THE INFLUENCE OF A VIRAL ENTERIC INFECTION ON‘
ABSORPTION OF CHLORTETRACYCLINE

Thesis for the Degree of M. S.
MICHIGAN STATE UNIVERSITY

Larry J. Wallace
1964:

THESIS

 

LIBRARY

Michigan State
University

 

ABSTRACT

THE INFLUENCE OF A VIRAL ENTERIC INFECTION ON
ABSORPTION OF CHLORTETRACYCLINE

by Larry J. Wallace

Research was conducted using 18 pigs assigned to the
following groups: (1) control, (2) control plus chlortetra-
cycline, (3) transmissible gastroenteritis (TGE) virus-
infected, and (4) TGE virus—infected plus chlortetracycline.

Signs of TGE were present within 12 hours after in-
fection. The disease was characterized by clinical signs,
leukopenia, and gross and microscopic lesions.

The virus of TGE had an inhibitory effect on absorp-
tion of orally administered chlortetracycline (CTC) on the
first two days of treatment. On the third day of treatment,
CTC blood levels of infected pigs approximated those of
control pigs.

Microscopic lesions in infected, CTC-treated pigs
included regeneration of intestinal epithelium on the third
and fourth days. Infrequently, small areas resembling pro-
liferation were observed in the intestinal epithelium during
and after the third day in infected pigs which did not
receive CTC.

Absorption of CTC appears to be involved in an active

transport system.

THE INFLUENCE OF A VIRAL ENTERIC INFECTION ON

ABSORPTION OF CHLORTETRACYCLINE

BY

Larry J. Wallace

A THESIS

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

MASTER OF SCIENCE

Department of Pathology

1964

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ACKNOWLEDGMENT S

The author is extremely grateful and indebted to his
long-time friend and advisor, Dr. C. K. Whitehair, of the De-
partment of Pathology for his assistance, suggestions, and
continued guidance during the planning and conducting of this
research project and writing of the thesis. Appreciation is
also expressed to Drs. C. C. Morrill and G. L. Waxler of the
Department of Pathology, and Dr. W. O. Brinker of the Depart—
ment of Surgery and Medicine, for their aid and constructive
criticisms.

Sincere thanks are given to the technical staff and
animal caretakers in the Department of Pathology who helped
in many ways during this research project.

In addition, the aid and constructive criticisms in
the histopathologic aspects of this research, by Dr. C. F.
Simpson, Pathologist, Department of Veterinary Science, Uni-
versity of Florida, are greatly appreciated. Appreciation is
also given to Mrs. Rosemary G. Rumbaugh and Mrs. Joan L.
Gibson of the Department of Veterinary Science, University of
Florida, for their assistance in sectioning and staining of
the tissues.

The author is most sincerely thankful and indebted to

his wife, Eileen, for her continual patience, understanding,

ii

and encouragement throughout the research, and for her assis-

tance in the preparation of this thesis.

iii

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . .
REVIEW OF LITERATURE . . . . . .
EXPERIMENTAL PROCEDURE . . . . .
RESULTS . . . . . . . . . . . .
Clinical Signs . . . . .
Gross Pathology . . . .

Gastrointestinal

Other Prominent Gross Lesions

Histopathology . . . . .
Stomach . . . .
Duodenum . . . .
Jejunum and Ileum
Colon . . . . .

Tissue Analysis . . . .

Tract

Chlortetracycline Blood Levels

Hematology . . . . . . .
DISCUSSION . . . . . . . . . . .
SUMMARY AND CONCLUSIONS . . . .
APPENDIX . . . . . . . . . . . .

REFERENCES CITED . . . . . . . .

iv

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14
14
15
15
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16
18
21
23
23
25
27
36
37

45

LIST OF TABLES

Table Page
I Identification of Pigs Assigned to the
Four Groups and Their Treatments . . . . . . 37
II Daily Average Body Temperatures, Milk and
Water Consumption of Pigs . . . . . . . . . 38
III Body Weights of Pigs . . . . . . . . . . . . . 39
IV Chlortetracycline Blood Levels of Non-

infected Pigs . . . . . . . . . . . . . . . 40

V Chlortetracycline Blood Levels of In-
fected Pigs . . . . . . . . . . . . . . . . 41

VI Daily and Total Dosage of Chlortetra-
cycline (CTC) Administered to Pigs
During the Experiment . . . . . . . . . . . 42
VII Average HemOgrams of Noninfected Pigs . . . . 43
VIII Average Hemograms of Infected Pigs . . . . . . 44

Figure

10.

LIST OF FIGURES

Infected and noninfected pigs on the
second day . . . . . . . . . . . . . .

Gross lesions of an infected pig as
compared to a noninfected pig . . . . .

Hypermucous secretion, desquamation and
erosion in gastric epithelium . . . . .

Slightly congested blood vessels in the
tip of a duodenal villus with normal
mucosal epithelium . . . . . . . . . .

Histopathologic lesions in the jejunum of
an infected pig . . . . . . . . . . . .

Same as Figure 5 at a lower magnification

A focus of neutrophils and degeneration in
a Peyer's patch . . . . . . . . . . . .

Recovery of intestinal epithelium in a
Group IV pig on day 4 . . . . . . . . .

Area of proliferation of intestinal
mucosal epithelial cells in a Group IV
pig . . . . . . . . . . . . . . . . . .

Graph illustrating average chlortetra-

cycline blood levels in infected and non-

infected pigs . . . . . . . . . . . . .

vi

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24

THE INFLUENCE OF A VIRAL ENTERIC INFECTION ON

ABSORPTION OF CHLORTETRACYCLINE

INTRODUCTION

Most drugs and other therapeutic agents are evaluated
in experiments using the normal animal. It is rather obvious
that in practice these products are not used in the normal
animal but are used by the clinician in the treatment of a
specific disease process. Therefore, it is of importance to
have information on the metabolism of drugs during the course
of a specific infection. This more closely approximates the
information of interest to the clinician.

Probably of most importance is the role of the gastro-
intestinal tract in the metabolism, especially the absorption,
of the orally administered drugs. Information on the influ—
ence of a specific lesion of the gastrointestinal tract on
the absorption of a drug would not only be of value for the
product tested but this information could be extrapolated and
applied in evaluating other drugs.

