THE I;-'FLUENCE C? A VIRAL
ENTERIC ENFECTION ON
CAROTENE METABOLISM

Thesis for We Deg?“ of M. S.
MICHIGAN STATE UNIVERSITY

Robert R. Maronp 0t
1965

THESIS

NIICI'I.
‘J 1gan SLIM-C
mvexsit V

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‘1

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inn". m A‘

ABSTRACT

THE INFIDENCE OF A VIRAL ENTERIC INFECTION
ON CAROTENE METABOLISM

by Robert R. Maronpot

Research was conducted using a total of 24 pigs in 3 experi-
ments to determine carotene metabolism during a specific viral enteric
infection, transmissible gastroenteritis CTGE).

Clinical signs and gross and microscopic lesions were not
noticeably different between infected animals fed carotene and those
not fed carotene. Fecal carotene excretion was significantly re-
duced within 24 hours after oral inoculation with the TGE virus.
Fecal carotene excretion was greater in pigs fed water-dispersible
beta-carotene beadlets than in pigs fed an oil-soluble beta-carotene.

The virus of TGE produced sprue-like lesions characterized by
villous atrophy in the jejunum and ileum of infected pigs. Diarrhea
‘was present within 72 hours after inoculation with the infective

agent and continued until the pigs were killed for necropsy.

THE INFLUENCE OF A VIRAL ENTERIC INFECTION

ON CAROTENE METABOLISM

3?

Robert R. Maronpot

A THESIS

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

MASTER OF SCIENCE

Department of Pathology

1965

ACKNOWLEDGEMENTS

I wish to express my sincere gratitude and appreciation to my
major professor, Dr. C. K. Whitehair, of the Department of Pathology
for his guidance, counsel and understanding throughout this research
and to Drs. G. L. Waxler and K. K. Keahey for their many favors and
courtesies.

I am extremely appreciative of the technical assistance given
by Robert A. Brooks and grateful to the Department of Pathology,
Muchigan State University, for providing facilities and supplies for
this research.

my sincere thanks go to my good friend and colleague, Dr. H. D.
Stowe, of the University of Kentucky, who first aroused my interest
in research and whose intellect has been a constant stimulus to do
good work and strive for excellence.

This work.was made possible through a postdoctoral fellowship

awarded by the National Institutes of Health, Bethesda, Maryland.

11

INTRODUCTION . . e e . .

REVIEW OF LITERATURE .

MATERIALS AND METHODS. .

EXPERIMENTS AND RESULTS.

Experiment 1.
Experiment 2.
Experiment 3.
DISCUSSION . . . .
SUMMARY. . . ... .

REFERENCES CITED .

TABLE OF CONTENTS

iii

10
10

12

16

22

26

27

10

11

12-

LIST OF FIGURES

Micrograms of carotene excreted. Experiment 1. . .
Total liver storage of vitamin A. Experiment 1 . .
Jejunum from a carotene, noninfected pig. . . . . .

Jejunum from a pig with TGE showing partial villous
atrophy O O O O O O 0 O O O O O O O O 0 0 O O O O O

Micrograms of carotene excreted. Experiment 2. . .
Per cent of carotene intake excreted. Experiment 2

Ileum fran a pig with TGE showing partial villous
Ctrophyeeeeeeeeeeeeeeeeeeeeee

Ileum fran a pig with TGE showing total villous
atrophy O O O O O O O O O O O O O O O O O O O O O O

Micrograms of carotene excreted. Experiment 3. . .
Per cent of carotene intake excreted. Experiment 3

Jejunal villous apex from a carotene, noninfected

pigeeeeeeeeeeeeeeeeeeeeeeee

Jejunal villous apex from a pig with TGE. . . . . .

iv

356‘)?

17
17
18

18
20

20

21
21

INTRODUCTION

Most studies on the metabolism of therapeutic agents and nutrients
are carried out using normal animals. Consequently, little basic
information is available on the metabolism of specific nutrients during
the course of an infection.

