamin A (Figure 4) was not present in the

keratinized tracheas of A deficient rats which had neither the mucus

nor cilia to remove purulent debris (Figure 5). The lack of epithelial

22

  
        

' - ‘ ‘
7"" 2'- ". A . .1. ' 3' . V.)
.\ ‘ " ‘... 5 ) - ' ' $
'.' o, '0‘, .~ . " '1 . s. . ’9
.3 "o‘ ’1. " ‘.-"- \ ‘.-.. .
7.; t. 7 ' A 3". .'I a ‘7 _1
. O O .
.. u'rb‘.’i" .'¢a
- r ~ -. . A
s ‘ I 4' .‘ . ‘,.fi.' M ‘
' ..o . p .
'L ‘3‘”! Z,“- 0- -, ‘.‘ .
.~" . 'cf‘>;A-J:.7l J..- l , utr'u.')." '7’
A,\ . ’-‘:.. '.' ‘ r.h‘,r- ,
,"_.", I. . o ‘ l ’,
- V \‘.L" - \“ e_‘
‘- a \
"I ‘z'
Q

Figure 3.
ventional rat.

Rhinitis in an A and E deficient, con—

Most inflammation involves the keratin-
ized turbinate, while the columnar epithelium remaining
on the nasal septum (arrow) is free of exudate.

H & E
stain; x 54.

 

Figure 4. Tracheitis in an E deficient, con-
ventional rat. Hyperplasia and folding of the epi-
thelium was a typical response of vitamin A supplemented
rats to ML pulmonis. H & E stain; x 54.

 

Figure 5. Tracheitis in an A deficient germfree
rat. Metaplasia and keratinization (arrow) was
present'rather than the hyperplasia seen in Figure 4.
Keratinization of 2 thyroid acini was also present
(K). H 8 E stain; x 54.

24

response of A deficient rats extended down to the bronchiolar level
(Figure 6). The gross appearance of pneumonia was characteristic and
always located in the apical, dependent portions of the lung (Figure 7).

The pattern of histopathologic lesions of mycoplasmosis closely
resembled the pattern of mycoplasma isolations (Table 4). The exposed,
germfree, A or A and E supplemented rats, from which mycoplasma was
not isolated, were free of respiratory lesions except for a mild inflam-
mation of the alveoli in some rats. Similarly the A and E supplemented,
exposed, conventional rats were also free of respiratory lesions except
for a small focus of pleuritis in 1 rat.

In the A and/or E deficient rats, from which mycoplasma was iso-
lated, the severity of the lesions was more individual than related to
a particular nutritional category. Severe fatal pneumonias as well
as mild upper respiratory infections were present in inoculated rats of
either of the 3 A and/or E deficient categories.

An opportunistic, ascending Pseudomonas infection of the keratinized
metaplastic urogenital tract of conventional A or A and E deficient
rats frequently caused unexpected deaths (Figures 8 and 9; Tables 5 and
6). All of the A or A and E conventional rats that were not exposed
to.M. pulmonfs but were found dead had evidence of bacterial infections.
These were primarily of the urinary tract but were also found in other
organs. Conversely, none of the non-exposed A or A and E supplemented
conventional rats were found dead.

Similarly, the exposed, conventional rats deficient in vitamin A
or E that were found dead had evidence of M. pulmonis or bacterial
infection. All had lesions of mycoplasmosis, and 6 of the 12 had con-

current bacterial infections primarily of the urinary tract.

25

Figure 6. Characteristic purulent bronchitis with bronchiectasis
and metaplasia of ML pulmonis infection in an A deficient. conventional
rat. The lumen was filled with purulent debris and a large focus of
mononuclear cells (arrow) is adjacent to the bronchiole. H & E stain;
x 54.

Figure 7. Pneumonic lungs of an E deficient conventional rat-

The apical portions (arrow) are t '
. ypicall the most ' ;.
in ML pulmonzs infections. y severely affected

 

Figure 6

 

Figure 7

27

.‘Yp -
.1“; 'k ‘

 

 

Figure 8. Multiple renal abscesses (arrow) in
an A and E deficient, conventional rat. White floc-
culent material was present in the urinary bladder.

 

Figure 9. Cystitis and keratinization in the
urinary bladder of an A deficient conventional rat.