This study was designed to determine the influence of
a specific viral infection (transmissible gastroenteritis)
and the resulting lesions on the absorption of chlortetra-
cycline (CTC). This information would have wide application

in clinical medicine as well as basic biomedical interest.

REVIEW OF LITERATURE

Interest in the absorption and distribution of chlor-
tetracycline (CTC) throughout the body is indicated by the
large amount of material which has been published on this
subject. In preparing this literature review, only those
references which seemed pertinent to serum and/or blood
levels of CTC have been cited. Although much has been
published on the general subject, very little has been
published on CTC absorption in swine. Information regarding
the influence of a specific enteric infection on absorption
of CTC is limited.

In one report on swine (Maddock._§__1., 1953) the
highest concentration of CTC in the serum was 0.83 mcg./m1.
which resulted from an oral dose of 4.5 mg./1b. Lepper gt _1.
(1949) reported that, when the drug was given orally, the
highest concentrations were obtained in the majority of indi-
vidual patients at the third and a few at the sixth hour.
Drill (1958) mentions that following oral administration of
the tetracyclines, peak concentrations after a single dose
are reached in about 2 to 4 hours and decrease slowly during
the succeeding 12 to 24 hours. Jones (1957) reported that

the tetracyclines are absorbed readily from the stomach and

first part of the small intestine to give a peak plasma level

within 2 to 4 hours in carnivorous animals. Putman g§.al.
(1953) compared serum concentrations between tetracycline and
chlortetracycline after 4 oral doses of 0.5 Gm. of each drug
every 6 hours. In their study there appeared to be a gradual
increase in the serum concentration of both drugs when
multiple doses were given. Whitlock and co—workers (1950)
gave children single oral doses of 11 mg./kg. and 22 mg./kg.
body weight. They found the peak level was attained slowly
somewhere between 1 and 4 hours after drug ingestion. This
peak level was low, but was maintained at a relatively
constant value until 6 and sometimes 8 hours after ingestion.
These same investigators stated that the absence of progress-
.ing or marked cumulation of Aureomycin in the serum after
prolonged oral administration, together with the failure of
increased oral dosage to produce significantly higher serum
levels, suggests some limiting factor in the ability of the
gastrointestinal mucosa to absorb the antibiotic. In a

study by Gray.g£.gl. (1953), dogs were given CTC orally at a
dosage of 5, 10 and 25 mg./kg. body weight. At the end of 4
hours the mean blood serum concentrations in micrograms per
ml. were 0.1 (0.1 — 0.2), 0.8 (0.1—2.0), and 2.2 (0.8 - 5.0)
respectively. They state that variability emphasizes the un-
reliability of such determinations as indices of CTC absorp—
tion from the gastrointestinal tract of the dog. Brainerd
.§§_al. (1951) reported that, following a 250 mg. oral dose

in humans, concentrations of CTC in serum reached a peak at

2 hours followed by a decrease at 4 and 6 hours. In this

same study it was found that at 6 hours there was relatively
little difference in serum concentration between oral doses
of 250 mg. and l Gm. Therefore, they concluded that there is
a maximal rate of absorption from the gastrointestinal tract
which can not be exceeded by increasing the dose four-fold.
Ibsen and Urist (1962) state that metal ions alter the ab-
sorption of CTC. They also state that tetracycline and
related compounds form complexes with calcium and other metal
ions and are deposited in all newly calcifying tissue and

can be used to label the growing skeleton.

Sirota and Saltzman (1950) found that 70% of Aureo-
mycin is bound to plasma components and, of that, approxi-
mately 85%Iis albumin-bound. As the free (therapeutically
active) component of the drug is dissipated by excretion and
metabolism, more of it is liberated from the protein-
aureomycin complex. They state that it is these character—
istics which explain the prolonged therapeutic blood levels
and relatively high urine concentrations obtained following
a given dose of the drug. Malek and Kolc (1960) studied
tissues by fluorescence using ultraviolet light and found
there can be no doubt that inflammatory hyperemia makes
possible increased penetration of CTC into the tissue and in-
creased resorption of CTC by the lymphatic system. They be-
lieved that the increase of CTC in the lymphatic vessels is
probably made possible by the binding of CTC on the proteins
of the inflammatory exudate, which causes CTC to be more
lymphotrophic. An additional factor is probably stasis of

the lymphatic system in the areas of the inflammation.

.lg vitro studies have indicated that CTC is most
stable near pH 2 (Dornbush and Pelcak, 1948). The serum
concentrations observed in chickens following oral adminis-
tration of penicillin, Aureomycin, and terramycin were much
lower than those that have been reported in man and other
animals (Smith, 1954).

Harrell and Heilman (1949) found that, regardless of
the duration of treatment in patients who were given 750 mg.
every 6 hours, the maximum concentration of CTC in serum
demonstrated by their technique was about 8 mcg./ml. They
report this would indicate that the rate of excretion is
rather constant and that repeated administration of 750 mg.
every 6-8 hours does not result in any "piling up" of CTC in
the serum.

Eisner gt _1. (1953) using guinea pigs found that
aureomycin concentration in the serum was not directly
proportional to the dose. They also found that when the
same doses were administered each day for nine consecutive

days, higher serum levels were found than with the single

doses.

EXPERIMENTAL PROCEDURE

Objectives.

1. To determine the blood levels of CTC treated pigs
after inducing a specific infection of the gastro—
intestinal tract.

2. To compare the blood levels of CTC in infected pigs
with noninfected control pigs.

3. To compare these results (under 1 and 2) with infor-
mation found in the literature.

4. To observe the clinical and pathological effects of

a viral enteric infection in the pig.

Housing and Care. Baby pigs were the experimental
animals used in this study. Two litters of third-generation
specific pathogen-free (SPF) Yorkshire pigs born August 1,
1963, were purchased from a local swine farm on August 9,
1963, and brought to the Department of Pathology's Disease-
Free Laboratory at Michigan State University. These pigs
were given a liquid iron compound orally by the owner at
three days of age to prevent anemia. No infection or gross
abnormality was present in the pigs or dams of either litter
at the time of purchase. The pigs were allowed to become
accustomed to a controlled environment and a milk ration for

3 days.

The two litters of 8-day-old pigs were assigned to 4
groups (Table I) by random numbers according to Goulden
(1956). Group I (Controls) consisted of 4 uninfected pigs
given no CTC. Group II consisted of 4 uninfected pigs given
CTC. Group III consisted of 5 infected pigs given no CTC.
Group IV consisted of 5 infected pigs given CTC.