For many years vitamin A deficiency has been associated with
infections of the respiratory and gastrointestinal tracts. Evidence
has been published on the interrelationship of vitmmin A and disease
resistance and severity (Ershoff, 1952; Ludovici and Axelrod, 1951).
This information has raised additional questions concerning the
therapeutic value and metabolism of carotene in an animal with a
specific infection. This research was an attempt to obtain information
on the metabolism of carotene during the course of an experimentally
produced gastrointestinal infection.

The objectives of this study were (1) to determine the metabolism
of beta-carotene during the course of transmdasible gastroenteritis
(TGE), (2) to determine the effect of TGE upon pigs fed a diet devoid
of vitamin A and vitamin A.precursors, and (3) to correlate TGE infec-

tion and carotene metabolism.in the pig.

REVIEW OF LITERATURE

In recent years a renewed interest has been stimmlated in the role
of vitamin A and its precursors in maintaining animal health. Of
special interest are the metabolism.and roles of these substances in
specific diseases, especially those involving the gastrointestinal
tract. Voluminous amounts of information concerning vitamin A and its
precursor, carotene, are available in such books as Moore's (1957).
This review deals primarilwaith the role and metabolism of carotene
in disease.

Only small amounts of vitamin A pass the porcine chorioallantoic
placenta and, consequently, at birth the baby pig has a low vitamin
A reserve. Hjarde (1961) and Heaney (1963) have reported that young
pigs are dependent upon colostrum and milk for their early source of
vitamin A.

Since the pig is an efficient converter of carotene to vitamin A
(Brande 23. 1., 1941), this vitamin A precursor may be fed to young
pigs to meet their vitamin A requirements. Rentges gt .a_l_. (1952a)
found that a young pig's'minimum requirement for a purified source
of carotene was 25 mcg. per kilogram of body weight daily. This
level allowed for good weight gain and adequate liver storage of
vitamin A. However, the Rational Academy of Sciences A National
Research Council (Nutrient Requirements of Swine, 1962) recommends
1.2 mg. of beta-carotene daily for growing pigs under 10 lb. of body

weight.

3

Evidence for the intestinal conversion of carotene to vitamin A
was given by Mattson, Mehl and Deuel (1947), who noted that vitamin A
first appeared in the small intestine wall following oral carotene
ingestion by vitamin Ardeficient rats. That same year Wiese, Mehl and
Deuel measured the in m transformation of carotene to vitamin A
in rat small intestine incubated in Ringer's solution. Employing
thoracic-duct fistulas, Goodwin and Gregory (1948) found increased
concentrations of vitamin A in goat and rabbit lymph 2 hours after
orally dosing them with carotene. More detailed information on the
mechanism.of carotene conversion to vitmEin A is provided by several
British investigators (Glover, Goodwin and Morton, 1948; Alexander and
Goodwin, 1950; Thompson 9; $1... 1950). In 1950, Thompson and Ron at
the National Institute for Research in Dairying in England determined
the intestine to be the site of carotene conversion to vitamdn A in
the pig. They concluded that carotene was converted to vitamin A
ester in the intestinal wall, transported by the lymph to‘the blood
stream.and carried to the liver and throughout the body by the blood.

Additional evidence for the intestinal-conversion viewWwas pre-
sented by Olson (1959) using CIA-labeled beta-carotene and by Popper
and Greenberg (1941), who photographed fluorescing vitamin A in the
intestinal mucosa 4 hours after vitamin A-deficient rats consumed a
carotene meal. "'

Although many body tissues can convert carotene to vitamin A in
small amounts (Bieri and Pollard, 1954; Olson, 1959; McGillivary,
1961), the small-intestine wall, probably the mucosa, is the primary

site of this conversion.

4

Some early workers have studied the interaction of carotene metabo-
lien and infection. Heymann (1936) studied the fecal output of caro-
tene in 24 healthy infants and in 17 infants with various types of
childhood infections. The normal absorption rate of 701 was diminished
to 351 in those infants with infections. Heymann hypothesized that
the decreased absorption associated with infection was due to a toxic
factor which directly inhibited fat absorption or affected bile pro-
duction. After studying more than 500 patients, Clausen (1933) noted
that carotene was poorly absorbed in the presence of fever or diarrhea.
Spielmmn ggflgl. (1946) reported that diarrhea and infection in calves
shortly after birth reduced absorption and utilization of carotene
regardless of the amount fed. When diarrhea was minimized, the calves
could utilize carotene more efficiently.