28

Table 6. Urogenital infections of conventional rats

 

 

Dietary

Vitamin Pyelo- Renal Cys-

Status Nephritis** nephritis Abscesses titis Vaginitis
A and E deficient* 1 3 4 1 4

A deficient* 0 3 0 2 l

E deficient* 0 0 0 0 l

A and E supplemented*' 0 0 0 0 O

 

*
Includes 12 inoculated and non-inoculated rats in each of the
4 groups.

**
Nephritis without pelvic or ureteral involvement.

Large, full, often hemorrhagic urinary bladders (Figure 10) were
found in 5, germfree, A deficient rats; thus, these were not due to
bacterial or fungal infection. No bladder stones were observed; however,
severe keratinization of the urethra from the bladder through the penis
was present (Figure 11). The amount of keratin was sufficient to
partially or completely block urine flow and cause the retention.

Squamous metaplasia and keratinization was present in the thyroid
of 17 of 48 A or A and E deficient rats (Figure 5). Usually 2 acini,
but up to 5 acini, were keratinized. The metaplastic acini were always
in the center of the thyroid, which is the embryonic location of the
ultimobranchial body.

Keratinization of the cornea and conjunctiva in A or A and E
deficient, conventional rats was usually accompanied by keratitis.
However, in the germfree group only 2 exposed A or A and E deficient
rats had keratitis, l of which had a unilateral panophthalmitis and

severe, necrotic inflammation of the lacrimal gland. One of the A

29

 

Figure 10. Penis, testes and large hemorrhagic
urinary bladder of an A and E deficient germfree rat.

 

Figure 11. Photomicrograph of a keratinized
urethra (large arrow) in the penis of an A and E
deficient, germfree rat. The outer horny layer (small
arrow) of the penis was present. Kerstinization of
the urethra contributed to enlarged bladders such as
in Figure 10. H & E stain; x 81.

30
deficient, germfree, exposed rats had a neutrophilic inflammation of
the salivary gland and ducts. Inflammation of the eyes or salivary
glands was not attributed to M2 pulmonis, although no other organism
was cultured from these rats.

The seminiferous tubules of the A deficient rats were hypocellular
and contained fusion cells characteristic of testicular degeneration
(Figure 12). The rats fed an E deficient diet still had spermatogenesis
(Figure 13).

Vitamin E deficiency lesions such as hyalinization and calcifica-
tion of skeletal and cardiac muscle occurred only in 17 of the 48 rats
fed E or E and A deficient rations (Figure 14). This was not related
to a metastatic calcification of renal tubules at the corticcmedullary
junction in 47 of 96 rats in all nutritional categories. Centrolobular
hepatic necrosis with hemorrhage also only occurred in the group of
17 E or E and A deficient rats (Figure 15).

The uteri of 2 conventional, E deficient rats were grossly
enlarged and yellow at euthanasia and necropsy at 150 days of age.
The uteri of 3 conventional, E deficient rats had many macrophages
filled with a yellow acid—fast pigment. This pigment was assumed to

be a lipofuscin, possibly ceroid.

 

Figure 12. Aplastic seminiferous tubules of an
A deficient, germfree rat. Fusion cells (arrow),
characteristic of degenerate testis, were present.
H & E stain; x 1350

 

Figure 13. Active spermatogenesis in the testis
of a conventional rat fed a vitamin E deficient ration.

H 8 E stain; x 135.

32

 

Figure 14. Degeneration and calcification of
the skeletal muscle of a germfree vitamin E deficient
rat. H 8 E stain; x 135.

 

Figure 15. Centrolobular hepatic necrosis (C)
in a vitamin E deficient germfree rat. The other
hepatocytes had some postmortem degeneration. H & E
stain; x 54.

DISCUSSION

Vitamins A and E in Resistance to Infection

The primary achievement of this research was to demonstrate the
difference in susceptibility of rats fed different levels of vitamins
A and E to Mycoplasma pulmonis infection. Although larger numbers of
rats and a specific experimental design are needed to show statistical
significance and an optimum level, supplementation of rats with triple
the National Research Council's recommendation for vitamins A and E
may provide a useful way to reduce the incidence of mycoplasmosis in
experimental rats and rat colonies.