The infected (Groups III and IV) and uninfected
(Groups I and II) pigs were housed in two separate isolation
rooms under similar environmental conditions. Temperatures
in each room were maintained at 82 F. The pigs were placed
in individual galvanized metabolism cages to allow recording
of feed and water consumption* and clinical observation of
each pig. Rectal temperatures were taken daily at 8 a.m.
Feed and water were offered to the pigs in crocks. A separate
caretaker was assigned to the pigs in each isolation room as

a precaution against spreading infection.

Ration. Pigs were fed pasteurized, homogenized,
whole milk fortified with 400 USP units of Vitamin D obtained
from the Michigan State University Dairy. The first two days
the pigs were offered 100 ml. of milk per feeding. This was
increased to 130 ml. per feeding on the third day and kept
at this level throughout the experiment. Feeding was done
at 8 a.m., 12 noon, and 4 p.m. Any milk left over from the

previous feeding was measured and recorded. The pigs had

 

*Feed and water consumption values are approximate due
to spillage and evaporation.

access to water at all times. At 8 a.m. and 4 p.m., the
amount of water remaining from the previous period was
measured and recorded and a fresh 300 ml. placed in each

clean crock.

Infective Agent. Transmissible gastroenteritis (TGE)
virus* was used as the infective agent. Virulence of the
original sample was determined by methods employed by Keahey
(1963). Virus material was maintained at —20 C, delivered
to the Disease-Free Laboratory on experimental day 0 and
allowed to thaw at room temperature. Immediately after
thawing, 2 ml. of virus material** were added to 10 ml. of
whole milk and given to each pig in the assigned groups. All
pigs consumed the virus—bearing milk within five minutes ex-
cept pig 14. The milk was administered to this pig orally

with a dose syringe.

Antibiotic. The antibiotic selected was crystalline
chlortetracycline (CTC).+ Dosage was 1 mg./50 Gm. live
weight, before each morning feeding.++ weights of the pigs

were taken each morning at 8 a.m., prior to feeding. The

 

*Original sample obtained through the courtesy of Dr.
E. O. Haelterman, School of Veterinary Science and Medicine,
Purdue University, Lafayette, Indiana.

**Virus material was given to assigned groups of pigs
at 8 a.m. on August 12, 1963.

+Chlortetracycline (Aureomycin), supplied by the
Agricultural Division, American Cyanamid Company, Princeton,
New Jersey.

++Chlortetracycline administration to the assigned
groups of pigs began at 8 a.m. on August 13, 1963.

assigned dose of CTC was weighed out on a Mettler Balance and
placed in size 00 gelatin capsules. Oral administration of

gelatin capsules containing CTC was by hand or by balling gun.

Tissue Analysis. Blood samples were collected from
the anterior vena cava, as described by Carle and Dewhirst
(1942), using heparinized 10 ml. luer—lok syringes with 1%
inch, 20 gauge needles. The samples were taken each day at
8 a.m., 10 a.m., and 2 p.m., with the 8 a.m. bleeding being
prior to the administration of CTC. Ten ml. of blood were
collected at each bleeding except at 8 a.m. when 11 ml. were
taken of which 1 ml. was utilized for hematologic study.

The 10 ml. of blood for CTC assay were placed in heparinized,
screwecap tubes and quick-frozen in dry ice within 4 minutes.
Hematologic studies included hemoglobin (Hb), packed cell
volume (PCV), total and differential leukocyte counts. At
the termination of the experiment the blood samples for CTC
assay were packed in dry ice and sent to Dr. L. A. Shor*, for
microbiological assays. These were conducted according to
the modified FDA cylinder—plate methods of Abbey and Hewel

(1962).

Pathologic Procedures. At 2 p.m. on experimental
day l and each day thereafter except experimental day 4, l

pig was selected from each group for necrOpsy. These pigs

 

*Assays were done through the courtesy of the
Agricultural Division, American Cyanamid Company, Princeton,
New Jersey.

10

were euthanatized with Lethol.* On experimental day 4.
necrOpsies were performed on all the remaining pigs. These
pigs were euthanatized by exsanguination (severing the
axillary blood vessels). Necropsies were performed immedi-
ately after euthanasia. Pigs chosen for necropsy on the
first, second and third days from the infected Groups III
and IV were those showing the most severe clinical signs.
Pigs were chosen from the uninfected Groups I and II at
random. Immediately prior to death, the pigs were weighed
and terminal blood samples were taken for hematologic study
and CTC assay. Tissue sections of approximately 2 cm. in
length were taken from the fundic region of the stomach,
duodenum, jejunum, ileum, and spiral colon. The sections
were collected at the same site for all pigs. The tissues
were placed in Zenker's fixative for 24 hours, washed for 24
hours, and stored in 80% ethyl alcohol until processed for
embedding in paraffin blocks. Sections were cut at 5 microns
and stained with Harris' hematoxylin and eosin. In addition
periodic acid-Schiff (PAS) stain was used, when necessary,
to demonstrate mucus and fibrinoid material. All histo-
pathological procedures used are described in the Manualgof
Histoloqic and Special Staininngechnics of the Armed Forces

Institute of Pathology, Washington, D.C. (1957).

 

* Lethol, Pitman—Moore Company Allied Laboratories,
Division of The Dow Chemical Company, Indianapolis, Indiana.

RESULTS

The details of the results are given in tables in
the appendix. The average body temperature, feed and water
consumption are given in Table II. The weight changes are
recorded in Table III, chlortetracycline blood values in
Tables IV and V, amount of chlortetracycline given to each

pig in Table VI and hemogram studies in Tables VII and VIII.