In cattle, horses, pigs and man not ingesting preformed vitamin A
carotene is the chief precursor of vitamin A. Since the vitamin A
status of the body is associated with resistance to infection and
stress (Keener‘gg 1., 1942; Boynton and Bradford, 1931; Harmon 25
‘31., 1963; Stowesand and Scott, 1964), preper and adequate carotene
metabolism may be of paramount importance to the organism's well being.

Transmissible gastroenteritis in young pigs is characterised by
histopathologic changes remarkably similar to those accompanying the
human malabsorption syndromes of tropical and nontropical sprue.

In the latter there is a shortening and blunting of the small-intestinal
villi, lengthening of the crypts of Lieberkahn and infiltration of the
lamina propria with granulocytes (Shiner, 1960; Padykula g£_gl,,

1961; Townley, Cass and Anderson, 1964). Electron-microscopic exami-

nation reveals that prior to these changes, the microvilli, which

5
comprise the brush border, are lost in part or completely (Padykula
__t_ __1._., 1961).

The histopathologic lesions of the small intestine in TGE, range
from a reduction in the height of the mucosal epithelium to necrosis
of the mucosa depending upon the animal's resistance and the duration
of the infection. In addition, the small-intestinal villi often
become swollen, and infiltration of the lamina propria with polymorpho-
nuclear leukocytes is seen on histopathologic sections (Wallace and
Whitehair, 1965; Doyle, 1958; Bay, Doyle and Hutchings, 1951).

Previous work and current clinical evaluation indicate that
vitamin A has a role in maintaining health in man and animals. The.
pathogenicity of the TGE virus has been well established. Thus, the
use of this infective agent seems apprOpriate to obtain additional

information on the metabolism of carotene in an experimentally pro-

duced infection.

MATERIALS AND METHODS

General p_l_a_t;. Experiments were conducted to compare the absorp-
tion of beta-carotene in pigs exposed to the TGE virus with that in
clinically normal pigs. The pigs were fed a synthetic vitamin A-free
diet with controlled amounts of beta-carotene added. Records of
body weight, feed consumption, fecal excretion and clinical signs were
maintained throughout the study. Blood, liver and fecal samples were
collected at selected intervals throughout the experiments for routine
hematologic examination as well as for carotene and vitamin A assay.
Tissue samples for histopathologic study‘were routinely taken from
the liver, kidneys and from selected areas of the gastrointestinal

tract at the time of necropsy.

Animals. Three-week-old Yorkshire pigs raised on the premises
were used for all experiments. The usual experiment consisted of an
entire litter of pigs using some as control animals and some as test
animals in accepted experimental design. The pigs‘were weaned at 2
weeks of age and given several days to become accustomed to the syn-
thetic diet. No infections or gross abnormalities were present in

either the pigs or the sows at the start of the experiments.

Housigg 55g,2553. [All pigs were housed in individual, grated-
bottom, galvanized cages and fed from crocks. The fecal collection
pans beneath the grating‘were covered with sheet vinyl plastic per-
forated with small holes to allow for separation of urine and feces.

6

7
Infected and control groups were housed in separte rooms but under other-
wise similar environmental conditions. The mbient temperature in each

room was 85 F.

Infective 5339;. The TGE virus, obtained from Dr. E. 0. Haelterman
of Purdue University, was employed as the infective agent. Its virulence
in the 3-week-old pig and the clinical signs produced are well charac-
terized by previous workers (Doyle, 1958 ; Bay, Doyle and Hutchings,

1951; Wallace and Whitehair, 1965).

Synthetic diet. A complete synthetic diet for young pigs was pre-
pared according to the formulation of Miller _e_t _a_l_. (1964), except

that vitamin A was not included in this diet.
Blood and tissue analyses.