Further research is needed to determine optimum levels of vitamins
A, E, the B complex, and C to use in the prevention of infections of
animals and man. Using the minimum levels to prevent deficiency lesisns
does not take into account the increased requirements during stresses
such as infection (Hayes, 1971), nor the decreased absorption when the
need is the greatest (Sirakumar and Raddy, 1971). Prophylaxis with
vitamins has advantages over prolonged antibiotic use in not upsetting
bacterial flora, predisposing the subject to fungal infections, or
producing antibiotic-resistant strains of bacteria. Vitamin A is already
useful in the prophylaxis and treatment of epithelial tumors (Bollag,
1970).

The factorial design of these experiments allowed the comparison
of vitamins A and E. The synergistic action of vitamin E with vitamin
A is reflected in the 2~week delay of the onset of vitamin A deficiency

33

34

lesions such as xerophthalmia. The higher hepatic vitamin A levels in
the rats supplemented with both vitamins A and E also reflects the
synergism. This is probably due to the prevention of oxidation of
vitamin A by the antioxidant nature of vitamin E (Crider at al., 1961).

Histopathologic evidence of vitamin A's role in mucus secretion
and epithelial maintenance confirms the previous reports of DeLuca and
wolf (1969) and Green and Mellanby (1928). The total serum electro-
phoresis suggests that vitamin A may be involved in the synthesis of
glycoproteins other than mucus, such as gamma globulins and other serum
proteins. However, there is not enough replication to be statistically
significant and the decreased total serum proteins, albumin and gamma
globulins, may only reflect the poor general health of the vitamin A
deficient rats.

The high incidence of ascending Pseudbmonas infections of the
urinary tract in the vitamin A deficient, conventional rats reflects
the importance of vitamin A in the resistance of rats to infection.
The importance of using germfree techniques to study vitamin A deficiency
or controlled infection in the vitamin A deficient animal is re-emphasized
to prevent concurrent infections which may confuse experimental results

(Lindsey, 1971).

Pathology

Squamous metaplasia and keratinization of thyroid acini have not
been reported in germfree A deficient rats, but have been reported in
conventional A deficient rats (Van Dyke, 1955).

DeLuca and wolf (1969) have reported that vitamin A is required in
glycoprotein synthesis, such as in mucus production in the gastroin-

testinal tract. One may speculate that, since thyroxine and thyroid

35
stimulating hormone are glycoproteins, their synthesis may require
vitamin A and they may be involved in thyroid keratinization as well
as in growth retardation observed in vitamin A deficiency. Similarly,
chorionic gonadotropin, follicle stimulating hormone, and interstitial
cell stimulating hormone are glyc0proteins and could be related to the
role of vitamin A in spermatogenesis.

One is tempted to ascribe the keratitis and panophthalmitis as
well as the salivary gland inflammation in 3 exposed, A deficient,
germfree rats to the ML pulmonis syndrome, but these lesions probably
should be considered incidental findings.

The spermatogenesis present in the E deficient rats as well as
our inability to obtain differences in hepatic vitamin E levels are
not consistent with a vitamin E deficiency. However, the presence of
muscle degeneration and hepatic necrosis only in E or E and A deficient
rats suggests that a borderline vitamin E deficiency was obtained.

The lipofuscin pigment in the uteri of 3 rats fed vitamin E deficient
rations also supports this opinion. I therefore suggest that hepatic
necrosis and muscle degeneration is not a vitamin A deficiency lesion
in the germfree rat, since it was not present in A deficient rats as
previously reported (Beaver, 1961), but was present in E deficient rats.

Vitamins A and E are required for the resistance of rats to
Mycoplasma infection, and further research may indicate how these
vitamins may be used in the control of mycoplasmosis in rat colonies
where eradication is not possible. This research also answers the
question posed by Lindsey et al. (1971) as to whether lesions of chronic
respiratory disease are enhanced by vitamin A deficiency or are purely

coincidental because of the prolonged periods of time the rats are

36
maintained on deficient diets. Chronic respiratory disease is enhanced

by vitamin A deficiency as well as vitamin E deficiency.

SUMMARY AND CONCLUSIONS

Ninety-six rats were used in 2 concurrent 2 x 4 factorial experi-
ments under germfree or conventional conditions. One-half of the 48
germfree rats were exposed to Mycoplasma pulmonis, as were one-half
of the conventional rats. These 4 groups of 24 rats were each further
divided into 4 groups of 6 rats fed either a (1) basal vitamins A and
E deficient, (2) vitamin A deficient, (3) vitamin E deficient, or (4)
vitamin A and E supplemented ration.