Clinical Signs

Definite clinical signs of TGE were present in all in-
fected pigs (Groups III and IV) after a 12-hour incubation
period. Body temperatures remained in the normal range for
all groups of pigs (Table II). This parallels the findings
of Reber and Whitehair (1955), who found that TGE infection
did not influence the average body temperature. Prominent
signs were vomiting in a few infected pigs, and diarrhea in
all of them. Vomiting persisted in some of the infected pigs
for 48 hours. Diarrhea persisted throughout the experiment
in all infected pigs. Feces of Group III pigs (infected,
non—treated) were watery with bloodetinged mucous strands
and varied from light greenish—yellow to light brown in
color. Feces from the Group IV pigs (infected, treated) were

similar in consistency and color to those of the pigs in

11

12

Group III until the third day. At that time they had more
consistency with a brown color and remained that way through-
out the experiment. The rear quarters and tails of all in-
fected pigs were pasted with a fetid, yellowish-brown fecal
material. No vomiting or diarrhea occurred in the non-
infected pigs. Anemia became apparent in all pigs (Tables
VII and VIII) as the experiment progressed. Dehydration
became apparent in the infected pigs as evidenced by a gaunt
appearance and dry skin (Fig. l). Dehydration was more
severe in Group III than in Group IV. This was noticed
especially during and after the third day when the latter
group showed signs of recovery in the way of alertness,
activity, increasing milk consumption and decreasing water
consumption (Table II). The water consumption of Group IV
remained essentially within the amounts consumed by the un-
infected groups as compared to Group III which had an
evident polydipsia especially on days 1, 3, and 4 (Table II).
Partial anorexia was evident in both infected groups during
the first 2 days after infection. All groups experienced

a slight weight gain during the experiment. To keep the
weight gains from being misleading they were calculated

as the difference between the terminal and day -l weights.
This kept the expected initial weight loss due to environ—

mental change from influencing the net results (Table III).

13

 

Fig. 1. Pigs l4 (TGE infected) and 16
(Control)on day 2. Pig 14 shows signs of dehy-
dration and has fecal material adhering to the
extremities and rear quarters.

 

Fig. 2. Pigs 18 (infection plus anti-
biotic) and 16 (Control) on day 2. In pig 18,
note the greatly distended gall bladder with dark
bile and a liver slightly lighter in color than pig
16 (A). Stomach distended with gas and slightly
congested blood vessels over the greater curva-
ture (B). The intestinal tract is distended with
gas (C). Pig 16 has normal appearing viscera.

l4

Gross Pathology

All pigs had some skin abrasions from cage trauma.
and pale mucous membranes as evidence of anemia. Carcasses
of uninfected pigs were in a normal condition of flesh.
Carcasses of infected pigs were in gaunt to emaciated con—
dition and were dehydrated, especially in Group III. Car-
casses of Group IV pigs appeared less dehydrated during and
after the third day. At the time of euthanasia the stomachs
of all uninfected pigs contained a normal milk curd under—
going digestion except pigs 7 and 13 whose stomachs contained
a more particulate curd. The colons of all uninfected pigs
contained yellow, pelleted feces. Pigs in the infected
groups all showed typical lesions of TGE (Fig. 2) in varying
degrees of severity that resembled the lesions reported by
Doyle (1958), Smith (1956) and Runnells gt a1. (1960). Not
all of the following lesions were necessarily observed in

each pig.

Gastrointestinal tract. The stomach was distended
with gas, and in some pigs there was moderate congestion over
the greater curvature of the stomach especially noticeable
in the serosa. The stomach contained solid to cheesy, bile-
stained, undigested, food material, in some instances ad-
hering to the gastric mucosa. A mild to moderate catarrhal
gastritis was present. Pig 8 (Group IV) had an ulcer, 1 cm.
in diameter, near the squamoglandular junction in the

esophageal region of the stomach. The pyloric sphincters

15

were relaxed to strongly contracted. The entire intestinal
tracts were flatulent and showed mild to moderate serosal
and mucosal congestion. Atony was especially prominent in
the jejunum and ileum. Intestinal contents had a foamy,
watery to mucoid consistency, and were clear to yellowish or
greenish—brown in color.

In some pigs bloodrtinged mucus was mingled with the
ingesta from the jejunum posteriorly. On the third and
fourth days no blood was present in the intestinal contents
of Group IV pigs and the intestinal contents were soft in

consistency and brownish in color.

Other_prominent gross lesions. The mesentery and
mesenteric lymph nodes were mild to moderately congested and
edematous. In several of the infected pigs the gall bladder
was slightly to markedly distended with a dark colored bile.
Moderately hemorrhagic areas were present in the subcutaneous
tissues and musculature of all (infected and uninfected) pigs
at the bleeding sites. The livers and kidneys of most pigs

were pale in appearance.

Histopathology

Lesions in the infected pigs paralleled those re—
ported by Bay, Doyle, and Hutchings (1951). However, due to
the nature of this experiment, the histopathology of the
gastrointestinal tract has been recorded, as there were
obvious differences between the two infected groups (III and

IV) of pigs. These differences were noted especially at the

l6

latter phase of the trial. Not all the lesions were neces-

sarily observed in each pig.

Stomach. Hypersecretion of mucus, coagulation
necrosis, erosion, desquamation and in some areas, reduction
in height of the gastric epithelial cells were observed
(Fig. 3). A constant finding was generalized congestion
with endothelial nuclei in some of the larger blood vessels
of the tunica muscularis appearing enlarged. There was
patchy distribution of fibrin in the lamina prOpria, pre-
dominantly around small blood vessels immediately below the
epithelial basement membrane. The lamina propria was edem—
atous and contained an increase in neutrophils, lymphocytes,
plasma cells and occasionally a few macrophages. Lympho-
cytic infiltration into the tunica mucularis was observed in
some pigs. Division figures did not appear to be present in
excessively high numbers in the mucosal and glandular

epithelium.

Duodenum. The most outstanding lesion was the con-
gestion of all blood vessels, especially at the tip of the
villi (Fig. 4). Pathologic alterations were not seen in the
mucosal epithelial cells in any pig. The lamina propria was
edematous and contained an increase in neutrophils, lympho-
cytes, plasma cells and occasionally macrophages. The same
inflammatory cells mentioned above were present in fewer

numbers in the submucosa.

l7

 

Fig. 3. Stomach from a Group III (TGE
virus)pig on day 3. Hypermucous secretion (A),
desquamation and erosion (B) in the gastric
mucosal epithelium, with leukocyte infiltration
in the lamina pr0pria. H.& E. stain; x 101.

 

 

 

Fig. 4.
virus) pig on day 3.
border (A).

Duodenum from a Group III (TGE

Normal mucosa with brush
Slight congestion of blood vessels
(B), edema (C), and leukocyte infiltration (D) in
lamina propria. H. & E. stain; x 507.