Blood analyses. Ten-milliliter amounts of blood were collected
from the anterior vena cava according to the method of Carle and
Dewhirst (1942). A11 pigs were bled prior to the time of inoculation,
48 hours after inoculation, and terminally. Total and differential
white-cell counts were carried out as described by Benjamin (1964).
Hemoglobin estimation was by the cyanmethemoglobin method (Benjamin,
1964). Serum vitamin A levels were determined by the method of
Neeld and Pearson (1963). This technique was evaluated by canpari-
son with the Carr-Price assay method (Kaser and Stekol, 1943) and was

found satisfactory for porcine serum.

Liver analyses. The livers from all pigs were weighed and analyzed

for vitamin A by the following modification of the trifluoroacetic

8
acid method (Neeld and Pearson, 1963): (1) preparation of aqueous
liver homogenates in a Waring blender, (2) alkaline saponification for
10 minutes at 40 0., and (3) substitution of a measured portion of
saponified aqueous homogenate for serum. Using the livers from 4 non-
experimental pigs, trials were conducted to compare this assay pro-
cedure with the techniques described by Diplock, Green and Bunyan-(1963).
Based on these trials this method proved satisfactory for liver vitamin

A assay.

Fecal analysis. .Carotene was extracted from an aqueous suspen-
sion of feces with petroleum ether (30-60 C.), and the optical density
was read in a Coleman Jr. spectrophotometer set at 450 mu against a
petroleum-ether blank. The carotene content was then calculated from
a previously prepared standard curve.

Using 4 nonexperimental pigs, pilot trials were conducted to
determine (1) if carotene is eliminated in the urine and (2) if it is
destroyed in the intestine. The results of these pilot studies indi-
cate that pigs with TGE do not eliminate carotene in the urine and
that carotene is not destroyed when incubated with the intestinal

contents of the infected pigs for 4 hours at 37 C.

Histgpathologic technique. The pigs were euthanatised by exsanguina-
tion (severing the axillary blood vessels). Tissues of approximately
2 cm. x 0.5 cm. were fixed in 10% formalin and processed for examina-
tion by procedures described in the Eggggl'gg Histolggic g§g_825cia1
Staining Technigues of the Armed Forces Institute of Pathology,

Washington, D.C. (1957).

9
To determine the histologic effect of distention of the intestine

with gas, air was injected into an intestinal loop of an anesthetized

pig. The pig was killed 4 hours later. Histologic examination of the

distended intestine suggests that mere distention of the intestine

with gas might cause the villi to shorten and become blunt.

EXPERIMENTS AND RESULTS

Egpgriment'l

Expgrimental plan.

4 groups as follows:

Groups

of‘the

72 hours after inoculation.

Group

Basal Control
(noninfected)

Carotene Control

(noninfected)

Basal TGE

Carotene TGE

No. of
Pigs

Ten 3~week-old pigs were randomly divided into

Diet

Basal vitamin A-free diet,
water, 30 m1. of skim milk
d.11ye

Basal vitamin A-free diet,
water, 30 ml. of skim milk
containing 1.2 mg. beta-
carotene* daily.

Basal vitamin A-free diet,
water, 30 m1. of skim milk
d‘ily.

Basal vitamin A-free diet,
water, 30 ml. of skim milk
containing 1. 2 mg. beta-
carotene* daily.

3 and 4 were inoculated at 3 weeks of age by administering 1 ml.

TGE virus suspension in milk.

after inoculation.

Results.

Clinical signs.

_ all 4 groups.

 

by Hoffmann-LaRoche, Inc., Nutley, N. J.

One pig from each group was killed

The remaining pigs were killed 120 hours

Ration consumption was essentially the same for

Groups 1, 2, and 4 had gains in body weight, while Group

*Assayed Dry Beta-Carotene Beadlets (Type 2. 4S)‘were supplied

10

11
3 (Basal, infected) lost weight. Vomiting and diarrhea started in the
infected pigs 12 to 24 hours after inoculation. Vomiting persisted for
48 hours. Diarrhea was still evident in the infected pigs 120 hours
after inoculation. A moderate leukopenia developed in the infected
pigs 48 hours after inocuation, which was essentially the same as
reported by Wallace and Whitehair (1965). Groups 1 and 2 had no ab-

normal clinical signs throughout the experiment.