Vitamins A and E prevented mycoplasmosis in the exposed rats;
therefore, vitamin supplementation at high levels may be a method of
reducing the disease in rat colonies. Vitamin A also prevented ascend-
ing Pseudbmonas infections of the urinary tract of conventional rats.
Vitamin A supplemented rats had higher levels of other glycoproteins
such as gamma globulins and other serum proteins than vitamin A
deficient rats.

Thyroid metaplasia and keratinization were present in vitamin A
deficient germfree rats. Hepatic necrosis and myocardial degeneraticn
with calcification previously attributed to vitamin A deficiency were
only present in vitamin E deficient germfree rats. Other vitamin A or

E deficiency lesions were similar to those previously reported.

BIBLIOGRAPHY

BIBLIOGRAPHY

Adams, L. E., Yamauchi, Y., Carleton, J., Townsend, L., and Kim, 0. J.:
An epizootic of respiratory tract disease in SpragueuDawley
rats. J.A.V.M.A., 161, (Sept. 15, 1972): 656,660.

Armed Forces Institute of Pathology: Manual of’Histolcgic and Special
Staining Technics. Washington, D.C., 1957.

Beaver, D. L.: Vitamin A deficiency in the germ-free rat. Am. J.
Path., 38, (1961): 335-349.

Bollac, W.: Vitamin A and vitamin A acid in the prophylaxis and
therapy of epithelial tumours. Int. J. Vit. Res., 40, (1970):
299-314.

Burek, J. D., Jersey, G. C., Whitehair, C. K., and Carter, G. R.: The
pathology and pathogenesis of Bordetella bronchiseptica and
Pasteurella pneumctropica infection in conventional and germfree
rats. Lab. An. Sci., 22, (1972): 844-849.

Bieri, J. G., McDaniel, E. G., and Rogers, W. E.: Survival of germfree
rats without vitamin A. Science, 163, (1969): 574—575.

Coates, M. E.: The Germ-Free Animal in Research, lst ed. Academic
Press, London, 1968.

Coleman, E. J., Zbijewska, J. R., and Smith, N. L.: Hematologic values
in chronic murine pneumonia. Lab. An. Sci., 21, (1971): 721-726.

Crawford, Y. E., Smith, P. E., Panos, C., and Lynn, R. J.: A Microbial
Enigma Mycoplasma and Bacterial L-Forms, lst ed. The world
Publishing Co., Cleveland and New York, 1966: 1-264.

Crider, Q. E., Alanpovic, P., and Johnson, B. C.: 0n the function and
metabolism of vitamin E: Vitamin E and antioxidants in the
nutrition of the rat. J. Nutr., 73, (1961): 64-70.

Daniels, A. L., Armstrong, M. E., and Hutton, M. K.: Nasal sinusitis
produced by diets deficient in fat-soluble A vitamin. J.A.M.A.,
81, (1923): 828-829.

DeLuca, L., and wo1f, 6.: Vitamin A and mucus secretion. Int. J. Vit.
ReSo’ 40, (1970): 284-2900

38

39

Evans, H. M., and Bishop, K. 8.: On the existence of a hitherto
unrecognized dietary factor essential for reproduction.
Science, 56, (1922): 650-651.

Fabianek, J., DeFilippi, J., Rickards, J., and Harp, A.: Micromethod
for tocopherol determination in blood serum. Clin. Chem., 14,
(1968): 456-462.

Fite, G. L.: The pathology of dietary liver necrosis - a preliminary
report. Ann. N. Y. Acad. Sci., 57, (1954): 831-838.

Fogh, J., and Fogh, H.: Morphological and quantitative aspects of
Mycoplasma-human cell relationships. Proc. Soc. Exp. Biol.
Med., 125, (1967): 423-430.

Getz, H. R., Long, E. R., and Henderson, H. J.: A study on the rela-
tion of nutrition to the development of tuberculosis. Influence
of ascorbic acid and vitamin A. Am. Rev. Tuberculosis, 64,
(1951): 381-393.

