NITRIYE. TOXECONS AND THE GASTRIC
ULCER COMPLEX EN SWINE

PART I. NITRETE TOXKOSIS
PART II. GASTRKC ULCER COMPLEX

Thesis for “he Degree oi: pl). D.
MICHIGAN STATE UNIVERSITY
Laumie W. NeEson
1965

 

mes-1c

   
 
  

LIBRARY

Michigan State
University

 

‘5 *v.-.‘ I -. - -- ~

This is to certify that the
thesis entitled

NITRITE TOXICOSIS AND THE GASTRIC ULCER

COMPLEX IN SWINE

presented by

Laurnie W. Nelson

has been accepted towards fulfillment
of the requirements for

Ph D degree mm

 

 

Major professor

Date—June A; 19 65

O~169

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IV‘.‘ '1
asalRALT

NITPITE TOXI SCSI N. THE GASTRIC ULCE?

PART I. NITRITE TCXICOSIS
Four erneriments were conducted using a total of #9 pigs to evaluate
and s*ndy nitrite toxicosis in swine. Thirty pigs in 2 different experi-
ments were given no to 500 1 .m. nitrite in their drinking water for
11 weeks. E0 major differences due to nitrite were observed in the

V

frowtn rate, dressir .g oercent -ées, organ weights. or white olooi cell,

hemoglobin or packed cell volume Values. In addition, serum and liver

vitamins A and E values were not i fluenced by ni rite. No nitrite

'D

r sidwes were found in the muscle tissue or vital organs. fighr

in
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‘

levels of nitrite occasionally caused listlessness, slight cyano
and letnargv in pi's shortly ait er eating and drinking. No lesions
wibutaole to prolonged nitrite ingestion were found on gross and
histocathologic exazinat ions. The incidence of gastric ulceration

‘.

one prolonged nitrite ingestion.

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Kineteen pigs in 1 different exueriments were used to study acute
nitrite toxicosis. The LDCQ of nitrite orally for Digs was calculated
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aq 30 to 3? mg. in. body weight. enemia L14 no elzect on tne L355.

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Laurnie J. Nelson
during the Q hours following nitrite dosing indicated a transitory
leukocytosis and an absolute neutrophilia, lymphocenia and eosinoocnia.
MethemOglobin and serum nitrite-nitrogen (NO:-N) values were not related
to different hemoglobin values in pigs. Decreased diastolic, systolic.
and pulse pressures and increased heart and respiratory rates were
noted after injection of nitrite into the stomach. Gross lesions in

acute toxicosis included brownish discoloration of the blood and tissues,

cyanosis, and hyperemia of the visceral and peripheral blood vessels.

PART II. GASTRIC ULCER COMPLEX
The eoizoolOgy of the gastric ulcer problem was studied using 1071
slaughter-age pigs fro om the Swine Evaluation Station, hichigan State
Univer51ty Swine Farm, and outside sources. The study indicated 373
(35.3%) had keratiniZation of the stomach, 115 (10.7%) had ulcers in the
eSOphageal region (hereinafter called esoohageal ulcers), and 33 (3.5a

had fundic ulcers. he 73 pigs from the Swine Evaluation station had

31)

the highest incidence of stow acn lesions t slaughter. Although all the

giirs were clinical lv normal, 39 of llfi with eSOphageal ulcers had free

*U

and clotted blood in their stomachs. The pigs subjected to the stresses

1

associ ted with growth exneriments and environm.-.t a1 and management

‘)

changes had the highest incidence of stomach lesions. Keratinization 01

the esophageal region was characterized by a parakeratotic proliferation

‘

o: the squar ous eo1tr eliun. This appeared to or cede necrosis and ulcer

formation. The esoxhareal ulcers and fundic ulcers appeared as separate
and unrelated entities.

No differences were noted in the ino“dence of stomacn lesions in

9? pigs given rations varying in vitamin A and carotene content. ne

Laurnie J. Nelson
research on stomach pH Values indicated that the average pH value was
2.5 in the normal stomachs, 3.8 in those with keratinization, and 6.2
in those with eSOphageal ulcers. Thus, low acidity, rather than hyper-

acidity, appeared to be related to the stomach lesions.

(3

andid

9.)

albicans was frequently cultured from stomach lesions. but

 

this organism was considered a secondary factor because ulcers could
not be experimentally reproduced. Using 12 pigs, it was established
hat g, albicans was capable of producing lesions following intravenous,
subarachnoidal, and oral routes of administration.
Twenty-four pigs were used in the study of prednisolone-induced
ulcers. Daily injections of prednisolone caused a high incidence of
stomach ulcers, increased gluconeoaenesis, inhibited inflamnatory

responses, and caused a leukOpenia with an absolute lymphopenia.

NITRITE TOXICOSIS AND THE GASTRIC ULCER

COMPLEX IN SXINE

PART I. NI'I'RITE TOXICOSIS

PART II. GASTRIC ULCER COMPLEX

By

laurnie W. Nelson

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Pathology

1965

6.,”
\59‘

ACKNONLEDGERLNTS

The author is grateful to Dr. C. K. Whitehair of the Department of
PatholOgy for his assistance and guidance during the planning and con-
ducting of this research project and writing of the thesis. Appreciation
is also expressed to Dre. C. C. Mo orrill, R. F. Langham, and G. L. daxler.
of the Department of Pathology. Dr. G. H. Conner, of the Department of
Surgery and Medicine, and Professor L. J. Bratzler, of the Department of
Food Science. for their aid and constructiVe criticisms.

Sincere thanks are also given to other members of the staff in the
Department of Pathology who helped in many ways in conducting this
research and to Miss Janice Fuller for typing the thesis.

The author is thankful to the Meat Inspection Division. Consumer
and Marketing Service, United States Department of Agriculture, for the
Opportunity to participate in their cooperative graduate prOgram with
Michigan St ate University. The use of a variety of facilities at Iiichigan
St ate University to eXpedite this research is acknowledged.

The author is most sincerely thankful and indebted to his wife
Margaret and children for their patience, understanding, and encourage-

ment throughout the research.

ii

TABLE OF CONTENTS

PART I. NITRITE TOXICOSIS

INTRODUCTION. . . . . . .

REVIEW OF LITERATURE . . . . . .
Properties of Nitrite . . .
Methemoglobinemia . . . . .
Nitrite Toxicosis in Swine .

Nitrite Toxicosis in Rats .

Nitrite Toxicosis in Other Species . . .

Relation of Nitrites to Public Health . .

OBJEC TIVE 3 o o o o o o o o o o 0
MATERIALS AN METHODS . . .

Experimental Animals . . . .

Facilities and General Information . . .

Analytical Procedures . . .
Specific Procedures for Each
RESULTS . . . . . . . . . . . .
Experiment I . . . . . . . .
Experiment II . . . . . . .
EXperiment III . . . . . . .
Experiment IV . . . . . . .
DISCUSSION . . . . . . . . . . .
SUMMARY . . . . . . . . . . . .

LIST OF REFERENCES . , . . . . .

Experiment

iii

Page

PART II. GASTRIC ULCER COMPLEX

INTRODUCTION. . . . . . . . . . . . . . . . .
REVIEW OF LITERATURE . . . . . . . . . . . .
Gastric Ulcers in Swine . . . . . . . .
Candidiasis and Ulcers . . . . . . . . .
Prednisolone-Induced Ulcers in Rats . .
OBJECTIVES. . . . . . . . . . . . . . . . . .
MATERIALS AND METHODS . . . . . . . . . . . .
Experimental Animals . . . . . . . . . .
Facilities and General Information . . .
Analytical Procedures . . . . . . . . .
Myoologic Examinations . . z . . . . . .
Specific Procedures for Each Experiment
RESULTS . . .-. . . . . . . . . . . . . . . .
Experiment I (Nature of Ulcer Problem) .
Experiment II (Candidiasis) . . . . . .
EXperiment III (Prednisolone) . . . . .
DISCUSSION. . . . . . . . . . . . . . . . . .
SUMMARY . . . . . . . . . . . . . . . . . . .

LIST OF REFERENCES . . . . . . . . . . . . .

iv

100
106
110

112

Table Page
l-l EXper im ent I. AVerage growth rate of pigs given

selected levels of nitrite in the drinking water. . . . . 25
1-2 EXperime nt I. AVe erage daily feed, water, and nitrite

consume tion for pi3s g iven nitrite in the drinking water

ill {group 5 Of eaC.l trial. 0 o o o o o o o o o o o o o o e 24)
1-? Experiment I. Slaughter data (averages) collected from

pigs given selected levels of nitrite in the drinh ing

“Va-tel“ o o o o o o o o o o e e o o e o o o o e o o o o o o 29
1-” Experiment I. Average vitamin.A and vitamin E values

of liver (meg. 3n.) ind serum (mcg./lOO ml.) from pigs

given sele te ed levels of nitrite in the drinking water. . 29
1-5 Eiuecirent I. Modified hemcxram of swi e given selected

levels of nitrite in the drinking water . . . . . . . . . 30
l-€ EXperiment II. Modified hemogram from representative

swineg n'en selected levels of nitr rite by stomach tube. . 32
l—7 Experiient II. Characteristic signs of pigs with acute

nitrite toxicosis. Pig 1 given 30 mg. of nitrite/lb.

b02131" l‘IpiSht bl}? StomaCh tllbe o o o e o o o o o o o o o o 0 3h
1-: Exierinent III. Average growth rate and organ weights

of iron -treated (Group 1) and nontreated (Group 2) pigs . 39
1-? Experiment III. Summary of modified hemOgrams (an. )

f ‘ 'ron-treated (Group 1) and nontreated (Groua 2W) igs

g1Ven selected levels of nitrite by stor ach tube. . . . . CO
-l$ Experiment IV. Plood pressure Values and reast and

respiratory rates of an anesthetized pig gi"en BU mg.

nitrite/1h. body weight by injection into tne stomach . . 0:
l—ll n~‘ner* I" Ts., "’“.. 2nd Sfi"1fl 10 -§ values

a“;; 'f . ‘ “ T r loca+ivrs from ares thetiZei pigs

5

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‘I '. -1. - ' -‘w - - ' 4-
iL. cod: Mela t by ingection into

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PART II. GASTRIC ULCER COMPLEX

Table
2-1 Manthly incidence of stomach lesions in slaughtered pigs .
2-2 Incidence of stomach lesions in slaughtered pigs from
various origins. O O C O O O O O O O O O O I O O O O 0 O 0
2-3 Incidence of stomach lesions in pigs on basal rations low
in vitamin A with various leVels of carotene cu~ vitamin
A supplemented . . . . . . . . . . . . . . . . . . . . . .
2-3 Average pH Values of stomachs from (A) piss fed rations
varying in carotene and vitamin A. and (B) pigs fed
Standard rations o o o o o e o o o o e o o o e e o o o o o
2-5 Incidence of liver condemnations in slaughtered pigs from
Various Orlglfl"; o o o o o 9 o o o o o o c o o o o e o o o
2-6 Monthly incidence of liver condemnations from slaughtered
pigs 0 O O O O O O O I O O O O O O O O l O I O O O 0 C O O
2—? EXperiment III. .ial 2. Average live weights, organ
weights. and blood values of pigs given prednisolone
intramuscularly . . . . . . . . . . . . . . . . . . . . .
2—3 Exceriment III. Trial 2. Incidence of stomach lesions

in pigs given preinisclone intramuscularly . . . . . . . .

Figure

1-1

1-4

1-5

1-6

1-8

1-9

1-10

1-11

LIST OF FIGURES

PART I. NITRITE TOXICOSIS

A pig given 200 p.p.m. nitrite in the drinking water
for 10 weeks. 0 O O O O C O O O O O O O O O O O ‘ O O O O

Carcasses of pigs given nitrite in the drinking water
for 10 weeks. Dosage levels from left to right:
200, 150, 100, 50, and 0 p.p.m. . . . . . . . . . . . . .

Stomach of Pig A, given 30 mg. nitrite/lb. body weight
by stomach tube for h consecutive days. Note hemor-
rhages and ulceration of the fundic region . . . . . . .

Liver, heart, and lungs of Pig h, which died after

being given 30 mg. nitrite/lb. body weight by stomach
tube for h consecutive days. Note the brownish color
of blood and lungs. . . . . . . . . . . . . . . . . .

Section from lung in Fig. 1-“. Note alveolar edema,
emphysema, and the congestion of the blood vessels.
H & E Stain; x 75 o o e o o o o o e o o o o o o o o o o 0

Section from liver in Fig. 1-4. Note the perilobular
infiltration of eosinophils and the fibroblastic and
bile duct proliferation. H & E stain; x 75 . . . . . .