Gross pathology. The following gross pathologic changes were seen
in the infected pigs: distention of the stomach and small intestine
with gas, mild to moderate congestion of the mesenteric blood vessels,
and yellow, watery fluid in the small and large intestines. In addi-
tion, 2 infected pigs had moderately engorged epigastric blood vessels.

No gross lesions were evident in the noninfected pigs.

Histopathology. Only the infected pigs had histopathologic lesions.
The lesions were confined to the small intestine and included the
following: partial to total villous strephy with loss of the epi-
thelial brush border, villous edema, congestion of the blood vessels
in the villi and in the tunica mmscularis, infiltration of the lamina
propria by neutrophils, eosinophils and macrophages. Infected pigs,
killed at 120 hours after inoculation, had a more pronounced cellular
infiltration of the lamina prOpria than those killed at 72 hours. Many
of the atrophic villi had cuboidal rather than columnar epithelium,
the more cuboidal epithelial cells being at the villous apex. The
villous atrophy'was confined to the jejunum and ileum.and'was most
marked in those intestinal segments greatly distended with gas (Figures

3, 4, 7, s, 11, 12).

12
Analyses. Comparison of fecal carotene excretion in Groups 3 and
4 is summarized in Figure l. Hepatic vitamin A stores are presented
in Figure 2. Serum vitamin A levels ranged from 40 to 58 mcg. per

100 m1. of serum, with no significant differences between any 2 groups.

Experiment _2.
Experimental plan. Eight 3~weekoold Yorkshire pigs were assigned

to 2 groups as follows:

No. of
Group Pigs Diet

1 - Carotene, Control 4 Basal carotene ration*,
water, 30 ml. of skim milk
d811Ye

2 - Carotene, TGE 4 Basal carotene ration*,
water, 30 m1. of skim milk
daily.

One milliliter of the virus suspension was mixed with the milk and given
to each pig in Group 2 as the inoculum.. One pig from each group was
killed 72 hours after inoculation; the remaining pigs were killed 120

hours after inoculation.
Results.

Clinical signs. Although feed consumption was uniformnwithin each
group of pigs, there was a distinct difference between the groups.
Both groups started consuming 150 Grams of ration per pig per day.
Group 1 pigs were consuming 250 Grams each at 120 hours after inocu-
lation, while each pig in Group 2 was consuming only 75 Grams. The
following table gives the mean body weight changes in kilograms for

each group:

 

*4.29 mcg. of beta-carotene per Gram of feed. The betaecarotene,
supplied by Distillation Products Industries, Rochester, N.Y., was a
24% solution in vegetable oil.

l3

 

 

 

 

 

 

 

 

 

 

 

MICROGRAMS OF CAROTENE EXCRETED
#8.
1800
1500 I 3.,
1200
Control
900 Group
600 ‘2.
300 .. :$..aeeaeeeeao.. InfeCted
G- .~.OOOOOOO Group
-72 -48 -24 0 24 48 72 96 120 hours
Preinfection Postinfection
Figure 1.
TOTAL LIVER STORAGE 0F VITAMIN A
2500
2000
1500
M8-
1000
500
Basal Carotene Basal Carotene
Control Control TGE TGE
Figure 2.

 

 

 

l4

   
      
 
   
   

"\(""".‘” .. .5
Figure 3. Jejunum from a carotene noninfected pig.
(A) normal villi, (B) glands of Lieberkehn. Hematoxylin and

eosin. x 75.

V
m‘
m

  

c“

.73:-

  

.
O

s ,
f

  
  
  

" 39s

  

w :3
..-*-
'4 us

    
  

‘

   
  
   
  
  
   

 

3“.“ .. -

32':

      

‘7
pa
‘0
.