Giddens, W. E., Jr., Whitehair, C. K., and Carter, G. R.: Morphologic
and microbiologic features of nasal cavity and middle ear in
germfree, defined-flora, conventional and chronic respiratory
disease-affected rats. Am. J. Vet. Res., 32, (1970): 99-114.

Giddens, W. E., Jr., Whitehair, C. K., and Carter, 6. R.: Morphologic
and microbiologic features of trachea and lungs in germfree,
defined-flora, conventional, and chronic respiratory disease-
affected rats. Am. J. Vet. Res., 32, (1970): 115-129.

Green, H. N., and Mellanby, E.: Vitamin A as an anti-infective agent.
Brit. Med. J., 2, (1928): 691-696.

Hayes, K. C.: On the pathophysiology of vitamin A deficiency. Nutr.
ReVQ, 29’ (1971): 3-6.

Hayes, K. C., Nielsen, S. W., and Rousseau, J. E., Jr.: Vitamin E
deficiency and fat stress in the dog. J. Nutr., 99, (1969):
196—209.

Herrick, J. B.: Vitamin E and selenium in animal nutrition. VMfSAC,
67, (1972): 568-571.

Herrick, J. B.: The influence of vitamin A on disease states. VM/SAC,
67, (1972): 906-909.

Hitchcock, C. R., and Bell, E. J.: Studies on the nematode parasite,
Gongylcnema neoplasticum (spiroptera necplasticum) and avitamin-
0813 A in the forestomachs of rats: comparison with Fibiger's
results. J. Nat. Cancer Inst., 12, (1952): 1345-1387.

Jericho, K. W. F.: Pathogenesis of pneumonia in pigs. Vet. Rec., 82,
(1968): 507-517.

40

Klainer, A. S., Beiael, W. R., and Atkins, W. K.: Determinations of
serum glycoproteins on cleared cellulose acetate strips. Am.
J. Clin. Path., 50, (1968): 137-141.

Langham, R. E., Zydeck, P. A., and Bennett, R. R.: Steatitis in a
captive Marcy garter snake. J.A.V.M.A., 159, (1971): 640-641.

Lemcke, R. M.: Association of PPLO infection and antibody response in
rats and mice. J. Hyg. (Camb.), 59, (1961): 401-412.

Lindsey, J. R., Baker, H. J., Overcash, R. G., Cassell, G. H., and
Hunt, C. E.: Murine chronic respiratory disease significance
as a research complication and experimental production with
Mycoplasma pulmonis. Am. J. Path., 64, (1971): 675-708.

Mason, K. E., and Bergel, M.: Maintenance of Mycobacterium lophurae
in rats and hamsters fed diets low in vitamin E and high in
unsaturated fat. Fed. Proc., 14, (1955): 442.

McLaren, D. S., Tchalian, M., and Ajans, Z. A.: Biochemical and
hematologic changes in the vitamin A-deficient rat. Am. J.
Clin. Nutr., 17, (1965): 131-138.

Means, J. H.: The Thyroid and Its Diseases, 2nd ed. J. B. Lippincott
Co., Philadelphia, 1948: 53-54.

Medway, W., Prier, J. E., and Wilkinson, J. S.: Textbook of Veterinary
Clinical Pathology, lst ed. The Williams and Wilkins Co.,
Baltimore, 1969.

Merchant, I. A., and Packer, R. A.: Veterinary Bacteriology and
Virology. 7th ed. The Iowa State University Press, Ames, Iowa,
1967: 251-256.

Michel, R. L., Whitehair, C. K., and Keahey, K. K.: Dietary hepatic
necrosis associated with selenium-vitamin E deficiency in swine.
J.A.V.M.A., 155(1), (1969): 50-59.

Mitchell, G. E.: Vitamin A nutrition of ruminants. J.A.V.M.A., 151,
(1967): 430-436.

Neeld, J. B., Jr., and Pearson, W. N.: Macro- and micromethods for
the determination of serum vitamin A.using trifluoracetic acid.
J. Nutr., 79, (1963): 454-462.

Nelson, J. B.: Respiratory infections of rats and mice with emphasis
on indigenous mycoplasmas. In Pathology of’Laboratory Rats and
Mice. Edited by E. Cotchin and F. J. C. Roe. F. A. Davis Co.,
Philadelphia, 1967: 259-294.