Eosinophilic infiltration of fundic region of stomach
shown in Fig. 1-3. H & E stain; x l87. . . . . . . . . .

Higher power of Fig. 1-7. Note the eosinOphils and
the edema of the fundic region. H & E stain; x 750 . . .

Experiment III. Signs of pigs given 32 mg. nitrite/lb.
body weight by stomach tube. (A) normal, (3) ataxia,
(C) weakness and ataxia, (D) unable to rise, (3) coma-
tose, and (F) gasping shortly before death. . . . .

Iron-treated and nontreated pigs. Note the paleness of
nose and ears of the nontreated pig on the left . . . .

Viscera of iron-treated and nontreated pigs shown in

Fig. 1-10. Note the paleness of the visceral organs and
blood of the nontreated pig on the left . . . . . . . . .

vii

28

35

36

36

37

Figure Page

1-12 Thoracic and neck area of 2 iron-treated pigs. The pig
on the right died after given 30 mg. nitrite/lb. body
weight by stomach tube. Note the brownish~colored
blood and blanched heart and lungs in the pig on the
right, in contrast to the control pig on the left . . . . 45

1-13 Visceral and neck region of 2 nontreated pigs. The pig
on the right died after given 32 mg. nitrite/lb. body
weight by stomach tube. Note the brownish watery blood
and the blanched lungs in the pig on the right, in
contrast to the control pig on the left . . . . . . . . . 45

FKRT II. GAgTPIC ULCVR COXPLEX

2-l A stomach of a pig with a keratinized esophageal region
and adhering mucus in the fundic region . . . . . . . . . 84
2-2 Photomicrograph of Fig. 2-1. Parakeratotic prolifera-

tion of the squamous epithelium of the esophageal region.
't-i & E Stain; x 75 O O O O O I O O D O O l O O O D O O I O 84

2-3 Parakeratotic proliferation of squamous epithelium on the
esophageal region of the stomach. Note the plaque forma-
£1011. H & P: Stain; X 75. o o I o o o n o o o o o o u o o 85

2-4 The stomach of a pig with a keratinized esophageal region.
At the squamous-glandular junction, small ulcers are
present a o o 0 I o o O o c o o v o o o I o o o I a o a o 85

2-5 ESOphageal region of a iig's stomach with ulcers and
attached blood clots. . . . . . . . . . .~. . . . . . . . 85

2-6 The stomach of a pig with a fibrinonecrotic lesion in
the esophageal region and adhering mucus in the fundic
region. . . . . . . . . . . . . . . . . . . . . . . . . . 86

2-7 Submucosa of esophageal region of stomach shown in Fig.
2-4. Note the neutrophils and lymphocytes. H & E
stain; x 187. O O O O O I 0 O O O D I O I Q 0 C C 0 D O U 88

2-8 Higher power of Fig. 2-7. Note the numerous neutrophils
and proliferating fibroblasts. H & E stain; x 750. . . . 88

2-9‘ , Stomach of a pig with 2 ulcers in the fundic region . . . 89

2-lO Photomicrograph of Fig. 2-9. Ulcerative gastritis of
the fundic region of stomach. Note coagulation necrosis
of the mucosa with infiltrations of inflammatory cells.
H & E stain; x 75 . . . . . . . . . . . . . . . . . . . . 8Q

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Porcine liver, weighing 2?~0 Gm. Note the yellowish
nodules and the rounded edges . . . . . . . . . . . . .

PhotomicrOgraph of Fig. 2-11. Subacute perilobular
eos inoo.ilic hepatitis in e si; liVer. H d E stain;

x 1%] . . . . . . . . . . . . . . . . . . . . . . . . .

\

Liver of a pig :Jh ch aad nun erous large ascarids in the
small intestine. Note the numerous whitish foci
(ascarid scars?) on the surface . . . . . . . . . . . .

A disseminating type lesion in a grossly typical ascarid-
scarred liver. Note the necrosis and the infiltration
of numerous inflammatory cells. H & E stain; x 137 , .

Yellow keratinized and eroded eSOphageal region of a
stomach of a pig. g, albicans was isolated from this

leSiOn. O O O 0 O I C I O O O O O O I O O O O O O O O O

. aloicans in the mucosal surface of a pig stomach.
omori' 5 silver methenamine stain; x 107. . . . . . . .

 

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Typical ch lamydospores of Q. albic ans grown on cornmeal
agar. Lactophenol preparation; x 750 . . . . . . . . .

Granuloma in the lung of a pig given Q. albicans intra-
venously. H & E stain; x 137 . . . . . . . . . . . . .

Granuloma with necrosis in the liver of a pig given Q.

Q

albicans intravenously. H i E stain; x 137 . . . . . .

Hi: er po er of 1g. 2-19. Note the neutrophils, lympho-
cytes and macr .a es. H a E stain; x 750. . . . . . .

u. alhicans in th granuloma 51
silver methenamine stain; x 75

 

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g. albicans in the meninges of a pig giv n the organism
in the subarachnoid Space. Gomori's silver methenamine
Stair)»; x 1750. D O O O O O O O O C O O O I O O O O O O 0

Stomachs from :redn1sclone-treated and control pi
Note the hemorrhage in the eSOpzageal region of t
stomach of the predn1501one-.reated pig on the le

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. Note the clot adhering to the

Closeu: 13. j
esit n near the junction of the esophageal
reg i

of
ulcerative
and cardia

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own in Fig. 2-19. Gomori's

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Figure

2-25

2-26

Page

Stomachs from prednisolone-treated and control pigs.
Hemorrhages and ulcerations are present in the fundic

region of the stomach of the prednisolone-treated pig

on the left . . . . . . . . . . . . . . . . . . . . . . . 102

Closeup of Fig. 2-25. Hemorrhages and ulcerations on
the fundic region of the stomach of prednisolone-treated
pig 0 C O O O O O O O ' 0 O O O O O O 0 O O C O O O O O O 102

PART 1. NITRITE'TOXICOSIS
INTRODUCTION

For many years, nitrite toxicosis was characterized by an acute
anoxia. More recently. reports have associated nitrites and nitrates
with a more chronic syndrome. The relationship of nitrites and nitrates
with a vitamin A interference syndrome led to a postulated etiology for
the increased incidence of the gastric ulcer complex in swine.

Numerous reports during the past. 10 years have indicated an
increased nitrite level in water and plant food supplies. Relatively
low levels were reported to cause nutritional interference syndromes.
This raised questions as to the safety of nitrite as an additive in
cured meat products and to the possibility of a nitrite residue in fresh
meat foods.

The primary purpose of this research was to produce acute and
chronic nitrite toxicoses in swine and to investigate a possible rela-
tionship to gastric ulcers. Attention was also focused on the clinical.
gross, and histOpathologic aspects of acute nitrite toxicosis. This
information bould be of value in verifying the LDEO' in determining the
hematolOgic response to a toxic chemical agent. in correlating methemo-
globin and serum nitrite levels, and in elucidating the metabolism of

nitrite.

REYESJ OF HTERA’I‘URE
Properties of Nitrite

The Pharmac0peia of the United States (1960) describes potassium
nitrite (KNOZ) as a whitish granular compound with a molecular weight
of 85.11. It is readily soluble in water and forms a slightly alkaline.
highly ionized solution. The American Meat Institute Foundation (1960)
characterized the nitrite ion as a highly reactive ion which can serve
as both a reducing and an oxidizing agent. Although it can be oxidized
to nitrate by powerful oxidizing agents such as permanganate, it usually
acts as an oxidizing agent as in the oxidation of hemoglobin to methemo-
globin. The curing reaction in meats is described as myoglobin plus
nitrite to form nitrosomyoglobin. Although tissue respiration has ceased
in the exsanguinated animal. fermentation and respiratory enzymes continue
to be active. During this period. glycogen is broken down, releasing
lactic acid. In this acidic environment an equilibrium is established
between nitrous acid and nitric oxide. Nitric oxide (NO) forms complexes
with hemOglobin, myoglobin and several of the porphyrin-containing
enzymes by attaching to the iron in the heme portion of the molecule.
Since myoglobin is the most prevalent pigmented compound in meat. some
of the nitric oxide combines to form nitrosomyoglobin. This red compound
is relatively unstable and therefore must be heated to form nitrosohemo-
chrome, which gives the characteristic pink color to cured meat.

In contrast to the above chemical reaction in meat, Nason (1962)
discussed how the nitrite ion is reduced by microorganisms into different
intermediate and end products. From the +3 oxidative state of nitrite

2

3
to the -3 oxidative state of ammonia. the nitrogen atom passes through
the following forms: +2 nitric oxide (NO); +1 nitrous acid (N20),
nitroxyl (HNO), hyponitrous acid (HZNZO) and nitramide (NOZ'NHZ);
o nitrogen (N2); -1 hydroxlamine (NHZOH) and -2 (NHZNHZ).

Winter (1962) studied nitrite metabolism in cattle. Although nitrite
was administered by drenching, both hydroxylamine and nitrite were
demonstrated in the blood serum. Hydroxylamine was formed from the
nitrite and was capable of converting hemoglobin to methemoglobin.

Blood ammonia values were not influenced by administration of either

nitrate or nitrite.

Pharmacodynamics

Beckman (1958) discussed the use of sodium nitrite in the treatment
of heart conditions in man. The breakdown of nitrite in the acid medium
of the stomach releases nitrous acid, which is irritating to the mucosa.

It was assumed. but not proven. that the nitrite was largely decomposed
in the gastrointestinal tract. About 70% of the absorbed nitrite
disappears in the body, probably through conversion to ammonia. The
remainder is excreted in the urine as nitrites and nitrates.

Nitrite relaxes the smooth muscles of all blood vessels. independent
of their innervation. Due to this relaxation, the most striking pharmaco-
dynamic effect is a fall in blood pressure. Because of the diminished
venous return to the heart, the stroke volume decreases but a compensa-
tory increase in heart rate preserves cardiac output. Toxicosis in man
is characterized by a well maintained diastolic pressure and a low systolic
pressure, which results in a small pulse pressure. This diastolic pressure

indicates that "nitrite shock" is not due to the pooling of blood in the

a
postarteriolar vascular bed but is due to the venous pooling in the lower

extremities and diminished venous return to the heart.

Serum Nitrite Determination

Diven gt gl. (1962b) suggested that total nitrate blood values were
a better indication of the amount of nitrate consumed by sheep than
methemoglobin values. Their research indicated that the nitrate blood
values were highest about 2 hours prior to the outward signs of intoxi-
cation. They (l962a) also devised a rapid method for the quantitative
estimation of serum nitrite- and serum nitrate-nitrOgen. These methods
were described for ruminant blood. and they indicated that a high degree

of accuracy for nitrite analysis was possible.

MethemOglobinemia
Finch (l9h8) characterized methemOglobin (HbFe+++OH) as a derivative

of hemoglobin in which the ferrous porphyrin complex was converted to
the ferric form. Since oxygen cannot combine with the ferric form,
methemoglobin is of no value in reSpiration. In the intact red blood
cell, methemoglobin is formed continuously, but it is also promptly
reduced to the functional ferrous form through action of reducing agents
such as ascorbic acid and glutathione which are normally found in the
blood.

Huennekens §t_§l. (1957) reported that the effectiveness of ascorbic
acid and glutathione in relieving methemoglobinemia was independent of
methemoglobin reductase and depended solely on the fact that these were
reducing agents. However, methemoglobin reduction was accomplished by

another methemOglobin reductase which depended on triphosphopyridine

5

nucleotide. generated by way of the "hexose monOphosphate shunt". This
enzyme required for its activity an added electron carrier for the normal
reduction of methemoglobin. Substitution of this electron carrier by
methylene blue explains the action of this dye in the treatment of

methemOglobinemia.

Nitrite Toxicosis in Swine
Sources of Nitrites

Ehgx. Wannunrp and Swahn (1953) recognized nitrite toxicosis in swine
fed byproducts of the cheese-making industry. Sodium nitrate was added
to milk in a cheese-making process to prevent certain bacterial fermen-
tations. When nitrate was reduced to nitrite, it inhibited the growth
of some bacteria. They reported the LD50 of sodium nitrite for swine as
below 90 mg./kg. of body weight.

Stormcrken(l953) noted that swine were more sensitive to sodium
nitrite than cattle and sheep. He postulated the minimum lethal dose

for swine to be 70 to 75 mg./kg. of body weight.