18

Jejunum and Ileum. The most severe lesions were ob-
served at this level of the intestinal tract and the greatest
difference in the two groups of infected pigs were evident in
this tissue. The lumen contained desquamated epithelial
cells, mucus, erythrocytes and fibrin strands. There was a
marked reduction in height of the mucosal epithelium with
coagulation necrosis, desquamation, and erosion. Goblet
cells, although present in the crypts, were almost entirely
absent in the surface epithelium. The lamina propria was
edematous, had small hemorrhagic areas, and contained in-
creased neutrophils, lymphocytes, macrophages and plasma
cells along with several areas of necrosis. The same cells
mentioned above, although in fewer numbers, appeared in the
submucosa and tunica muscularis. Necrotic debris was ob—
served in the lumens of several glands. An outstanding and
constant finding was in the terminal portion of the villi,
immediately under the basement membrane, where large accumu-
1ations of fibrin were observed around the small blood
vessels (Fig. 5 and 6). Some of the blood vessels appeared
thrombosed. Numerous pyknotic nuclei were found in the
mucosal and glandular epithelium. Small foci of neutrophils
along with degenerating leukocytes and nuclear debris were
present in the reaction centers of the lymph nodules in
Peyer's patches (Fig. 7). A marked difference between the
infected groups (III and IV) became evident on the third day.
In pig 8 of Group IV, areas suggesting proliferation of

mucosal epithelial cells were observed along with an increase

l9

 

Fig. 5. Jejunum from a Group III (TGE
virus) pig on day 4. Lumen contains cellular
debris, leukocytes and fibrin (A). Marked re-
duction in height of epithelium (B), with fibrin
and leukocytes around a small blood vessel (C).
H. & E. stain; x 507.

 

Fig. 6. Jejunum from a Group III (TGE
virus) pig on day 4. Erythrocytes, fibrin and
leukocytes in the lumen (A), erosion and necrosis
of epithelium (B). Leukocyte infiltration in
lamina propria (C). H. & E. stain; x 101.

20

 

Fig. 7. Peyer's patch from an infected
pig on day 4 showing a focus of neutrophils and
nuclear debris (A), and degenerating leukocytes
(B). H. & E. stain; x 507.

21

in the number of goblet cells and an increase to almost
normal height of the intestinal epithelium. These changes
were also found on the fourth day in pigs 10 and 15 (Group
IV, infection and antibiotic) with slight improvement over
the third day (Fig. 8 and 9). Small areas resembling pro-
liferation were infrequently found in the surface mucosal

epithelium of Group III pigs.

Igglgn. During the first two days there was very
little alteration of the mucosal epithelium. On the last
two days of the experiment, changes similar to those in the
jejunum and ileum were observed but with less severity. The
lumen contained desquamated epithelial cells, necrotic
debris, leukocytes, erythrocytes, fibrin strands and mucus.
Epithelial mucosa was reduced in height, eroded and contained
areas of coagulation necrosis. Throughout the entire period
of infection there was moderate edema in the lamina propria
and infiltration with increased numbers of neutrophils,
lymphocytes, macrophages and occasionally plasma cells. All
blood vessels were moderately to severely congested. The
pathologic changes in the colon were, in most cases, patchy
in distribution.

Inclusion bodies were not seen in any part of the

gastrointestinal tract.

18

Jejunum and Ileum. The most severe lesions were ob-
served at this level of the intestinal tract and the greatest
difference in the two groups of infected pigs were evident in
this tissue. The lumen contained desquamated epithelial
cells, mucus, erythrocytes and fibrin strands. There was a
marked reduction in height of the mucosal epithelium with
coagulation necrosis, desquamation, and erosion. Goblet
cells, although present in the crypts, were almost entirely
absent in the surface epithelium. The lamina propria was
edematous, had small hemorrhagic areas, and contained in-
creased neutrophils, lymphocytes, macrophages and plasma
cells along with several areas of necrosis. The same cells
mentioned above, although in fewer numbers, appeared in the
submucosa and tunica muscularis. Necrotic debris was ob-
served in the lumens of several glands. An outstanding and
constant finding was in the terminal portion of the villi,
immediately under the basement membrane, where large accumu-
lations of fibrin were observed around the small blood
vessels (Fig. 5 and 6). Some of the blood vessels appeared
thrombosed. Numerous pyknotic nuclei were found in the
mucosal and glandular epithelium. Small foci of neutrOphils
along with degenerating leukocytes and nuclear debris were
present in the reaction centers of the lymph nodules in
Peyer's patches (Fig. 7). A marked difference between the
infected groups (III and IV) became evident on the third day.
In pig 8 of Group IV, areas suggesting proliferation of

mucosal epithelial cells were Observed along with an increase

l9

 

Fig. 5. Jejunum from a Group III (TGE
virus) pig on day 4. Lumen contains cellular
debris, leukocytes and fibrin (A). Marked re-
duction in height of epithelium (B), with fibrin
and leukocytes around a small blood vessel (C).
H. & E. stain; x 507.

 

Fig. 6. Jejunum from a Group III (TGE
virus) pig on day 4. Erythrocytes, fibrin and
leukocytes in the lumen (A), erosion and necrosis
of epithelium (B). Leukocyte infiltration in
lamina propria (C). H. & E. stain; x 101.

20

 

Fig. 7. Peyer's patch from an infected
pig on day 4 showing a focus of neutrophils and
nuclear debris (A), and degenerating leukocytes
(B). H. & E. stain; x 507.

21

in the number of goblet cells and an increase to almost

normal height of the intestinal epithelium. These changes
were also found on the fourth day in pigs 10 and 15 (Group
IV, infection and antibiotic) with slight improvement over
the third day (Fig. 8 and 9). Small areas resembling pro-
liferation were infrequently found in the surface mucosal

epithelium of Group III pigs.

Colon. During the first two days there was very
little alteration of the mucosal epithelium. On the last
two days of the experiment, changes similar to those in the
jejunum and ileum were observed but with less severity. The
lumen contained desquamated epithelial cells, necrotic
debris, leukocytes, erythrocytes, fibrin strands and mucus.
Epithelial mucosa was reduced in height, eroded and contained
areas of coagulation necrosis. Throughout the entire period
of infection there was moderate edema in the lamina propria
and infiltration with increased numbers of neutrophils,
lymphocytes, macrophages and occasionally plasma cells. All
blood vessels were moderately to severely congested. The
pathologic changes in the colon were, in most cases, patchy
in distribution.

Inclusion bodies were not seen in any part of the

gastrointestinal tract.