 

   

"1 35' '3': ' ' .
Ezfl"'°‘ ‘- "°‘ ‘ - ‘ r. a '3; .,I
Figure 4. Jejunum from a pig with TGE showing partial

villous atrophy.. (A) increased length of the glands of
Lieberkahn, (B) cellular infiltration of the lamina propria,
(C) blunted, atrOphic villi. Hematoxylin and eosin. x 75.

15

Initial Wei gilt Terminal m
Group 1 2.9 kg. 3.4 kg.
Group 2 2.4 kg. 2.0 kg.

The clinical signs of vomiting and diarrhea were evident 24 to 48 hours
after inoculation in the infected pigs. In this experiment only 1 pig
in Group 2 vomited. All infected pigs developed a persistent, watery
diarrhea which lasted for the duration of the experiment. Forty-eight
hours after inoculation there was moderate leukopenia in the infected
pigs. Control pigs had no abnormal clinical signs throughout the

experiment.

Gross pathology. The only gross pathologic changes seen at the
time of necropswaere a mild congestion of the mesenteric blood vessels
in 3 of the infected pigs and gas-filled small intestines in all the

pigs in the infected group.

Histopathology. Three of the 4 infected pigs had significant
histopathologic lesions in the small intestines. The lesions included
a shortening and blunting of the intestinal villi in the jejunum.and
ileum, leukocytic infiltration of the intestinal lamina prOpria, and
a morphologic alteration of the intestinal epithelium from simple
columnar to simple cuboidal, with loss of the epithelial brush border.
In 1 of the infected pigs the villi were almost nonexistent in some
portions of the ileum. All pigs in Group 1 and 1 of the pigs in Group
2 had no significant histopathologic alteration in the gastrointestinal

tract .

16
Analyses. A comparison of the fecal carotene excretion for the
control and infected groups is presented in Figures 5 and 6. The mean
vitamin A content of the livers was 2742 meg. for Group 1 and 2358 mcg.
for Group 2. There was no apparent difference between the serum
vitamin A values of the pigs in Groups 1 and 2. The serum vitamin A

values ranged from 33 to 46 mcg. per 100 ml. of serum.

Expgriment‘g
Experimental plan. Experiment 3 was a replicate of Experiment 2

using 3 pigs in each group. All animals were killed 96 hours after

inoculation.
Results.

Clinical signs. Feed consmnption was essentially the same for
both groups of pigs. Two of the infected pigs had a loss of weight,
1 infected pig had no weight change, and the control pigs all gained
‘weight. In the infected group, diarrhea was evident in 1 pig in 48
hours after inoculation with the TGE ‘virus, and 1 of the infected pigs
had leukocytosis with a left shift. The 3 pigs in the control group

had no abnormal clinical signs throughout the experiment.

Gross pathology. The infected pigs were all characterized by
gas-filled small intestines which contained a yellowish fluid. A
diphtheroid membrane covered the tongue and lined the esophagus of
2 of the infected pigs. Hypermotility of the small intestine was

still present at necropsy in 2 of the infected pigs.

Histopathology. Partial to complete villous atrophy was evident

in 2 of the 3 infected pigs. The villi were blunted and the brush

17

 

80
n g.
60

 

120

100

 

MICROGRAMS OF CAROTENE EXCRETED

 

 

I

Liigure 6 a

Control
Group
40
20 Infected
“.00...OOQOOOOOW.“.....OO. .0000...
. «mu «on... Group
-24 O 24 48 72 96 120 hours
Postinfection
Figure 5.

 

 

Per Cent

PER CENT OF CAROTENE INTAKE EXCRETED

 
   

 

 

-24 0 24 48* 72 96** 120 hours
Postinfection

 

 

 

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v
.afli‘
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v‘ .
'l‘h
. a -’\OC‘. 0".
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.‘v

Fv$' .
Igw~z. ,
3s. .
”A
‘:
‘5‘ w-

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“ -N~_"-J

..

(A) atrophic villi, (B)

Hematoxylin and eosin. x 75.

18
Ileum from a pig'with TGE showing

partial villous atrophy.