Cooked mangels and mangolds. Robinson (l9h2), in New Zealand. reported
the death of 200 pigs of all ages in a herd of 600 which were fed freshly
pulled mangels that were cooked about 2 hours the previous evening. I
The pigs died % to 2 hours after the morning feeding. The post—mortem
finding of methemoglobinemia was typical of nitrite poisoning. He also
indicated that mangel poisoning in cattle was associated with the forma-
tion of nitrite from nitrate.

Also in New Zealand, McIntosh. lielson and Robinson (l9h3) experi-

mentally produced nitrite poisoning in pigs by feeding boiled mangolds:

6

l to 3 Gm. NaNO in mangolds per day to pigs of about 20 kg. in weight

3

produced signs of poisoning, but they recovered.

gzigg. In Canada. Gwatkin and Plummer (l9u6), concerned over salt poisoning
in swine. found that pickling brine that contained potassium nitrate and
nitrite was more toxic than other potassium salts. Under certain con-
ditions the nitrate was reduced to nitrite by bacterial action and 500

to 800 p.p.m. of nitritefihflx demonstrated in some pickled products. Nhile
oral doses of 30 to 90 Gm. KNO

or 5 Gm. KNO were lethal for small pigs

3 2
(20 to #5 pounds), these dosages caused only a transient methemoglobinemia
in pigs weighing 70 pounds. The only post-mortem finding was methemo-

globinemia.

herring. Hvidsten (1955) reported the use of sodium nitrite as a method of
preserving factory herring. since herring were used as a protein supplement
in swine rations. Feeding 200 Gm. per animal per day of a herring meal
which contained up to 2% sodium nitrite had no influence on methemoglobin
values in the blood, feed efficiency, or quality of the pork. When the
herring meal contained hfi nitrite. and cod liver oil was mixed into the
feed, no vitamin A was deposited in the liver. He postulated that the

vitamin A was destroyed by contact with cod liver oil and nitrite.

Plant sources. Laing and McIntosh (l9h7), in New Zealand, indicated that
sudden deaths in cattle and pigs were recorded occasionally during the pre-
vious 12 years. The signs included staggering. rapid breathing, convulsions,

and death within an hour. hey also indicated that animals could eat sublethal

7
amounts of nitrites daily without suffering harmful effects. No resis-
tance was built up by the animals: the same dose would kill an animal
that had been on a ration containing nitrite or an animal on a nitrite-
free ration. Besides methemoglobinemia, they' described a dilatation of
the blood vessels throughout the body and an increased heart rate. Death
was attributed to suffocation.

Case (l95h) reported abortion in a drove of sows pastured on rape
and oat pasture. Although some sows aborted 2 weeks before full term,
others at full term had weak or dead pigs. The nitrate content of the
rape was 5.52% and of the cats 0.53%.

Smith, Lovell, Reppert, and Griswold (1959) were among the first to
report nitrate poisoning in pigs in the,veterinary literature. The most
prominent signs were labored breathing, loss of appetite, polydypsia,
and convulsions. Losses were attributed to the pigs' eating damp straw,
since when the straw was removed death losses stopped. Toxicity of the
straw was explained on the basis of the reduction of nitrate to nitrite
in the wet straw. The lesions found at necrOpsy included acute gastritis,
edema of the lungs, conjunctivitis, and purplish discoloration of the
abdomen.

Wunder and Schultz(l960) described nitrate poisoning in pigs caused

from eating lambsquarter (”hena odiun album) and redroot pigweed

 

(Amaranthus retzgflexus). The signs were rapid breathing and watery
diarrhea. Necropsy revealed dark-colored blood, perirenal edema and

subcapsular ecchymotic hemorrhages in the kidneys.

8

flgtgg. Winks, Sutherland and Salisbury (1950), in Queensland, reported
heavy losses on 2 farms where pigs were fed soup which was prepared by
cooking beef and offal in well water. Since the nitrate content of the
well watervwnn32970 and l7hO p.p.m., the mortality of the pigs was thought
to be due to nitrite poisoning. These workers ascertained that 40 to
80 mg. NaNO2 orally per kg. of body weight caused increased methemOglobin
values and that 90 mg./kg. of body weight killed the pigs within 2 hours.

Bjornson gt gl. (1961) described outbreaks and sources of nitrate
intoxication in cattle and swine in North Dakota. From July, 1959. to
July, 1960, #7 cases of nitrate poisoning were diagnosed by the North
Dakota State University workers. On one farm where swine losses occurred,
the water from 4 wells contained #80, 510, 200 and 205 p.p.m. KNO3
equivalents. Methemoglobinemia was a constant finding at necrOpsy of
the animals. These authors also indicated that nitrates in the diet
depleted the amount of vitamins A, D, and E.

Case (1963), in Missouri, reported serious losses of pigs on many
farms. One outbreak involved pigs in an SPF program in which the
extreme susceptibility of young animals to nitrite toxicosis was observed.
Although nitrite toxicosis can be fatal, Case described a nutritional
interference syndrome due to the nitrites which resulted in poor weight
gains, diarrhea, and generally unthrifty animals. He also emphasized
the importance of having water supplies analyzed for nitrates. For
instance, on one farm the well contained 250 p.p.m., and the pond 30

p.p.m., KNO equivalents, whereas the drainage ditch contained little or

3

no nitrates. On the basis of his clinical observations in swine, he

considered water with 30 to 50 p.p.m. nitrate-nitrogen (NOB-N) as risky

9
and water with 50 to 200 p.p.m. NOB-N and over as possibly causing heavy
acute death losses. He also reported that nitrites were at least 10

times as toxic as nitrates.

Contaminated concentratgg. Recently, Bush and Matteson (1963) described
what they believed to be nitrate toxicosis in cattle and swine in Indiana.
In swine the average mortality was 10 to 15%, but in some droves reached
90%. Chronicity with this problem resulted in unthriftiness and sporadic
deaths. Although methemoglobinemia was not found, the necropsy findings
included gastroenteritis, gastric and duodenal ulcers with free blood

in the lumen, varicolored livers with areas of necrosis, ascites, subcu-
taneous edema, and pulmonary emphysema. These authors suggested that
dicalcium phosphate with 20,000 p.p.m. nitrate and soybean meal with as
.igh as 73,503 p.p.m. nitrate were the chief sources. RemoVal of the
animals from rations high in plant-origin protein and replacement with

rations high in animal-origin protein stopped the losses on most farms.

Nitrite Toxicosis in flats
PatholOgy

 

Hueper and Landsberg (l9h0) described pathologic changes in the
organs of rats produced by chronic nitrite poisoning. One of their
experiments was devoted to sodium nitrite with 0.5 Gm. of NaNO2 being
added daily to a meat ration for each rat. This experiment began with
6 rats, 2 of which died during the first 2 weeks and 2 more of which
were killed after u weeks. The other 2 rats were maintained on this
diet for 18 weeks. Clinical signs included cyanosis, listlessness, and

a marked loss of weight. The necrOpsy findings included brownish

10
discoloration of the blood and lungs, a soft dark liver, dark red kidneys
and coffee-ground-colored mucosal erosions in the stomach. Histopatho-
logic examination revealed degenerative vascular and parenchymatous
lesions in the heart, liver, brain, kidney and testes. As an explana-
tion for the develOpment of these lesions, they concluded that nitrite
caused vasodilatation which in turn caused a slowing of the blood flow

and a stagnant hypoxemia.

Interference with Vitamin A Nutrition

Garner £2 31. (1958) reported a depletion of vitamin A in rats and
swine fed 2% KNO3 in the ration. O'Dell, Garner and Muhrer (1960), while
studying the effects of nitrate in the rations of rats, noted a rapid
depletion of vitamin A and the precipitation of a vitamin E deficiency.

Smith (1961) found that nitrite toxicosis was not restricted to
methemoglobinemia but involved other oxidative processes. Since vitamin
A and carotene are susceptible to oxidative destruction, it was conceivable
that nitrite oxidized and destroyed vitamin A in the tissues. Smith
postulated that in chronic cases of nitrite toxicosis protein enzymes
necessary for conversion of carotene to vitamin A were altered and this
resulted in an impaired vitamin A metabolism.

In rats, Emerick and Olson (1962) found that feeding nitrite but
not nitrate significantly lowered liver storage of vitamin A from orally
administered sources of preformed vitamin A, but not from injected sources.
Nitrite in the ration also lowered vitamin A storage in the liver when

carotene was fed.

ll

fiitrate Toxicosis in Other Species

 

Acute Toxicosis

Saussol (1836), one of the first to record nitrate toxicosis,
described poisoning of lambs by the nitrate of potash. The lambs licked
the rocks and obtained sufficient amounts to cause death.

Mayo (1895) reported observations of cattle that had consumed
cornstalks high in nitrate or were given large amounts of nitrate salts
in a drench. This author described the darkened blood, which was due to
methemoglobin, as an indication of nitrite poisoning.

Newsom g3 11. (1937) incriminated oat hay as the source of nitrates.
Thorp (1938) and Bradley, Eppson and Beath (1940), other earlier workers
on oat hay poisoning in ruminants, recommended methylene blue as an
effective antidote. Besides cornstalks and oat hay they incriminated
wheat, barley, and various weeds as containing sufficient nitrate to be
poisonous.

In Canada, Davidson, Doughty and Bolton (1941) described signs in
affected cattle as an accelerated pulse, quick breathing, trembling,
staggering, and apparent blindness. The animals would cease to eat and
would lie in a sternal position. Cyanosis of the tongue and sclera was
followed by death with little or no struggling. Since sublethal quan-
tities were consumed daily for a long period of time without producing

toxic effects, these workers reported that nitrates had little or no

cumulative effect.

12

Nutritional Interference Syndrome

Case (1957) discussed nitrate intoxication in livestock in Missouri.
Because nitrite was considered 10 to 15 times more toxic than nitrate,
animals drinking from farm ponds containing over 5 p.p.m. nitrite often
suffered from nitrite intoxication. He also pointed out the dangers of
nitrate accumulation by plants during droughts and heavy nitrogen fer-
tilization. Although the cow herds were well cared for, abortion and
stillborn calves were a serious problem. Blood analyses of these calves
revealed methemoglobinemia. Vitamin A deficiency was evident in the dams
and the hay contained 0.7% nitrate, which exceeded the safe limits.

Sippel (1960) found that a constant level of 5 p.p.m. or above of
nitrite in the water could interfere with vitamin A metabolism and result
in abortion, sterility, poor performance and other ills probably due to
a vitamin A deficiency. He also indicated that many of the affected
animals would never fully recover.

McIlwain and Schipper (1963) reported that nitrites and nitrates
inhibited the conversion of carotene to vitamin A in calves. One eXpla-
nation incriminated the nitrite ion as causing irritation to the mucous
membranes of the gastrointestinal tract and in this manner acting as a

blocking agent to carotene conversion.

Relation of Nitritesgto Public Health

 

Used as a Meat Additive
Histozy. Kerr gt_al. (1926) studied the use of nitrite in curing meats.
Although nitrates were used for many years, these authors found that a

small amount of nitrite produced the thermally stable meat pigment

13

characteristic of cured meats. In their extensive analytical studies,
nitrite levels were higher in the nitrate-treated meat than in the
nitrite-treated meat. From this they concluded that a small amount of
nitrite produced the desired results and was no more dangerous to public
health than the meat treated with nitrates. Because of this classical
work, the U. S. Department of Agriculture (1960) issued regulations
allowing the use of nitrite in cured products provided that,

"The use of sodium nitrite, potassium nitrite, sodium

nitrate or potassium nitrate, or combination of nitrite

and nitrate, shall not result in the presence of more

than 200 parts per million of nitrite in the finished

product."
Toxicosis. Orgeron 2t,al. (1957) described 10 cases of methemoglobinemia
in man resulting from eating weiners and bologna high in nitrites. The
clinical symptoms included nausea, vomition, profuse sweating, and an
intense cyanosis of the fingertips, nose, and ears. Of 131 samples of
bologna and weiners collected and analyzed, 17 contained nitrite concen-
trations in excess of 200 p.p.m., with the highest containing 6570 p.p.m.
Although the violating plant was using a prepared curing mixture, they
had recently purchased the curing ingredients separately. Instead of
using the curing mixture which contained not more than 10% sodium nitrite
and sodium nitrate, it appeared that pure potassium nitrite was used in
preparation of the products containing high nitrite content. These
authors also reported similar incidents in Florida, where 3 children
became ill and 1 died after eating uncooked weiners. They stressed the
employment of responsible employees on jobs where curing mixtures are

formulated and emphasized the importance of state and Federal regulations

concerning meat additives, such as nitrite.