Neutra, M., and Leblond, C. P.: The Golgi apparatus. Sci. Amer., 220,
(1969): 100-107.

Olson, J. A.: The metabolism of vitamin A. Pharm. Rev., 19, (1967):
559.

41

Pirie, A., and Overall, M.: Changes in the lens epithelium of the rat
Exp. Eye Res., 13, (1972): 88A-89A.

in vitamin A deficiency.

Raica, N., Jr., Stedham, M. A., Herman, Y. F., and Sauberlich, H. E.:
In The Fat-soluble

Vitamin A deficiency in germ-free rats.
Edited by H. F. DeLuca and J. W. Suttie. The Uni-

Vitamins.
versity of Wisconsin Press, Milwaukee, Wisconsin, 1964: 283-289.

Rogers, W. E., Jr., Bieri, J. G., and MtDaniel, E. G.: Vitamin A
deficiency in the germfree state. Fed. Proc., 30, (1971):

1773-1778.

Scrimshaw, N. 8.: Synergistic and antagonistic interactions of nutri-
Fed. Proc., 25, (1966): 1679-1681.

tion and infection.

Singsen, E. P., Bunnell, R. H., Matterson, L.D., and Kozeff, A.:
Studies on encephalomalacia in the chick. Poult. Sci., 34,

(1955): 262-271.

Sirakumar, E., and Reddy, V.: Absorption of labelled vitamin A in
children during infection. Brit. J. Nutr., 27, (1971): 299-304.
Veterinary Pathology,

Smith, H. A., Jones, T. C., and Hunt, R. D
4th ed. Lea & Febiger, Philadelphia, 1972.

Glycoproteins. Ann. Rev. Biochem., 39, (1970): 599-637.

Free radical lipid peroxidation damage and its inhibi-
Fed. Proc., 24, (1965): 73-77.

Spiro, R. G.:

Tappel, A. L.:
tion by vitamin E and selenium.

Trapp, A. L., Keahey, K. K., Whitenack, D. L., and Whitehair, C. K.:
Vitamin E-selenium deficiency in swine. Differential diagnosis
and nature of field problem. J.A.V.M.A., 157, (1970): 289-300.

The use of plastics in the design of isolator systems
(1959): 29-36.

Effect of withdrawal of vitamin A on
Proc. Soc.

Trexler, P. 0.:
Ann. N. Y. Acad. Sci., 78,

Turner, R. G., and Loew, E. R.:
leukocyte and differential count in the albino rat.

Expo 3101:; Medo , 28’ (1930-31): 506-5100
Experimental thyroid metaplasia in the rat: Preliminary

vanDyke, J. B.:
Arch. Path., 59, (1955): 73-81.

report.
wallach, J. D.: Nutritional diseases of exotic animals. J.A.V.M.A.,

157, (1970): 583-599.
Tissue changes following deprivation

Wblbach, S. B., and flows, P. R.:
of fat soluble A vitamin. J. Exp. Med., 42, (1925): 753-777.

Zucker, T. F., and Zucker, L. M.: Nutrition and natural resistance to
In Modern Nutrition in Health and'Disease. Edited by

infection.
.M. G. Whhl and Robert S. Goodhart. Lea & Febiger, Philadelphia,

1968: 600-609.

VITA

I, Harold William Tvedten, was born in Milwaukee, Wisconsin, on

I graduated from Fordson High School in Dearborn,

October 21, 1946.
Michigan, in 1964, and then attended Henry Ford Community College in

In 1968 I received a 3.8. degree with high

Dearborn for 2 years.
distinction in experimental biology from the University of Michigan,

I was granted the DVM degree in June 1971 by Michigan

Dearborn Campus.

State University.
Following graduation from veterinary school, I practiced veteri-

nary medicine in a 4-man small animal practice in Ann Arbor, Michigan.

In January 1972 I began a Master’s degree program in veterinary pathology
at.Michigan State University, being funded by a postdoctoral fellowship
from the Upjohn Company.

On June 8, 1972, I married Gretchen Legreid Flo, DVM, who was an

assistant professor of orthopedics and neurology at Michigan State

university.

42

MICHIGAN STATE UNIVERSITY LIB RRRRRR

\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ \\\\\\\\\\\\\

 

3 1293 02504 9978