Peyer's patches.

 

. .‘ :4
‘ b
1‘ O
' ..‘£
“‘1‘”

 

Ileum from a pig with TGE showing

Figure 8 0
total villgus atro
of Lieberkuhn.

(A) atrophic villi, (B) glands

Hematoxylin and eosin. x 75...

phy-

l9
border was absent from many of the villi. The diphtheroid membrane
foundcovering the tongue and lining the esophagus of 2 infected pigs

was composed largely of sloughed epithelium, neutrophils and fungi.

Analyses. Figures 9 and 10 illustrate the fecal carotene excre-
tion for this experiment. The mean hepatic vitamin A content was
essentially the same for both groups of pigs. The control pigs had
3014.mcg. of vitamin A as a group mean,‘while the infected pigs had
2929 meg. Serum vitamin A values.ranged from 36 to 46 meg. per 100
ml. of serumnwith no apparent differences between the control and

infected groups.

20

 

 

 

 

 

 

 

 

 

 

 

MICROGRAMS 0F CAROTENE EXCRETED
120
100
Control
80 . Group
"u... Infected
60 m a... Group
Mg.
40
20
0
-24 0 24 48 72 96 hours
Postinfection
Figure 9.
PER CENT 0F CAROTENE INTAKE EXCRETED
10 .QO
0..
.a
3 __, ‘—1::=..¢=L_.
-p éfl’ '
g 6 a"
L) Jf’
H 49’
a 4
d9
2
0
1 -24 O 24 48 72 96 hours
' Postinfection

 

 

 

i__
Figure 10.

 

Figure 11. Jejunal villous apex from a carotene,
noninfected pig. (A) normal columnar epithelium, (B)
brush border. Hematoxylin and eosin. x 768.

         

. .. ‘ .
Figure 12. Jejunal villous apex from a pig with
TGE. (A) cuboidal epithelium without brush border, (B)
cellularinfiltration of the lamina propria. Hematoxys
lin and eosin. x 768.

       

h-“

DISCUSSION

These experiments indicate that there is reduced fecal carotene
excretion in pigs infected with TGE virus. Pilot studies (see page
8) indicate the following: (1) TGE infected pigs do not eliminate
carotene "in the urine and (2) carotene is not destroyed when incubated
with the intestinal contents of TGE infected pigs. In view of these
findings, it is probable that pigs with TGE absorb more carotene
than noninfected pigs. The increased liver stores of the infected pigs
with carotene supplementation (Group 4) in Experiment I tend to con-
firm this possibility. The serum vitamin A levels give no indication
of an increased carotene absorption in pigs with TGE. This is to be
expected, however, since it is well known that serum vitamin A levels
remain in a normal range as long as there is an abundant hepatic
vitamin A reserve (Hentges 53; $1., 1952b; Caster and Mickelsen, 1955).

The infected pigs fed the basal ration (Group 3) in Experiment
1 had adequate hepatic vitamin A stores as compared to the controls
(Groups 1 and 2). Hence, they were not dependent upon dietary caro-
tene to meet their vitamin A needs. In view of this, it is not
surprising that the inclusion of carotene in the diet did not affect
the susceptibility of pigs to TGE, nor did it affect the severity of
the infection.

The histopathologic lesions in the jejunm and ileum of the
animals with TGE are remarkably similar to those described in the
human malabsorption syndromes of tropical and nontropical sprue

22

23
(Shiner and Doniach, 1960; Padykula ggwgl., 1961). For convenience,
the intestinal lesions were classified into the categories of partial
villous atrophy and total villous atrophy according to Shiner and
Doniach (1960). In these experiments the pigs‘with TGE often had
both categories of villous atrophy and in some areas there were normal
villi. Whether the TGE virus itself is responsible for the villous
atrophy remains to be determined.

The villous atrOphy seen in these efipcriments was most pronounced
in the gas-distended segments of the small intestine. A pilot study
(see page 9) suggested that mere distention of the intestine with gas
might cause the villi to become shortened and blunted. However, this
does not explain the loss of the brush border (microvilli), the cue
boidal epithelium, cellular infiltration of the lamina propria and
congestion of the mesenteric blood vessels in infected pigs. Multiple
factors, including TGE virus, distention of the intestine with gas,
vascular congestion, and other unknown factors, are possibly responsible
for atrophy of the villi.