1h

Contaminated Water Sources
Nate: nitgite content. Although numerous reports have associated nitrate
toxicosis in man and animals to drinking water, only the more pertinent
references are considered in this review.

In Kansas, Metzler and Stoltenberg (1950) surveyed municipal and
rural water supplies and found 19.8% of 2635 Kansas wells contained more
than 10 p.p.m. N03-N, with the highest concentration of 486 p.p.m. By
sampling b municipal wells at selected intervals, they found seasonal
fluctuations in the nitrate levels with the highest level in winter.

They also pointed out the importance of having rural, especially school,
water supplies tested.

Campbell, Davis. and Myhr (1954), in Saskatchewan, reported losses
of cattle on a farm where the water contained 2970 p.p.m. nitrate. This
water source was an old well and was located at the edge of a barnyard
where cattle wintered. These authors attributed the source of pollution
to manure.

Johnston (1955) surveyed usu Ontario wells and found 18.8% of the
dug wells and 5% of the drilled wells contained NOB-N at levels of over
10 p.p.m. This author incriminated seasonal leaching of tepsoil, vegeta-
tion, fertilizers and nitrate deposits as sources of the pollution.

Faulhaber (1956), in California, reported a survey in which the
same wells were sampled at selected intervals over a period of 10 years
or more. Although the highest nitrate level reported was 106 p.p.m.,
this author believed that the nitrate content of individual wells was
increasing with time. He attributed this increase to heavy fertilization

of citrus crops and to sewage disposal of nearby cities and towns.

15

In Missouri, Case (1957) reported that many ponds contained an
excess of 5 p.p.m. NOB-N. Drought had necessitated the use of ponds for
water supplies. Case (1963) further discussed the nitrate hazard of
water supplies in Missouri. In one survey over 50% of the wells con-
tained over 10 p.p.m. NOB-N, and more than 25% contained over 100 p.p.m.
He also estimated that 90% of the rural water supplies in Missouri did
not meet the drinking water standards set by the U. 3. Public Health
Service. Although the nitrate problem in water was severe in Missouri,
Iowa, and Nebraska, the problem also occurred in Canada, California,
Texas, Montana, Oklahoma, Wyoming, Alabama, Florida, and Georgia. Coli-
form organisms from surface contamination were believed to change the
nitrates to the much more toxic nitrites. This implied the increased
danger of nitrite toxicosis from water in contaminated stock tanks, even

though the source of water was free of coliforms.

Toxicosis. Comly (19b5), in Iowa, was one of the first to diagnose
cyanosis in infants due to excessive nitrates. He reported 2 cases in
infants fed formula water from wells containing 140 and 90 p.p.m. NOB-N.
Examination of the wells revealed such poor construction that surface
drainage contaminated the water. He also indicated that this methemo-
globinemic condition occurred commonly in Iowa. In 1905, 91 wells were
sampled, and the water of 56% contained OVer 10 p.p.m. N03-N. Besides
the nitrate pollution. 755 Of the privately owned Iowa wells were contami-
nated with coliiorm microorganisms.

Following Ccmly's report, Ferrant (1943} reported on 2 cases of
nitrate toxicosis in infants. The wells from which the formula water

was used contained 497 and 180 p.p.m. nitrates, respectively.

16
Faucett and Miller (19Q6), in Kansas, reported 3 cases of methemo-
globinemia resulting from using well water of high nitrate content in
infants' formulae. The first 2 cases occurred in twins, and the well
water contained 70 p.p.m. NOB-N. The third case was attributed to a well

'v a g . a . ‘ ‘
having an tag-J content 0: 33C p.p.m.

Summary

Numerous reports have clearly established the toxicity of nitrites
to man and animals. The early reports dealt primarily with acute nitrite
toxicosis; however, the more recent reports suggested nutritional inter-
ference syndromes as a manifestation of chronic nitrite toxicosis. The
indiscriminate use of nitrites in meat food products and the accumulation
of nitrites in drinking water and plant foods have caused serious illness

and even death of man and animals.

OBJECTEVES

The objectives of this research were:

1. To determine the influence of prolonged nitrite ingestion upon
experimental animals, as measured by (3) growth rates, (3) death losses,
(3) carcass quality, (2? organ weights, (E) clinical signs, (F) hema-
tology, and (C) gross and microscopic pathology.

2. To correlate the gross and microsc0pic lesions associated with
nitrite ingestion with the occurrence of stomach ulcerations in pigs.

3. To determine the toxic effects of nitrite when given to anemic
and nonanemic pigs.

4. To determine and record the relationship of serum nitrite to
methemoglobin concentration.

5. To obtain information on the pharmacodynamics of nitrite when

given to anesthetized pigs.

17

MATERIALS AED METEQES
gaperimental Animals
The pig was used as the experimental animal because it was (1)
reported as tho species most susceptible to nitrite toxicosis, (2) large
enough to permit ready clinical observations and collection of samp1-s for
patholOgic examinations, (3) readily available at a minimum of expense,
and (a) reported to have a correlation between prolonged nitrite inges-

tion and the gastric ulcer complex.

Facilities and Gegergl Informgtion

For the nitrite experiments, #9 pigs were used and were maintained
in Barn Number 5 on the Veterinary Research Farm, Michigan State Uni-
versity. In Experiment I, on chronic nitrite toxicosis, 30 pigs were
used. Trials 1 and 2 were conducted using 15 pigs in each trial. The
acute nitrite toxicosis studies, designated as Experiments II, III, and
IV, included h, 13, and 2 pigs, respectively. Random numbers (Dixon and
Massey, 1957) were used to assign pigs to the various treatments. Nitrite
stock solutions were made up as follows: Experiment I - l8.h7 Gm. of
potassium nitrite (KNOZ) q.s. 1000 ml. water constituted a 10,000 p.p.m.
nitrite concentration, or 10 mg. of nitrite per ml. of the stock solution;

and Exoeriments II and II - 18.h7 Gm. of KNO q.s. 500 ml. water consti-

2
tuted a 20,000 p.p.m. nitrite concentration, or 20 mg. of nitrite per m1.

of the stock solution. Fresh nitrite stock solutions were made up daily.

18

l9

Analyticgl Procedures

Hemat010gy. Blood samples were collected from the anterior vena cava,
using ethylene diaminotetraacetic acid (EDTA) or heparin as an anti-
coagulant. The samples were collected at selected intervals, depending
upon the experiment. Hemoglobin was determined by the cyanmethemOglobin
method, packed cell volume values were determined by the micro method
(capillary tube), and white blood cell counts were made using Turk's
diluting fluid and a hemacytometer. At the time blood samples were
collected, blood smears were made, then stained with Wright's stain and

differential white blood cell counts determined.

Mgthemoglobin. Methemoglobin was determined by the method of Evelyn and

Malloy (Hawk, Oser, and Summerson, 195“).

Seggg nitritg determinatiog. Serum nitrite-nitrogen (NOZ-N) values were
determined according to the method of Diven gt g1. (1962a), except that

concentrated mercuric chloride was used to precipitate the serum protein.

Sezgm vitamins A and E determinations. Vitamin A was determined according
to the procedure of Sobel and Snow (19u7). And vitamin E was determined

with a macromodification of the Emmerie-Engel procedure employing mild
saponification with ascorbic acid as an antioxidant (Hawk, Oser, and

Summerson, l95u).

Live; vitamins A and E determinations. Liver vitamin A was determined
according to a method of Gallup and Hoefer (19flo) modified to include a

saponification step with the protection of vitamin A from oxidation using

20
pyrogallol as an antioxidant. Vitamin E was determined by a total
saponification-digestion method with a standard Emmerie-Engel reaction

carried out on an aliquot of the unsaponifiable extract.

Meat nitrite determination. The nitrite concentration was determined by
the standard analytical procedure of the Association of Official Agri-

cultural Chemists (1955).

Hisopgthology. Routine sections were collected, preserved in buffered
10% formalin, and were stained with hematoxylin and eosin. The histo-
logical procedures were according to the United States Armed Forces

Institute of Pathology Manual of Histologic and Special Stainipg Technics
(1957).

 

Specific Procedures fog Each Expgriment
Experiment I (Chronic Toxicosis)

Izigl 1. Fifteen Yorkshire crossbred pigs, 8 weeks of age, were divided
into 5 groups of 3 each and placed in separate pens. weights of the pigs
were recorded at selected intervals. Using a modified paired-feeding
technique, the pigs were fed the Michigan State University swine grower
ration. The amounts of feed and water were recorded daily.

In the drinking water, nitrite was given to the groups in the
following amounts: Group 1, none: Group 2, 50 p.p.m.: Group 3, 100
p.p.m.: Group 4, 150 p.p.m.; and Group 5, 200 p.p.m. This was accomplished
by adding 0, 20, MO, 60 and 80 ml., respectively, of the 10,000 p.p.m.
stock solution q.s. #000 ml. of water. During the experimental period,

the pigs were fed and watered twice per day and were observed for signs

21
of toxicosis. After 10 weeks, 1 pig from each group was removed and
sent to the Meats Laboratory for slaughter. The remaining 10 pigs were
slaughtered at 11 weeks, when the experiment terminated. Although the
animals were weighed when delivered to the Meats Laboratory at noon on
the day prior to slaughter, preslaughter weights at 6 a.m. the following
day were also recorded. At slaughter, blood samples were collected and
the weights of the livers, kidneys, hearts, spleens, and dressed car-
casses were recorded. Tissues which were placed in plastic bags, sealed
and frozen at -20 F. for nitrite analyses included: ham muscle, kidney,
liver, heart, spleen and lung. Liver samples were saved in a similar
manner for vitamins A and E determinations. Sections from the following
organs were placed in 10% buffered formalin for routine histological
examination: fundic region and cardiac region of the stomach, duodenum,
lung, liver, kidney, spleen, heart, superficial inguinal or supramammary

lymph node, and medulla oblongata.

Trial 2. Fifteen Yorkshire crossbred pigs, b weeks of age, were purchased
from a nearby farm. Procedures were similar to those in Trial 1, with

the exceptions that higher levels of nitrite were given and methemoglobin
values on the pigs were determined. In the drinking water, the nitrite
was given in the following amounts: Group 1, none: Group 2, 200 p.p.m.;
Group 3, 300 p.p.m.: Group R, #00 p.p.m.: and Group 5, 500 p.p.m. This
was accomplished by adding 0, 80, 120, 160, and 200 ml., respectively,

of the 10,000 p.p.m. stock solution q.s. #000 ml. of water. One pig

from Group 4 (#00 p.p.m.) died after 6 weeks. The remaining 1n pigs

were sent to slaughter after 11 weeks.

22
Besides the collection of a blood sample from each pig at slaughter,
blood samples were collected the fourth and seventh days of the experi-
ment. At each time these samples were collected, the water and feed
were withheld 18 hours. Then feed and nitrite water were given and
blood samples collected 2 hours later. In addition to the routine

hematology, methemoglobin values were determined.

EXperiment II (Acute Toxicosis)

Four Yorkshire crossbred pigs were used, Pigs Nos. 1 and 2 were
purchased from a nearby farm, and Nos. 3 and h were raised at the Veteri-
nary Research Farm. Each day, the pigs were weighed and an initial blood
sample was collected. After oral dosing with the calculated amount of
nitrite, blood samples were collected each hour for # hours for routine
hematology and methemoglobin analyses. The nitrite was administered by
using a small plastic stomach tube and a 30 ml. glass syringe. After
administration of the nitrite, 20 ml. of water were placed in the syringe
and injected through the plastic tube into the stomach to assure that
none of the nitrite solution remained in the tube. For 4 hours after
dosing, the pigs were observed and the clinical signs recorded. At
necrOpsy the tissues preserved for histological examination were the

same as those listed for EXperiment I.

Experiment III (Anemia)
Thirteen Yorkshire crossbred baby pigs were born November 25, 1963,
and left with the dam throughout the experimental period. At 1 week of

age, all were weighed and ear notched. At this time 7 were given 2 ml.

23

of an iron preparation (Armidextran, Armour & Co.) intramuscularly.

37.
(0*
k]
(“w

and 21 days of age, weights were again recorded. The group of pigs
given the iron injections are designated as iron-treated and the other
group as nontreated.

Initial blood samples plus samples at selected intervals were
collected. In addition to routine hematology and methemOglobin analyses,
serum NOZ-N concentrations were also determined. At death, or when
euthanatized, each pig was necrOpsied and gross changes were recorded.
Sections preserved for histologic examination from Pigs 5, 6, 30, and
50 included heart, spleen, kidney, and liver. Spleen and liver weights

were recorded for all pigs.