22

  
   

rig. -.' ‘ejunum from a Group IV (infec—
tion plus antibiotic) pig on day 4. Increase in
height of mucosal epithelium with increasing
numbers of goblet cells (A). Leukocyte infil-
tration into the lamina propria (B). Necrotic
debris in the lumen of some glands (C). H. & E.
stain; x 101.

 

Fig. 9. Jejunum from a Group IV (infected
plus antibiotic) pig on day 4 with an area of pro-
liferation of mucosal epithelial cells (A). H. &
E. stain; x 507.

23

Tissue Analysis

Chlortetracycline (CTC) Blood Values. Results from

this aspect of the experiment gave some interesting differ-

ences between Groups II (CTC, no TGE virus) and IV (CTC,

plus TGE virus) (Fig. 10 and Tables IV and V).

1.

Group II: Peak CTC blood values were reached between
the second and sixth hours after oral administration
of the antibiotic. A'build-up of CTC was evidenced
by higher peak levels each day. A marked drop in

CTC blood level from the 2—hour bleeding on one day
to the 0-hour bleeding of the following day is clearly
evident. Measurable levels of CTC were still present
in the blood 24 hours after oral administration. It
is of interest to note that the daily amount of CTC
in the blood did not correspond to an increasing
daily dose of CTC to individual pigs within the
group, as is illustrated in pigs 17 and 13 on days 3
and 4 respectively (Table VI).

Group IV: This group of pigs presented an entirely
different result. High peak CTC blood levels were
not reached initially. For the first 26 hours after
oral CTC administration the blood levels continued

to rise at a low level as compared to Group II. The
first decrease in CTC blood level occurred between
the 2- and 6-hour bleedings on the second day and re-
mained the same until the 0-hour bleeding on the

third day. On day 3, the CTC blood level increased

24

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25

progressively until it almost equalled that of the Group II
pigs at the 6—hour bleeding. By use of the Rank Sum Test
(Walker and Lev, 1953), there was no difference in CTC blood
levels between Groups II and IV on day 3. From the 6-hour
bleeding of day 3 to the 0-hour of day 4, the CTC blood level
in this group decreased more than it had between any previous
bleeding periods. It is difficult to understand why the CTC
blood level decreased between the 0— and 2-hour bleedings on
day 4; however, it did begin to increase between the 2- and
6-hour bleedings. Pig 18 was omitted from the determinations
(Fig. 10) because of having only one measurable CTC blood

level prior to euthanasia (Table V).

Hematology

To facilitate evaluation of the results, the hemo-
grams of noninfected pigs (Groups I and II) were averaged
together as were the hemograms of the infected pigs (Groups
III and IV) (Tables VII and VIII). A pronounced anemia oc-
curred during the experiment as indicated by the low Hb and
PCV values. Hemoglobin and PCV values were higher in the in-
fected pigs from about the second through the fourth days,
which can be attributed to the pigs' dehydrated condition.
Hemoglobin values have been previously reported which were
higher in pigs infected with TGE than in noninfected pigs
(Reber and Whitehair, 1955). Nucleated erythrocytes (NRBC)
remained essentially within the normal limits for pigs of

this age (Schalm, 1961). However, by the end of this

26

experiment the increase in NRBC was probably due to the
anemia. A leukopenia was evident in the infected pigs

(Groups III and IV) 24 hours after they were infected with
the TGE virus. This was supported by the Rank Sum Test
(Walker and Lev, 1953). Total and differential leukocyte
counts varied widely as can be expected for pigs of this age.
Immature leukocytes (lymphoblasts, prolymphocytes, myeloblasts
and metamyelocytes) were found in varying numbers in all pigs
throughout the experiment. Macrocytes were seen occasionally.
Polychromasia, hypochromia and numerous platelets were a
constant finding. As illustrated in Table VIII, the leuko-
penia in Groups III and IV actually persisted during the
first 3 days. A leukopenia was present in the noninfected
pigs on the second day. Of extreme interest is the progres-
sive neutropenia present on the second day and persisting

throughout the experiment in the noninfected pigs (Table VII).

DISCUSSION

General. The results of this experiment demonstrate
the inhibitory effects of a viral enteric infection on the
absorption of orally administered chlortetracycline. The
CTC blood values of infected pigs, although low initially,
did not decrease precipitously over a 24-hour period, as did
the CTC blood levels in the noninfected pigs. In the non-
infected pigs it is obvious that CTC was rapidly metabolized
and eliminated. The almost continuous rise in CTC blood
levels of infected pigs can be explained in part by the fact
that about 70% of CTC is bound to protein components of an
inflammatory exudate, making a CTC-protein complex, (Sirota
and Saltzman, 1950). Inflammatory hyperemia makes possible
increased penetration of CTC into the tissue and increased
resorption of CTC by the lymphatic system (Malek and Kolc,
1960). Some of the changes in the gastrointestinal tract
produced by the TGE virus (generalized hyperemia and edema
throughout the gastrointestinal tract) therefore would en-
hance CTC penetration into tissue and binding to protein
components of the inflammatory exudate.

The build-up of CTC in the blood of the Group II
(CTC, no TGE virus) pigs is similar to research done by

Brainerd §§.al. (1949). They found a build-up of CTC in the

27

28

serum of humans on a continuous oral dosage of l Gm. of CTC
every 4 to 6 hours. These same researchers stated that this
effect was noted as early as after the second dose of the drug.
but usually was more marked after several days.

In the past much work has been done to show what ef-
fects different levels of dietary calcium have on the absorp-
tion of CTC. It is also well known that calcium and the
tetracycline antibiotics can form a complex by the mechanism
known as chelation. Therefore one might assume that the
calcium in the milk diets of these pigs may have some alter-
ative effect on CTC absorption. In man it has been found
that concomitant administration of milk does not influence
the absorption of CTC as does aluminum hydroxide gel
(Bartholomew and Nichols, 1950). Therefore, since there is
a great similarity in the digestive processes in the porcine
and human species, it is not unreasonable to believe that
calcium would not influence CTC absorption in the pig. It
has been suggested that calcium absorption is involved in an
active transport system (Palmer and Thompson, 1961). De-
pending on the absorptive mechanisms by which calcium ab-
sorption is involved in this active transport, perhaps the
CTC calcium complex could also be actively absorbed from the
gastrointestinal tract. The above statement is merely an
assumption as it would depend on the chemical binding of
calcium in the active transport mechanism and in the CTC-
calcium binding. Certainly CTC must in part be involved in