Certain biochemical alterations, especially decreased ensyme
activity in the intestinal epithelium, accompany sprue in humans
(Padykula,g£flgl., 1961). .Diminished activity of enzymes suggests
disturbances in energy production and release which would affect
certain aspects of active transport. Decreased activity of a chemical
substance which normally inhibits excessive carotene absorption might
explain the decreased carotene excretion seen in these experiments.
However, no such chemical substance has been isolated. Without

further investigation, this is mere speculation.

24

Clinicaligiggg. ‘While TGE in pigs under 1 week of age is usually
fatal, older pigs generally survive (Doyle, 1958). In the 3-week-old
pigs used in these experiments, the clinical signs of infection with
TGE varied in severity. Diarrhea was the major clinical sign in all
infected animals. In addition, vomiting, reduced feed consumption,
‘weight loss and leukopenia were noted. The leukocytosis with a left
shift in l infected pig (Experiment 3) was probably due to a secondary

bacterial complication.

QEQEE’Bathology. The most marked and consistent finding in the
infected pigs was a distention of the small intestines with gas and
fluid. As was pointed out previously, this may have a more or less
direct relationship to the histopathologic changes seen in TGE. The
intestinal hypermotility noted in 2 infected pigs (Experiment 3)
would contribute to the diarrhea. Congestion of the mesenteric and
epigastric blood vessels is in agreement with the findings of other

workers (Doyle, 1958; Bay, Doyle and Hutchings, 1951).

Histgggtholggz. Villous atrophy was first described in detail
by Shiner and Doniach (1960), who classified 3 types: partial, sub-
total, and total. In describing the villous atrophy in these experi-
‘ments the partial and total categories were used.

Partial villous strephy in pigs with TGE was characterized by i
a shortening and blunting of the villi to as mmch as one third of the
normal height. These villi became thickened and the lamina propria
was infiltrated with numerous leukocytes. The epithelial lining was
diminished in height and the brush border was absent. In addition,

the glands of Lieberkuhn were relatively lengthened and the blood

25
vessels in all layers were congested. Total villous atrOphwaas simi-
1ar to the above but with the villi shortened to less than one third
of the ncrmal height and covered with a simple cuboidal epithelial

lining.

Chemical pathology. These experiments indicate that hepatic
vitamin A stores were not noticeably affected by TGE virus infection.
If the pigs had been more deficient in vitamin A at the start of the'
experiment, more group differences might have been anticipated.

In Experiment 1 the hepatic vitamin A reserves were lower than in the
2 succeeding experiments. The most pronounced difference in terminal
hepatic vitamin A stores was recorded in this experiment.

In Experiment 1, fecal carotene excretion was absolutely higher
than in the 2 succeeding experiments. A possible explanation for
this might be that in Experiment 1 the carotene was administered in
the daily skimmed milk while in the next 2 experiments, carotene was
mixed with the ration. In addition, dry, water-dispersible, carotene
beadlets were used in Experiment 1 and oil-soluble carotene was used

in Experiments 2 and 3.

SUMMARY

Research was conducted using a total of 24 pigs in 3 experiments
to determine carotene metabolism during a specific viral enteric
infection, transmissible gastroenteritis (TGE).

Clinical signs and gross and microscopic lesions were not
noticeably different between infected animals fed carotene and those
not fed carotene. Fecal carotene excretion was significantly reduced
within 24 hours after oral inoculation with the TGE virus. Fecal
carotene excretion was greater in pigs fed water-dispersible beta-
carotene beadlets than in pigs fed an oil-soluble beta-carotene.

The virus of TGE produced sprue-like lesions characterised by
villous straphy in the small intestines of all infected animals.

Diarrhea was present within '72 hours after inoculation with the

infective agent and continued until the pigs were killed for necropsy.

26

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