Experiment IV (Pharmacodynamics)
Two Yorkshire crossbred pigs, each weighing 45 pounds, were pur-

chased from a nearby fa.h. Each pig was given 50 mg. Sparine (Jyeth)

~
9‘

q

intramuscularly, followed by an intravenous injection of 6% sodium
pentobarbital to produce surgical anesthesia.

In the first big, the anterior vena cava, both carotid arteries,
and femoral artery were cannulated. A tracheal tube was inserted into
the trachea bv surgical means. A Grass Polygraph C-channel recorder
(Model 5, Grass Instrument Company, Quincy, Massachusetts) recorded
the blood pressure values, EKG. and reSpirations. For the blood pressure
recordings. Channel 1 and 5P1 amplifier with a Statham in} pressure
transducer was used. Using Lead 2, the EKG was recorded by Channel 2
and 5P4 amplifier. Hespirations were recorded by Channel 3 and a 5Fl

. . . ..,, . . m p. . A I
amplifier with a PLjA low pressure air transaucer. Lhe nitrite ()0 mg./lb.

body weight) was dissolved in 10 m1. of water and administered directly

2%
into the stomach which was eXposed by laparotomy. After collecting an
initial blood sample. subsequent samples were collected at 30-minute
intervals from the anterior vena caVa, carotid artery, saphenous vein,
and femoral artery.

In the second pig, the carotid artery was cannulated and the
tracheal tube inserted. A laparotomy exposed the stomach and the abdomi-
nal blood vessels. Again, the same recording devices were used. Blood
samples were drawn initially and at 30-minute intervals from the posterior
vena cava at the diaphragm and posterior to the kidneys, the renal vein,
and the aorta posterior to the kidneys. The nitrite (30 mg./lb.body
weight) was injected into the stomach.

For collection of the blood samples, heparin was used as an anti-
coagulant. Methemoglobin and serum NOZ-N values were determined. Routine
hematoIOgy was determined at each bleeding interval on the blood sample
from the carotid artery of the first pig and from the aorta of the
second pig. At death, both pigs were necropsied and observed for

gross changes.

are"? I
A‘. I

gxperiment I
Growth

is data on the growth rate of pigs given various levels of nitrite
in their drinking water are summarized in TABLE l-l. In each of the 2
trials, no major differences are noted for the various treatments. All
pigs gained well during the experimental period. Figure 1-1 is presented
to illustrate the size and general condition of a pig given 200 p.p.m.

itrite in the drinking water for 10 weeks. The average daily feed.

water, and nitrite consumption of pigs given the highest level of nitrite

in each trial is presented in TABLE l-2.

Analyses

\
Slaughter data. The data collected at slaughter are given in TABLE 1-3.

‘-: “., ,. '_ +1" V7 . t V
or difzerences are noted in cue 'ressing percen-aae or in ~he

No ma

(

liver, kidney, heart or spleen weights. Gross examination of the car-
casses and primal cuts failed to reveal any significant alterations in
the color or other quality characteristics. Figure 1-2 illustrates the
carcasses of pigs given various amounts of nitrite in their drinking

water for 10 weeks.

'7

nematolos'. The hematological data from the pigs of Trials 1 and 2 are
given in TABLE l-5. No major differences are noted in the heme-
globin (Hb.) or packed cell volume (PCV) values or the white blood cell

(HBC) values at slaughter. The NBC Values of blood collected on days

25

26

TABLE l-l.--Experiment I. Average Growth Rate of Pigs Given Selected
Levels of Nitrite in the Drinking Water.

 

 

 

 

 

 

 

-—-:-

 

Treatment Average Average Average
Group No. of (p.p.m. Initial Final Wt. Daily Gain
No. Pigs N02) wt.(ib.) (1b.) (1b.)
Trial 1 (77 deval—
1 3 O h? 186 1.87
2 3 50 #9 189 1.88
3 3 100 46 184 1.8“
4 3 150 #7 182 1.81
5 3 200 h? 191 1.90
Trial 2 (77 days)
1 3 0 15 107 1.20
2 3 200 17 98 1.05
3 3 300 17 105 1.14
a 2 #00 lb 108 1.20
5 3 500 15 103 1.16

 

TABLE 1—2.--Experiment I. Average Daily Feed. Water. and Nitrite Con-
sumption for Pig Given Nitrite in the Drinking Water in
Group 5 of Each Trial.

Week of Feed Water Int ke of N02 (mg,2
Experiment (lb./day) (gal./day) Total7day Per lb.
Trial 1, Group 5 (200 p.p.m.)

 

l 3.0 0.62 496 9.1
2 h.3 0.82 688 10.0
3 h.“ 1.1h 912 11.1
h b.6 1.28 1020 10.8
5 “.7 1.33 106“ 10.1
6 5.h 1.33 --- ---
7 6.0 1.33 --- "'
8 7.6 1.71 1366 10.4
9 8. O 1.71 I.-- ---
10 9.0 1.71 1366 8.2
11 9.0 1.71 1366 7.5
Trial 2, Group 5 (500 p.p.m.)
1 0.7 0.29 580 28.3
2 1.4 0.33 660 26.1
3 2.0 0.Q8 960 30.2
4 2.4 0.62 1240 30.0
5 2.7 0.75 --- ---
6 2.7 0.86 1720 30.7
7 2.7 0.90 --- ---
8 3.0 1.00 --- ---
9 3.8 1.00 --- ---
10 0.0 11.10 -- --~
ii 5.0 1.20 2&00 28.2

 

b and 7 from pigs in Trial 2, given nitrite, indicate a leukocytosis
with an absolute neutrOphilia. Nethemoglobin (MetHb.) values up to

0.5 Gm./100 m1. of blood are noted in Groups h and 5 during Trial 2.

I

Serum and liyer_yitamins A and E determinations. Liver and serum vitamins

 

A and E values are presented in TABLE l-e. Differences are noted between

treatments but could not be correlated with nitrite consumption.

Nitrite residue determinatgpns. No nitrite residues were present in the

lungs, kidneys, spleen. liver. or heart from the pigs in Trial 1.

Signs
The pigs given 200 p.p.m. nitrite and above were occasionally
cyanotic, dyspneic, listless, and lethargic within 30 minutes after
eating and drinking. These signs usually subsided within 2 hours and the
pigs appeared normal again. One pig in the #00 p.p.m. group died the

8th week of the experiment following signs of acute nitrite toxicosis.

Gross Lesions
At necrOpsy. the pig that died during the experiment had a
brownish discoloration of the blood, edema of the lungs. and congestion
of the visceral and peripheral blood vessels. At slaughter. some of the
remaining 11g: revealed akeratinization of the stomach, ulcers in the
cardial region of the stomach, and scarring of the liver. These lesions

were noted in the control pigs as well as in the nitrite-treated pigs.

Microsc0pic Lesions
No significant pathological changes were found. except in sections

from the stomachs and livers of some of the pigs. The lesions in the

 

Fig. l-l.-~A pig given 200 p.p.m. nitritegin the drinking
water for 10 weeks.

It gs;

   

 

Fig. 1F2.--Carcasses70f pigs given’fiit£§£3”ifi‘€fié‘aiifikihg
water for 10 weeks. Dosage levels from left to right: 200, 150,
100. 50, and 0 p.p.m.

29

TABLE l-3.--EXperiment I. Slaughter Data (Averages) Collected From
Pigs Given Selected Levels of Nitrite in the Drinking Water

 

W L

Treatment Live

 

 

Group No. of (p.p.m. Wt. Dressing ____Q§g§flvWelghts (Gm.)
No. Pigs N02) (1b.) 6 Liver Kidneys Heart Spleen
Trial 1
1 3 0 169 7a 1511 313 273 129
2 3 50 171 75 1528 307 267 112
3 3 100 166 74 1&57 328 250 110
4 3 150 162 75 1415 272 250 105
5 3 200 172 75 1563 333 273 117
Trial 2
1 3 0 102 83 1000 195 192 82
2 3 200 92 83 1016 170 183 59
3 3 300 99 84 926 190 169 76
u 2 400 102 83 1035 187 220 92
5 3 500 97 8b 983 157 173 73

 

TABLE 1-4.--EXperiment 1. Average Vitamin A and Vitamin E Values of
Liver (mcg./Gm.) and Serum (mcg./lOO ml.) from Pigs Given
Selected Levels of Nitrite in the Drinking Water.

 

 

.1 W

Group No. of Treatment Vitamin A Vitamin E

 

 

 

No. Pigs (p.p.m. N02)‘ Liver Serum Liver Serum
Trial 1
1 3 0 186 u3 10 116
2 3 50 th 63 11 380
3 3 100 221 41 12 26
A 3 150 196 01 9 386
5 3 200 253 be 11 71
Trial 2
l 3 0 69 41 5 245
2 3 200 6b 58 u 193
3 3 300 93 62 6 205
a 3 400 58 52 5 2A1
5 3 500 7“ 39 5 225

 

30

33%.... mason mm Umuoeflnoo moans: 3035 «EN. 3.00:0 ooh.“ 805% :93 6.50: ma “038.30: .50.?» and Humour.

l

 

 

 

 

H 0 H 00 H HN 000.0H m.N0 0H.0 N.0H 000 N m
H 0 N 00 H 0N 000.HN 0.00 00.0 0.0H 000 N 0
m 0 H H0 0 NN 000.0H 0.H0 00.0 0.0H 000 0 m
0 0 0 00 N 0N 000.HN 0.00 00.0 0.0H 00N N N
m 0 N 00 H 0H 00H.0N 0.N0 00.0 0.0H 0 N H
neenmseHm 04
H 0 H 00 0 H0 00N.0m 0.Hm 00.0 0.0 000 0 m
0 0 0 00 0 N0 000.Nm 0.0m 0H.0 0.0 000 0 0
H 0 0 00 0 0m 000.0N 0.0N 00.0 0.0 000 m 0
N 0 0 00 0 mm 00H.mN 0.0N 00.0 m.m 00N m N
0 0 0 00 H mm 000.NN 0.00 00.0 0.0 0 m H
an mac
H 0 H N0 0 mm 000.0N 0.0N 00.0 0.0 000 m n
N 0 H 00 e 00 000.00 0.00 0H.0 0.0 000 0 e
H 0 0 00 0 H0. 00H.0N 0.0N 00.0 0.0 000 0 e
H 0 0 00 H mm 000.H0 0.0N 00.0 0.0 00N m N
0 0 H mm 0 N0 000.NN 0.0N 00.0 0.0 0 m H
m H H 00 m 0H 1-: 0.NH 00N N m
m 0 H 00 n 0N -s- 0.HH 00H N e
0 0 m 00 0 6H 000.HN 0.00 uuu 0.mH 00H m m
m H m 00 0 «N 000.0N 0.0: -u- 0.HH 00 m N
N 0 H or H NH 000.0N m.H4 --a 0.0H 0 N H
neeswseHn 0e
.nauwauywu
:Heem eeem 6:62 neezq neem 2:0 H.2ee m H.He 00H H.Ha 00H HNoz emHi .62
HHH neeeeo HeHeeeneeena 002 \nHHeov >00 \.50 \.sov .e.e.0v Ne .62 neeeo
002 .0006: .00 “argumene

 

 

.eeeex meerHeo 0H eeanHz we nHeeeH eeeeeHen eerHo eearm No seemeeex neHMHeez .H ecosHeeexmuu.muH mHmee

31
stomach were characterized by a parakeratotic proliferation of the squa-
mous epithelium in the cardiac region. The ulcers in the same region
were characterized by a break in the continuity of the epithelium,
lymphocytic and neutrOphilic infiltration. and the presence of erythro-
cytes on the ulcerated surface. The lesions in the liver were charac-
terized as a chronic eosinophilic hepatitis and consisted of a perilobular
infiltration of eosinOphils with an accompanying proliferation of fibro-

blasts and bile duct epithelium.

Experiment II
Analyses

~The blood values from pigs given the initial dose of nitrite by
stomach tube are given in TABLE 1-6. Except for the animal that died,
the methemoglobin values reached a peak in l to 2 hours after dosing.
At death. all the hemOglobin was oxidized to methemoglobin in Pig 1.
The average total W8C counts increased to the highest level in l to 3
hours after administration of nitrite. During the first h hours after
dosing. the neutrOphil count increased and the lymphocyte and eosinophil
counts decreased. The blood counts of Figs 3 and h revealed 20 and 22
eosinOphils per 100 NBC prior to dosing with nitrite but 4 hours later
only 1 eosinophil per 100 NBC was found in l pig and no eosinophils in
the other. This observation is of importance to the interpretation of
the microsc0pic findings. The blood values given in TABLE 1-6 are
essentially the same as those obtained when Figs 2, 3, and h were given

the same dosage of nitrite on 3 additional successive days.