some active transport system. Previously cited work has

29

clearly demonstrated that only a certain amount of CTC is ab-
sorbed at a given time. If this absorption was entirely
passive, then with all the exposed blood vessels from epi-
thelial erosion and necrosis, hyperemia and inflammatory
exudate which were present in this viral infection, it would
seem that the initial CTC blood levels in the infected pigs
should have been higher than they were. On the third day,
at which time the mucosal epithelium was first observed in
the recovery phase, the CTC blood levels were found to in-
crease markedly in the infected (Group IV) pigs, almost
equalling the CTC blood level in the noninfected (Group II)
pigs. In man it has been found that the brush border on the
free surface of the absorbing cells in the small intestine
increases the absorbing area of the intestinal cell about 14
times (Schiff, 1961). This should also, at least in part,
hold true for the pig. Thus some part of the gastrointestinal
epithelium must be involved with active absorption of CTC
and apparently this viral infection interfered with an active
transport mechanism.

The decreasing CTC blood level observed in the Group
IV pigs from the 6-hour bleeding of the third day to the 0-
hour bleeding of the fourth day could mean that less CTC was
bound to the protein of the inflammatory exudate. Therefore,
CTC was being metabolized and eliminated from the body faster
than it had been previous to this time. From this it could
be interpreted that less inflammatory exudate was present

due to the apparent recovery of the pigs. However, in the

30

histopathologic sections the edema was extensive in the

lamina propria throughout the experiment.

Clinical. The clinical signs of TGE were present
within 12 hours after administration of milk containing the
virus. This is perhaps on the early side of the usual incu-
bation period for this disease, but is within the range
commonly seen. The change in character of the feces in the
Group IV pigs was due to the CTC and perhaps can be explained
in part by work done by Reber (1955). He tested the effect
of CTC on isolated segments of normal porcine jejunum and
found it to have a varied effect upon the motility of smooth
muscle but, in general, decreased amplitude of contractions,
followed by inhibition of motility, was observed. With
enteritis, intestinal peristalsis is increased. It is
possible that, as the epithelium was recovering, the effects
of CTC absorption in some way also slowed peristalsis, there-
by not only allowing for more fluids to be absorbed, but also
allowing for the CTC to be in contact with the mucosal epi-
thelium longer, and thus further enhancing the recovery and
changing the character of the feces. Dehydration is usually
seen in pigs with this disease from the loss of body fluids
and was undoubtedly enhanced in this experiment by frequent
bleeding of the pigs. Because of essentially normal water
and food consumption by Group IV pigs, dehydration from loss
of body fluids at the end of the experiment was not as

severe as in Group III pigs.

31

From the results of this experiment, the clinical
improvement in the Group IV pigs was evident. As the intes-
tinal epithelium began to recover, this allowed better
absorption and assimilation of nutrients, thus strengthening
the pigs in their attempt to overcome the disease. Also the
presence of CTC undoubtedly inhibited the stress of secondary
bacterial infection. The weight gain in the Group III pigs
was totally unexpected and perhaps can best be explained on
the basis that these pigs were kept in a controlled environ-
ment. Had it not been for this type of environment, it is
doubtful that any weight gain would have occurred. The con-
trolled environment is undoubtedly the reason for no deaths
occurring in the infected pigs, especially those in Group
III. Perhaps the controlled environment was why no deaths
occurred in the Group IV pigs. However, with this group re-
ceiving CTC to control a secondary infection, their chances
for survival would be much greater. Even though the infected
pigs were not stressed from various environmental conditions,
they were markedly stressed from daily repeated bleedings.

The severe anemia present in all pigs by the end of
this experiment was primarily a result of the multiple daily
bleedings. Blood loss from the enteritis in the infected
pigs also influenced the anemia. A leukopenia was observed
especially during the first 24 hours after infection and
should aid in a differential diagnosis. However, under field
conditions and after the initial leukopenia, a secondary

bacterial infection may occur and make the hemogram more

32

difficult to interpret. Hemograms vary markedly as the age
changes in baby pigs and must therefore be interpreted care-
fully.

In the past CTC and several other chemotherapeutic
agents have been used in attempts to treat this disease and
have proved to be of little or no value. Based on the re-
sults obtained from this experiment, it appears that CTC has
certain beneficial effects in treating perhaps the secondary
effects of this viral enteric infection.

.lg.yiggg activity of CTC has shown that the majority
of both Gram-negative and Gram-positive bacteria are inhibited
by less than 1 microgram per m1. (l:1,000,000) (Kanegis.§t
.gl., 1950). The average CTC blood level in the infected
pigs was 0.47 mcg./ml. at the 6-hour bleeding on the first
day of treatment. A higher blood level would be preferred
such as the 1.2 mcg./ml. in the noninfected pigs at the 2-
hour bleeding on the first day of treatment. The need for
a higher CTC blood level is obvious, since the infected pigs
are experiencing a leukopenia and other stress factors due
to the TGE virus, the severity of which would be much greater
under field conditions. Chlortetracycline probably doesn't
have any direct effect on the TGE virus, but it might inhibit
a secondary bacterial infection which in most instances plays
an important role in the deaths of infected pigs in a
weakened condition. Chlortetracycline blood levels from the
treatment regimen followed in this experiment were not high

initially, but at least the intestinal epithelium may have

33

been helped in its attempt to recover. Better results may
have been observed if the dose of CTC used in this experiment
were increased or given more frequently, especially in field
outbreaks. It is not unreasonable to believe that death
losses in baby pigs from this disease could be reduced if
oral administration of this antibiotic could be started as
soon as the signs are manifested. Along with this, sound
management practices should be enforced to relieve other

stress factors.

Gross and Microscopic Lesions. Lesions observed at
necrOpsy generally paralleled those seen by other researchers
who have studied the effects of TGE in pigs. The gall
bladders were found to be distended with dark greenish-brown
bile in both groups of infected pigs. The livers in some of
the infected pigs, along with being pale, also had a slight
yellowish tinge. The big difference in this experiment was
the change in the character of the intestinal contents during
and after the third day. Perhaps this change occurred in
part because the intestinal epithelium was recovering from
the effects of the TGE virus under the influence of CTC as
was shown in Fig. 8 and 9.