Umwms

 

 

 

 

 

 

0 0 0 00 0H 00 00N.0 0H 0.0 0.0 .0
0 0 0 0m 0 00 000.0H 0N 0.0 H.0 N
0 0 H N0 0 mm 000.0H 0N 0.0 0.0 H
H 0 0 00 0 00 000.0H NN 0.0 0.0 0 00 H
H 0 0 H0 N 00 000.0H mm 0.H 0.mH a
0 0 0 00 0 . Hm 000.0H 00 N.N 0.NH a
N 0 0 0N NH 00 000.HH 00 0.0 0.NH 0
0 H 0 00 MH N0 00N.0N 00 0.0 0.HH N
0N 0 0 we 0H NN 000.0H H0 0.0 0.NH H
NN 0 H 00 HH NN 000.0H Ne N.0 0.0H 0 00 0
0 0 N 00 a 00 000.0H 00 N.0 0.0H 0
H 0 0 00 NN 00 000.0H 00 0.H 0.NH 0
H 0 0 00 m 00 000.0H 00 0.0 0.NH 0
0 0 0 mm 0H 00 000.0H H0 0.0 0.0H N
0H 0 0 00 N 00 00N.0H H0 0.0 0.HH H
0N 0 0 H0 N 0N 000.0H 00 0.0 0.0H 0 0N 0
0 0 0 0N 0H 00 000.0N HN 0.0 N.0 0
0 0 0 0 N0 00 000.0H HN 0.0 0.0 e
0 0 H 0H NN m0 000.0N 0N N.H 0.0 0
0 0 0 00 0N H0 000.0N 0N 0.N 0.0 N
0 0 H H0 0H 00 00N.0H NN N.N N.0 H
0 0 0 00 H 00 000.0H 0N 0.0 0.0 0 0N N
sumom 000m ocoz gash» pawm 2x0 H.950 ”WV A.H£ OOH H.HE 00H awnwmoa H.9H .02
H00 000060 HeHeceeetaHo 000 \nHHe00 >00 \Jeuv \.s0v nee0< \Noz Marv 0H0
. 000 .0000: .nm eneem peerseena
enie eeeaeee m0 meHneHa
mo mmmbmn mewooamn ampHm @CHzm opwwmusemmaucn nonm EmnmoEwm nmfimwwom .HH pcmanmaxmnl.OtH m4m<~

Signs
The signs listed in TABLE l-7 are typical of all the pigs dosed with
nitrite by stomach tube; only the intensity and termination varied. Pigs
l and 4 died with signs of acute nitrite toxicosis. Pigs 2 and 3 were
euthanatized at the end of the experiment. Death usually occurrred

within # hours, and by 6 hours the pigs generally appeared normal.

Gross Lesions
At necrOpsy, Pigs l and 4 had extreme cyanosis and brownish dis-
coloration of the blood and tissues. The stomach of Pig h had hemorrhages
and ulcerations in the fundic region (Fig. 1-3). The livers of Figs 3
and h had numerous whitish foci 1 cm. in diameter and extended into the
parenchyma. Fig. 1-4 illustrates these foci and also the evidence of

methemoglobinemia.

Microscopic Lesions
Histopathologic examination of the lungs of the pigs dying of acute
nitrite toxicosis had areas of emphysema, edema, and congestion (Fig. l-S).
The livers of Figs 3 and h contained numerous areas of perilobular
eosinophilic infiltration and fibroblastic and bile duct proliferation
(Fig. 1-6). Numerous eosinOphils and edema were present in the fundic
portion of the stomachs of Pigs 3 and 4 (Figs. 1-7 and 1-8). Hemorrhages

and ulcerations were also present in the stomach of Pig h.

TABLE l-7.--Experiment II.
Nitrite Toxicosis. Pig 1 Given 30 mg. of Nitrite/lb.
Body Weight by Stomach Tube.

 

Minutes
After Dosing

34

Characteristic Signs of Figs with Acute

 

 

L

 

Signs

 

0
15
30
00

100

Normal

Salivation

Defecation, stretching

Urination

Ataxia, restless, skin blanched
Vomition, dyspnea. abdominal pain
Urination. abdomen tucked up, quivering
Vomition. salivation

Marked ataxia. weakness
Urination, buccal mucosa cyanctic
Marked dyspnea. side paddling
Severe convulsions

Severe dyspnea

 

 

/
ml I . I A l t l I I I. i! Ll ll HA
lwy .. u n .m w. A“ u x 1 Mml‘ ‘1 1H .J‘ml "JHH'H'W MHH‘H‘ ‘ ‘ .

 

Big. l-3.--Stomach of Pig h, given 30 mg. nitrite/lb. body
weight by stomach tube for b consecutive days. Note hemorrhages
and ulceration of the fundic region.

 

 

 

 

vFig. 1-u.--Liver. heart, and lungs of Pig 4. which died after
being given 30 mg. nitrite/lb. body weight by stomach tube for u
consecutive days. Note the brownish color of blood and lungs.

 

Fig. l-5.--Section from lung in Fig. 1-1+.Noi'e’a71Woi’eT
edema. emphysema and the congestion of the blood vessels. H & E
stain; x 75.

 

 

Fig. l-6. --Section frOm liver in Fig. 1-4. Note thEWperi-
lobular infiltration of eosinophils and the fibroblastic and bile
duct proliferation. H & E stain; x 75.

 

Fig. 1-7.:-Eosinophilic infiltration of rundie region 31‘
stomach shown in Fig. 1-3. H & E stain; x 187.

 

. ’1“
fig: 1-8 :Higheripowerg of—FVig? 147 .Wthe eosinoplfils
and the edema of the fundic region. H 8: E stain: x 750.

 

38

Eerriment III
Growth
The data on weight gains for the iron—treated and nontreated pigs
are given in TABLE 1-3. The pigs given iron intramuscularly gained 2

pounds per pig more than the nontreated group during the 2-week period.

Analyses
Organ weights. Spleens of the iron-treated pigs were larger than the
spleens of the nontreated pigs per unit body weight. There were no

major differences in the average liver weights of pigs from the 2 groups

Hematolocv. Hemoglobin. methemoglobin, PCV and serum nitrite-nitrOgen

 

(NOZ-N) values and the total and differential NBC counts are given as
averages for each group in TABLE 1-9. Prior to dosing with nitrite,
the average hemoglobin value for the iron-treated pigs was 10.8 Gm. and
for the nontreated pigs h.0 Gm./lOO ml. of blood. The PCV values are
in.direct pr0portion to the hemoglobin values, with an average of 37.0%
for the iron-treated and 15.5% for the nontreated pigs. Methemoglobin
and serum NOZ-N values were not detected prior to dosing with nitrite.
The average total WBC counts are slightly lower for the nontreated group
but the differential NBC counts for the 2 groups indicate no major
differences. The nucleated red blood cells (NRBC) per 100 NBC counted,
however. averaged 1U for the nontreated group and 3 for the iron-treated
group.

The methemoglobin values parallelled the hemoglobin values and were

both much higher in the iron-treated group than in the nontreated group.

\A)
\(l

A“! T“ Q 73,... ' ‘ \ A ~ "‘ «0“
.ABL; l--.---iperinart III. average JTOch Rate
+ +
V U

 

 

 

 

and gan feights
of Iron-Trea ei (Group 1) and Non rested (Group 2) Figs.
r— ’ m .00.-..- ._-_ ___1 - *«r—"r" -....:.;

 

Days of Age At Necronsy _ __
Group No. of 7 l; 21 Liver Spleen
Number Pigs {1b.\ (1b.) (7“ ) (Gm. (Gm.)

l 7 5.5 9.7 12.3 199 15

“O
O\
-n
(.0
(l)

\O
b. J
C)

.5 176 9

 

 

 

 

o o o oq N mm N com-N mmN o. N.. N.0 oqN . N
o o N NN m .N o coo.N. MN 0N N.N N.N ooN . .
o o . .m n ma ON omo... Noe «. 0.. N.m on. m N
o o . Ne m Ne q ooa.N. «x. mm ..N 0.0. on. e .
o o . mN N mN NN on.N me .. 0.. ..m V 09 m N
o o N N. . .m 0 0.N-m. mON om 0.0 N.0 00 q .
o c c .N N mN N. coo-w Nom. 4. m.. q.m 00 e N
o o . ¢q N mm m oo.-o. mom 0N m.q 0.0 00 m .
o o o No . Nm c. 0mm.N Nee. m. 0.. q.m on m N
o o 0 mm o .q n omN.m. NoN 0N N.N 0.0 on N .
o o 0 mm . mq .. omN.N NNN N. m.o o.m m. m N
o o N 00 . Nm n cmN-c. 0N. om m.o N.0 m. N . -
.£H\muwhuwz .wE NM
0 o o mq m mm a :5..N. mNN 0. 0.. 0.N om. m N
a o . a. 0 cm m ONo.N. o mm N.0 ..o. om. m .
.p.\mu.uu.z .we on
o o o mm N mq o. 0mm.N ow m. m.. m.m cm. N N
o c 0 mm . oo a ooq.N. m. «m m.¢ N.0. om. N .
.n.\ou.pu.z .we mN
o o . oo o mN ¢. CNN-N o m. 0.0 0.. o o N
. o . oN . NN m oqq.w 0 NM c.o 0.0. o N .
wuwuufiz :u.3 wowmoa muowom
a.mom Oman ocoz scabg nmum 22m 0mm A.EEu A.~E oo.\.woav ANV A..E OCH A.~E oo. ucfiwcc mmfim .oz
wmum \m..uo z-Noz >00 \.euv \.aov nouN< .0 .02 nacho
. w :50 w :wuo u
.00 u u . .. w..o om. -o.o=z on; sauwm .pzoaz .00 m~.::.:

 

II."

.0029 nomsoum an muwuufiz 4o m.m>mq wouom.mm sw>wu wwmm AN msouov kumoue
ncoz com A. asouov vuummuhncouw Eouwn.m$¢qm5dhwosom Umwmwvflnmo zuflafidw .HHH ucmeumaxmnu.onH mqm48

,

41
In general, methemOglobin values were highest at 2 hours after dosing in
the pigs that survived. In pigs that died, values were the highest at
the time of death. In contrast to the methemoglobin values. the serum
NOZ-N values were highest at 1 hour after dosing with nitrite. No corre-
lation was noted between the serum NOZ-N values and methemOglobin values
in a specific pig at a given time after dosing with nitrite.‘ In general,
the total NBC counts increased after nitrite treatment in the iron-
treated pigs much more than in the nontreated pigs. An absolute neutro-

philia was also noted.

Signs

ihe signs observed were similar to those described in TABLE l-7
(Experiment II); only the intensity varied. Fig. 1-9 illustrates some
of the characteristic signs of acute nitrite toxicosis. Although death
followed convulsions in most of the pigs. a few fully recovered spon-
taneously without treatment within 2 to 3 hours. Blood samples from
pigs that recovered contained low levels or no methemoglobin at 7 hours
after nitrite administration. The LD5O of nitrite, dosed by stomach
tube to swine. was 30 to 32 mg./lb. body weight. No major differences

were noted in the LD for the iron-treated or nontreated groups.

50
Gross Lesions
Fig. l-lO illustrates the paleness of the skin. nose and mucous
membranes of the nontreated pig in contrast to the pink color of the
iron-treated pig at necrOpsy. The viscera of the nontreated pig wer-
pale, and the blood was watery and thin (Fig. l-ll). The pigs dosed

with nitrite had brownish discoloration of the blood and tissues, and

42

 

Fig. 1-9.--£xpe;iment III. Signs of pigs given 32 mg. nitrite/1b.
body weight by stomach tube. (A) normal, (3) ataxia, (C) weakness and
ataxia, (D) unable to rise, (E) comatose, and (P) gasping shortly before
death.

43

 

. —. . r
; " T31 ' S "
» iL; i .\ ,_ ‘ '

I, .

Fig. l—10.--Iron-treated and nontreated pigs. Note the pale-
ness of nose and ears of the nontreated pig on the left.

 

 

Fig: l-ll.--Viscera of ironLtreated and nontreated pigs shown
in Fig. l-lO. Note the paleness of the visceral organs and blood
of the nontreated pig on the left.

as
the lungs appeared blanched. Only the intensity of the brownish dis-
coloration varied with the iron-treated and nontreated groups (Figs.

1-12 and 1-13).

Microscopic lesions
On histopathologic examination of the lungs of pigs with acute
nitrite toxicosis, congestion and edema were present, and in other

organs eni tissues, only congestion was present.

Exceriment IE
Analyses

Some of the pharmacodynamic effects of 30 mg. nitrite/lb. body
weight injected into the stomach of anesthetized pigs were recorded
until the time of death. Information on blood pressure values and leart
and respiratory rates is summarized in TABLE 1-10. The systolic and
diastolic bloc”J r: sure‘values decreased rapidly after injection of
nitrite into the stomach. Since the systolic pressure decreased more
than the diastolic pressure, the pulse pressure also decreased. The
heart and respiratory rates, in general, increased during the experi-
mental period, except near death, when both decreased rapidly. TABLE
l-lO also illustrates that death is apparently due to respiratory failure
rather than to cardiac failure. Electrocardiographic tracings were
essentially the same before and after nitrite administration.

The hemOglobin, methemoglobin and serum NOZ-N Values for various
locations are given in TABLE l-ll. The methemoglobin values increased
during the experimental period, and no major differences were noted in

the values determined from arterial or venous blood. The serum NOZ-N

 

Fig. l-lZ.--Thoracic and neck area of 2 iron-treated pigs.
The pig on the right died after given 32rng. nitrite/lb. body weight
by stemach tube. Note the brownish-colored blood and blanched
heart and lungs in the pig on the right, in contrast to the con-
trol pig on the left.

 

 

Fig. 1-13. --Visccral and neck region of 2 nontreated pigs.
The pig on the right di ed after given 32 mg. nitrite/lb. body
weight by stomach tube. Note the brownish watery blood and
blanched lungs in the pig on the right, in contrast to the con-
trol pig on the left.

x /

4O

TARLE l-lO.--Experiment IV. Blood Pressure Values and Heart and Respira-
tory Rates of an Anesthetized Pig Given 30 mg. Nitrite/lb.
Body Weight by Injection into the Stomach.

 

 

 

 

Hinutes

After Blood Pressure 1mm. Hg) Heart Rate Respirations
In;eetion Systolic Diastolic Pulse Lper Minute). [per Minute),

0 150 120 30 13a 25

3 135 105 30 150 24

5 105 30 25 180 20

1 75 50 25 174 22

20 7S 5; 25 156 26

so so 55 25 150 30

b5 80 60 20 150 28

60 80 60 20 174 26

9O 95 75 20 193 35

120 85 65 20 234 36

130 75 55 20 250 30

1L4 60 L5 15 230 20

1&5 60 05 15 230 O

150 50 35 15 130 o

151 O O O 0 O

 

0.. - -~
um Kioofl -alues of Lioci

TABLE l—ll.-—Exneriment IV. Fb., Fetxb.. and 3?”
:olLected in Sifterent Locations from anesthet’zei Pigs Given
30 mg. Nitrite/lb. Ecdy weight by Injection into the Stomach

W

 

 

Dig Minutes After Injection
:3. Bleeding Site 0 30 60 90 120 150
Hemoglobin (0m.7100 ml.)
1 Carotid artery 9.6 10.0 9.9 11.0 12.0 12.0
2 Aorta 8.8 10.3 10.5 --- --- ---
Methemoglobin (Gm./100 ml.)

1 Carotid artery 0.0 2.0 2.9 3.9 “.7 5.1
Femoral artery 0.0 1.7 2.9 u.u h.h 5.“
Anterior vena cava 0.0 2.0 3.2 4.2 0.4 5.4
Saphenous vein 0.0 1.5 3.0 “.0 “.9 5.3

2 Aorta 0.0 0.7 1.5 --- --- ---
Posterior vena cava 0.0 0.7 l.7 --- --- ---
Renal vein 0.0 0.7 1.7 --- --- ---

Serum NOZ-J (mcg./100 m1.)

1 Carotid artery 0 390 130 330 150 150
Femoral artery 0 450 200 390 150 170
Anterior vena cava .0 310 130 270 90 100
Saphenous vein 0 200 100 70 90 130

2 Aorta 0 170 150 --- --- ---
Posterior vena cava 0 70 7S --- --- -—-
Renal vein 0 25 35 --- -- ---

1+7
values were highest 30 minutes after administration of nitrite and were
higher in the arterial blood than in the venous blood. The blood from
the renal vein contained considerably less serum NOZ-N than either the
aorta or posterior vena cava. Further, since NOZ-N was present in the
urine, it was concluded that some of the nitrite is; eliminated in the

urine.

Gross Lesions
Marked cyanosis, congestion of the visceral and peripheral blood
vessels, and brownish discoloration of the blood and tissues were

evident at necrOpsy.

DISCUSSION
gzperiment I

Prolonged nitrite ingestion had no significant effect on growth,
dressing percentages or organ weights; on the white blood cell, hemo-
globin or packed cell volume values; or on the serum and liver vitamins
A and E values. No nitrite residues were present in the lungs, kidneys,
livers, spleens, or hearts. Pigs given higher levels (300, 400, or
500 p.p.m.) of nitrite in the water were occasionally listless, cyanotic,
and lethargic shortly after eating and drinking. Case (1963) reported
nutritional interference syndromes in pigs given water containing 50
p.p.m. nitrate-nitrogen (NOB-N) and also that nitrite was at least 10
times more toxic than nitrate. By conversion of the nitrite to NOB-N
and dividing by 10, 16 to 17 p.p.m. nitrite should have caused the
interference syndromes in this study. Some of the pigs in this experi-
ment were given 30 times this calculated dose, and there was no evidence
of chronic toxicosis. The pig that died while consuming #00 p.p.m.
nitrite in the water had evidence of methemoglobinemia but evidence
of chronic toxicosis either grossly or microscopically was not present.
These facts indicate that the chronic nitrate-nitrite toxicosis under
farm conditions was not reproduced under these experimental conditions.
This might suggest that toxic factors other than nitrite may partially
be responsible for the toxicosis observed by other workers.

The gastric ulcerations and other subacute to chronic lesions
believed due to nitrite ingestion by Bush and Matteson (1963) were not
observed. The gastric ulcerations and chronic eosinOphilic hepatitis

1.3

1;.9
observed at necropsy of these exnerimental animals were similar to those
seen routinely at slaughter. In addition. no increase in the incidence
of these conditions was noted in the pigs given nitrite in their drinking
water. In regard to the possibility of a cumulative effect by
nitrite, he pigs given 500 p.p.m. nitrite in their drinking water con-
sumed daily nearly an LDSO amount of nitrite if it had been given in a
single dose. This lack of any accumulative effects of daily conSumption
of nitrite supported the research of Laing and McIntosh (l9u7).

The liver vitamins A and E values from the pigs in this experiment
were not significantly different from the controls. This information is
in contrast to the reports of Hvidsten (1955) and Bjornson §£.él- (1961),
who suggested that nitrite interfered with vitamins A and E metabolism.
The liver vitamins A and E determinations revealed more consistent

values than the serum determinations.

Exceriments III IIII and IV
General ObserVations

These experiments on acute nitrite toxicosis suggested the LD5O
for swine was apprOXimately 30 to 32 mg. nitrite/lb. body weight. This
confirmed the investigations of Stormorken (1953). It was particularly
significant that no differences were noted in the L050 for the non-
treated (anemic) and iron-treated (nonanemic) pigs of EXperiment III.

While the signs of acute nitrite toxicosis included those of anoxia,
the data in Experiment IV suggested the ultimate cause of death as
respiratory rather than cardiac failure. The gross pathologic observa-
tions included brownish discoloration of the blood and tissues and con-

gestion of the visceral and peripheral blood vessels.

\I\

O
Hematology

Several hematological findings associated with nitrite dosing by
stomach tube merit discussion. First, the total white blood cell counts
increased during the first hour but then decreased in 4 to 7 hours to
near the predosing value. Secondly, the number of circulating neutro-
phils increased during the first 4 hours after dosing. The total white
cell counts decreased to nearly the predosing values in b to 7 hours,
which indicated an absolute neutrophilia. Thirdly, the number of circu-
lating lymphocytes decreased during the first 4 to 7 hours after nitrite
dosing and, therefore. an absolute lymphOpenia was noted. Lastly, 2
pigs had high eosinophil counts prior to dosing with nitrite, but 4
hours later the eosinOphil counts were near zero. Subsequent histo-
pathologic study of the gastric mucosa indicated the infiltration of
numerous eosinophils. These histopathologic findings were not present
in the pigs with low eosinophil counts prior to dosing with nitrite.
Therefore, further investigations into the role of eosinophils in toxi-

coses such as nitrite are suggested.

MethemOglobin

In all the pigs that surVived, the highest methemOglobin levels
were found 1 to 2 hours after dosing with nitrite. In the pigs that
died of acute nitrite toxicosis, the highest methemoglobin values were
found at death. Pigs with low hemoElobin values had lower methemoglobin
values after the same dosages than pigs with high hemoglobin values.
However, a direct proportion of methemoglobin to hemoglobin was noted
in each group of pigs given the same per pound dosages of nitrite. This

would indicate that the natural body protective mechanisms. as described

51
by Huennekens §t_gl. (1957), may have reduced the methemoglobin to the
functional hemoglobin. These latter observations were confirmed in this
study as there appeared to be a point of maximum methemoglobin formation
unless the natural protective mechanisms were overwhelmed. If over-
whelmed, the methemoglobin formation continued and death occurred. This

body protective mechanism appeared to control the proportion of methemo-

globin to hemoglobin, regardless of the amount of hemoglobin.

Serum Nitrite-Nitrogen

The results of the serum nitrite-nitrOgen (NOZ-N) determinations
indicated that the highest values occurred about 30 minutes following
dosing with nitrite. As expected, these high serum values occurred prior
to the highest methemoglobin Values. However, the serum NOB-N values
were not in proportion to the amount of the methemoglobin that eventually
formed. The iron-treated pigs with an average hemoglobin value of
10.8 Gm./lOO ml. had lower serum NOZ-N values than nontreated pigs with
an average hemoglobin value of 4.0. Methemoglobin values were highest
in the iron-treated pigs. From this, it is concluded that the serum
NOZ-N values are not indicative of the amount 0r preportion of methemo-
globin that is eventually formed. These findings indicate the need for
further investigational work with nitrite reduction products, especially

hydroxylamine and ammonia.

Pharmacodynamics
The pharmacodynamic effects of nitrite upon anesthetized pigs
included decreased diastolic, systolic, and pulse pressures and increased

heart and respiratory rates. No major differences were noted in the

52
electrocardiograms. The differences in serum NOZ-N values in the renal
vein from values in either the aorta or posterior vena cava and the
presence of NOZ-N in the urine indicated that some of the nitrite was
excreted unchanged by the kidneys. This experiment indicated the possi-
bilities of usi.g pigs under anesthesia for future work in studying

acute toxicoses.

SUMMARY

Your experiments were conducted using a total of #9 pigs to evaluate
and study nitrite toxicosis in swine. Thirty pigs in 2 different experi-
ments were given up to 500 p.p.m. nitrite in their drinking water for
ll weeks. No major differences due to nitrite were observed in the
growth rates dressing percentages, organ weights, or white blood cell,
hemoglobin or packed cell volume values.' In addition, serum and liver
vitamins A and E values were not influenced by nitrite. No nitrite
residues were found in the muscle tissue or vital organs. Higher
levels of nitrite occasionally caused listlessness, slight cyanosis,
and lethargy in pigs shortly after eating and drinking. No lesions
attributable to prolonged nitrite ingestion were found on gross and

O

histopathologic examinations. The incidence of gastric ulcerations
was not increased by the prolonged nitrite ingestion.

Nineteen pigs in 3 different experiments were used to study acute
nitrite toxicosis. The L050 of nitrite orally for pigs was calculated
as 30 to 32 mg./lb. body weight. Anemia had no effect on the LD50'
Signs of acute nitrite toxicosis were primarily those of anoxia and
death was apparently due to respiratory failure. Hematological studies
during the u hours following nitrite dosing indicated a transitory
leukocytosis and an absolute neutrOphilia, lymphOpenia and eosinopenia.
hethemOglobin and serum nitrite-nitrogen (NOZ-N) values were not related

to different hemoglobin values in pigs. Decreased diastolic, systolic,

and pulse pressures and increased heart and respiratory rates were

5b
noted after injection of nitrite into the stomach. Gross lesions in

acute toxicosis included brownish discoloration of the blood and tissues,

cyanosis, and hyperemia of the visceral and peripheral blood vessels.