Histopathologic changes were similar to those re—
ported by Bay.gt.§l. (1951). Only specific lesions observed
in the infected pigs will be discussed in detail. The most
severe lesions were in the jejunum and ileum. Coincidentally,

these areas of the intestinal tract also revealed the

34

outstanding differences between the untreated (Group III)
and treated (Group IV) pigs. In some areas of the gastro-
intestinal tract the TGE virus caused severe reduction in
height of the gastrointestinal epithelium and in other areas
caused total erosion. This reduced the surface area for
absorption and probably altered the enzymes which function
in transport of substances across the mucosal epithelium.

A common histopathologic observation was the ap-
pearance of fibrin, predominantly around the small blood
vessels, within the lamina propria. It was seen most fre-
quently around the smaller blood vessels immediately beneath
the basement membrane. The reaction closely resembles that
of a myoarteritis as described by HOppS (1961), followed by
a fibrinoid necrosis and later thrombosis of the affected
blood vessel. This same reaction is probably incorporated
in the necrdbiotic changes observed by Bay g§_al. (1951).
The periodic acid-Schiff (PAS) stain gave evidence of a
mucopolysaccharide derived from the ground substance in the
fibrinoid material. Some of the superficial epithelial
necrosis and erosion was due to thrombosis of blood vessels
within the lamina propria.

On the third day of the experiment, in the Group IV
pigs areas suggestive of proliferation, increase in height
of the surface mucosal epithelium and an increase in numbers
of goblet cells were significant indications that the epi-
thelium was in a recovery phase. These same findings were

present on the fourth day but were more advanced. In work

35

done by Abrams.§§_al. (1963), it was stated as a recognized
principle that one of the accompaniments of mucosal injury is
an abundance of desquamated cells associated with an exudate.
They also mentioned that the sharp increase in cellular pro-
liferation in areas of inflammation would be viewed as a
secondary adjustment essential to epithelial integrity.

The surface mucosal epithelial recovery, observed in
the pigs of Group IV, possibly were from the beneficial ef-
fects of CTC. Apparently the inhibition of secondary
bacterial invaders provided an environment in which the epi-

thelium could recover from the effects of the TGE virus.

SUMMARY AND CONCLUSIONS

Research was conducted using 18 pigs assigned to the
following groups: (1) control, (2) control plus chlortetra—
cycline, (3) transmissible gastroenteritis (TGE) virus-
infected, and (4) TGE virus—infected plus chlortetracycline.

Signs of TGE were present within 12 hours after infec-
tion. The disease was characterized by clinical signs,
leukopenia, and gross and microscopic lesions.

The virus of TGE had an inhibitory effect on absorp—
tion of orally administered chlortetracycline (CTC) on the
first two days of treatment. On the third day of treatment,
CTC blood levels of infected pigs approximated those of
control pigs.

Microscopic lesions in infected, CTC—treated pigs in-
cluded regeneration of intestinal epithelium on the third
and fourth days. Infrequently, small areas resembling pro-
liferation were observed in the intestinal epithelium during
and after the third day in infected pigs which did not re-
ceive CTC.

Absorption of CTC appears to be involved in an active
transport system.

Infected pigs given CTC orally appeared to be in
better clinical condition at the end of the fourth day as

compared to the untreated, infected pigs.

36

APPENDIX

TABLE I—-Identification of Pigs Assigned to

the Four Groups and Their Treatments

 

Group No. Pig No. Treatment
I 3,5,7,l6 Control — No CTC, no TGE
virus.
II 4,12,13,17 CTC, l mg./50 Gm. body

weight. One capsule(s)
at O-hour on 4 succes-
sive days), no TGE virus.

III l,2,9,ll,l4 No CTC, TGE virus only.
IV 6,8,10,15,18 CTC, l mg./50 Gm. body
weight. (One capsule(s)

at 0—hour* on 4 succes-
sive days), plus TGE virus.

*O-hour is 8 a.m.

37

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40

TABLE IV--Chlortetracycline Blood Levels of

Noninfected (Group II) Pigs

Blood Sample Time and Level (mcg./ml;)

 

 

 

 

Day Pig No. 0—Hour* 4;:Hour 6-Hour Terminalf*
l 4 neg. 0.65 0.65 0.58
12 neg. 1.13 0.83 ——-—
13 neg. 0.75 1.35 ——--
l7 neg. 2.25 1.35 -—-—
Average neg; 1:20 1.05 0.58
2 12 0.37 1.44 1.28 0.93
13 0.35 1.25 1.05 ----
17 0.35 0.98 0.57 —---
Average 0.36 1;;6_ 0.97 0.93
3 13 0.35 1.28 1.52 -—--
17 0.25 1.42 1.13 1.00
Average 0.30 1.35 1.33 1.00
4 13 0.51 1.45 1.45 1.52
Average 0.51 1.45 1.45 1.52

 

*Dosage was 1 mg./50 Gm. body weight on 4 successive

days after 0-hour sample.

**Not included on the graph (Fig. 10).

 

41

TABLE V--Chlortetracycline Blood Levels of

Infected (Group IV) Pigs

Blood Sample Time and Level (mcg.Zml;)*

 

 

 

 

Day Pig No. 0-Hour** 2—Hour 6-Hour Terminal+
l 6 neg. 0.41 0.39 0.48
8 neg. 0.23 0.34 ----
10 neg. 0.32 0.32 —-—-
15 neg. 0.51 0.83 ----
l8++ neg. neg. neg. —---
Average neg. neg430.37 neg.-0.47 0.48
2 8 0.27 0.52 0.76 ----
10 0.32 0.30 0.35 ----
15 1.50 1.40 0.96 ----
18++ neg. ---- 0.44 0.44
Average neg.-0.70 0.74 0.63 0.44
3 8 0.65 0.82 1.10 0.98
10 0.58 0.98 1.35 ----
15 0.84 1.55 1.50 ----
Average 0.69 1.1;_ 1.32 0.98
4 10 1.22 1.02 0.89 0.78
15 0.80 0.89 1.15 1.10
Average 1.01 0.96 1.02 0.94

 

* . . .
Negative responses not con31dered as 0 for computation.

**Dosage was 1 mg./50 Gm. body weight on 4 successive
days after 0-hour sample.

+Not included on the graph (Fig. 10).

++Confirmed by re-assay, dropped from experiment, thus
average at 6-hour (day 2) is 0.69.

42

 

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