LIST OF R& EPEI'CES

American ‘Weat Institute Foundation. 1960. The science of meat and
meat products. N. H. Freeman and Co., San Francisco.

Association of Official A:tricultural Chemists. 1955. Official methods
of analysis of the association of official agricultural
chemists. 3th ed., mashington, D.C.

D ckman, H 19‘”. Drums; .neir nature, action and use. N. B. Saunders
Bjornson, C. 8., McIlwain, P. K., Eveleth, D. F., and Bolin, F. i.

lQol. Sources of nitra e intoxication. Vet. Med., 56:1?8.
Bradley, 3. 9., Eppson, H. F., and Beath, O. A. 1940. Methylene blue

as an antidote for poisoning by oat hay and other plants
cor taining nitrates. J.A.V.H.A., Quzhl.

Bush, J. A., and Iatteson, R. E. 1963. A syndrome in liVestOCk fed
hi7h levels of nitrates. Mod. Vet. Prac., u’ (8): 50.

. 1954. MethaemOglobi-
ate contents of well

Ccmpbell, J. 8., Davis, A. N., and Myhr, P. J
nernia o: livestock caused by high rd
water. Canad. J. of Comp. Med., 18

Case, A. A. 1954. Corn stock intoxication. Sheep «reeder, 7uzlh.

-------. l957. Some aspec s of nitrate intoxication in livestock.
(Iono‘vri‘o'lioltog .130: 323.

-------. 1903. The nitrate problem. lat. hog Farmer, Bull. Dldzl.

Comly, E. H. 1945. Cyanosis in infants caused by nitrates in well
water. J. Am. M. A., 129:112.

Davidson, W. 8., Doughty, J. L., Bolton, J. L. l9hl. Nitrate poisoning
livestock. Canad. J. Comp. Med., 5:303.

Diven, R. 8., Pistor, W. J., Reed, R. 3., Trautman, R. J., and Jatts,
R. E. 1962a. The determination of serum or plasma nitrate
and nitrite. Am. J. Vet. Res.,.23:h97.

Diven, P 8., Reed, R. E., Trautman, R. J., Pistor, W. J., and Watts,
' R. 3. 1962b. Experimentally induced nitrite poisoning in
sheep. Am. J. Vet. 395., 23:494.

Emerick, F. J., and Olson, 0. E. 1962. Effect of nitrate and nitrite
on vitamin A storage in the rat. J. Nutr.. 75:73.

J!
‘J‘.

9.6

Faucett, R. L., and Miller. H. C. l9&6. Methemoglobinemia occurring
in infants fed milk diluted with well water of high nitrate
content. J. Pediat., 29:593.

Faulhaber, C, E. 1956. Nitrates in underground waters. Cal. Citrog.
bl:206.

Ferrant, M. 1956. Kethemoglobinemia. Two cases in newborn infants
caused by nitrates in well water. J. of Pediat., 29:585.

Finch, C. A. l9h8. Methemoglobinemia and sulfhemOglobinemia. New
Eng. J. Med., 239:470.

Gallup, N. D., and Hoefer, J. A. 19Q6. Determination of vitamin A in
liver. Ind. Eng. Chem. Anal., 18:288.

Garner, G. 8., O'Dell, B. L., Radar, P., and Muhrer, M. E. 1958.
Further studies on the effects of nitrate upon reproduction
and vitamin A storage with rats and swine. J. Ani. Sci.,
17:1213.

Gwatkin, R., and Plummer, P. J. G. 1946. Toxicity of certain salts of
sodium and potassium for swine. Canad. J. Comp. Med., 10:183.

Hawk, P. 3., Oser, B. L., and Summerson, w. H. 1954. Practical
physiological chemistry. 13th ed. McGraw-Hill Book Co.,
Inc., New York.

Huennekens, F. M., Caffrey, R. w., Basford, R. E., and Gabrio. B. H.
1957. E. chrocyte metabolism. IV. Isolation and properties
of methemoglobin reductase. J. Biol. Chem., 227:261.

Hueper, N. C., and Landsberg, J. w. 1940. Experimental studies in
cardiovascular pathology. I. Pathological changes in organs
of rats produced by chronic nitrite poisoning. Arch. Path.,

29:633.

Hvidsten, H. 1955. Studies on the effect of nitrite in pig feed. Acta
Agr. Scand., 5:2h5.

JOhnston, R. A. 1955. The incidence of nitrates in rural Ontario well
waters. Cmiwd. J. of Pub. Health, 46:30.

Pflerr, R. H., Marsh, C. T. N., Schroeder, W. F., and Boyer, E. A. 1926-
The use of sodium nitrite in the curing of meat. J. Agr.
Res., 33:5Ql.

Laing, A. D. M. 0.. and McIntosh, J. c. 19m. Nitrite poisoning in
cattle and pigs. New Zealand J. Agr., 74:377.

57

Mayo, N. S. 1895. Cattle poisoning by nitrate of potash. Kansas Agr.
Exp. Sta. Bull., 99:3.

Mollwain, P. K., and Schipper, I. A. 1963. Toxicity of nitrate nitro-
gen to cattle. J.A.V.M.A., 1&2:502.

McIntosh, I. C., Nielson, R. L., and Robinson, N. D. 19h3. Mangel
poisoning in pigs. New Zealand J. Agr., 66:3bl.

Metzler, D. F., and Stoltenberg, H. A. 1950. The public health sig-
nificance of high nitrate waters as a cause of infant cyanosis
and methods of control. Kansas Acad. Sci. Trans., 53:19b.

Nason, A. 1962. Symposium on metabolism of inorganic compounds. 11.
Enzymatic pathways of nitrate, nitrite, and hydroxylamine
metabolisms. Bact. Rev., 26:16.

Newsom, I. E., Stout, E. N., Thorp, F. J., Jr., Barber. C. w.. and
Groth, A. H. 1937. Oat hay poisoning. J.A.V.M.A., 90:66.

O'Dell, B. L., Erek, Z., Flynn, L., Garner, 8. 8., and Muhrer, M. E.
1960. Effects of nitrite-containing rations in producing
vitamin A and vitamin E deficiencies in rats. J. Ani. Sci.,
19:1280.

Orgeron, J. D., Martin, J. D., Caraway, C. T., Martino, R. M., and
Rauser, G. H. 1957. MethemOglobinemia from eating meat with
high nitrite content. Pub. Health Rep., 72:189.

Robinson, J. D. 1992. Mangel poisoning in pigs. New Zealand J. Agr.

65:139.
Saussol, M. 1836. The poisoning of several lambs by the nitrate of
potash. The Veterinarian, 9:530.

Sippel, N. L. 1960. Nitrate poisoning. Vet. Med., 55(5):2 .

Smith, G. S. 1961. Vitamin A and dietary nitrates, effects of high
nitrate feeds on vitamin A. Vet. Med., 56:225.

Smith. H C-. Lovell. V. E.. Reppert, R., and Griswold, D. 1959.
Nitrate poisoning in swine. Vet. Med., 5hz5e7.

The estimation of serum vitamin A

SObSl’ A o E. , aY—vj 311034, 3. Do 19u70
l dichlorohydrin. J. Biol. Chem.,

with activated glycero

‘Ir C‘.
1/1:tl7.

tormorken, H. 1953. Methemoglobinemia in domestic animals. Proc.
XVth Int. Vet. Cong. ’t. 1, 1:501.

 

,—

5

Thorp, F.

nited States Armed Forces Institute of Pathology. 1957. hanual of
histologic and Special staining technics. Washington, D.C.

United States Department of Agriculture. 1960. Regulations governing
the meat inspection of the United states department of agri-
culture. United States Government Printing Office, Washington,
D.C.

United-States Pharmacoaoeial Convention, Inc. 1960. PharmaCOpeia of
the United States. 16th rev. Mack Publishing Co., Easton, Pa.

wanntorp, H., and Swahn, O. 1953. The toxicity of whey for swine in
conjunction with bacterial reduction of nitrate to nitrite.
Proc. XVth Int. Vet. Cone., pt. 1. 1:996.

Jinks, N. R., Sutherland, A. K., and Salisbury, R. 1 0. Nitrite
poisoning of pigs. Queensland J. Agr. Sci., 7

Winter, A. J. 1962. Studies on nitrate metabolism in cattle. Am. J.
V'eto R88. ’ 23:500.

Nunder, R. D., and dchultz, a. A. 1960. Nitrate poisoning in swine.
Iowa State U. Vet., 22:99.

PART II. GASTRIC ULCER COMPLEX

INTRODUCTION

The increased incidence of gastric ulcers in swine stimulated this
investigation on the epizoology. clinical manifestations, gross and
microsc0pic pathology, and mycology. Reports have suggested a relation-
ship of the gastric ulcer complex to gastric hyperacidity, to vitamin A
deficiency, to the "eosinophilic hepatitis" problem, and to candidiasis.
With the increased use of antibiotic feeds, some reports suggested
Candid; albicans as a possible etiologic agent in the gastric ulcer
complex. Other reports regarded it as a secondary invader.

In rats, prednisolone is known to produce ulcers which are similar
to the peptic and steroid-induced ulcers in man. Since this steroid is
a powerful gluconeOgenic drug. the stress of increased metabolism followinq

injection may simulate the metabolic stress in rapidly growing pigs.

REVIEJ OF LITERATURE
A large volume of literature has accumulated on the various aspects
of gastric ulcers and prednisolone therapy. An attempt has been made to
review selected references referring primarily to swine; however, a few

references relating to prednisolone-induced ulcers in rats are included.

Gastric Ulcers in Swine
Association with Infections

Rosenow (1923) was one of the first to report a study of stomach
ulcers in swine, calves, cows, sheep, and dogs. Although many different
organisms were cultured from the stomachs, Streptococcus spp. were iso-
lated in pure culture or predominating numbers from many of the ulcers.
Jensen and Frederick (1939) reported on an 8-year survey of the stomach
ulcers of slaughter-age swine at a packing plant, and again, species of
Streptococcus were considered as the etiolOgic agents.

Dodd (1960), in New Zealand, described an ulcerative gastritis and
an anemia condition caused by gyostrongylus 333313;. It was clinically
characterized by a retarded growth rate and diarrhea. This condition
most seriously affected the adult breeding sows, and many deaths resulted

from hemorrhage from 1 or more deep gastric ulcers.

Association with Nutritional Deficiencies
Obel (1953), while studying toxic liver dystrophy in swine, discussed
cardiel stomach ulcers as a cause of fatal gastrorrhagia. Although the
stomach lesion was not believed to be characteristic of the liver disease,

a slightly higher incidence of ulcers was noted in experimental groups

60

61
fed high levels of cod liver oil. In some groups the incidence of cardial
ulceration was 22%, and this was attributed to unsaturated fatty acids
and peroxide formation in the absence of sufficient vitamin E.

Hvidsten (1955), while studying the effects of nitrites in pig
rations, described hemorrhages in the stomachs of pigs fed high levels
of sodium nitrite and cod liver oil. This author reported a lack of
vitamin A deposition in the liver.

Pallaske (1960) and Nieberle and Cohrs (1962) described parakera-
totic lesions of the cardial region of the stomach and related these
lesions to vitamin A deficiency or to toxicoses related to a vitamin A
deficiency.

Barron and O'Brien (1963), in Ireland, observed a similar syndrome.
In 2 fatal cases, the livers contained 39.8 and 58.9 1.0. of vitamin
A/Gm., which they considered suggestime of a deficiency. At necrOpsy
the large intestines contained blood-stained ingesta and the stomachs
were ulcerated in the cardisl area. They also found keratinization of
the nonglandular portion of the stomach which was described as thickened,

roug , corrugated and yellowish-brown in color.

Incidence Studies

Jensen and Frederick (1939) in their 3-year study of slaughter-age
swine reported the rate of ulcers in the 20,000 pig stomachs examined
as 5%, and the highest incidence occurred in the late winter and spring
months. Kernkamp (19u5) investigated the incidence of ulcers in Minne-
sota and found 2.38% of 754 swine affected. Berg (1960) discussed a
similar ulcer problem at the Iowa Boar Testing Station and indicated a
definite increase in the incidence of gastric ulcers in swine from 1956

to 1960.

62

Perry (1962) reported a 25% incidence of gastric ulcers in 164 swine
slaughtered at the Indiana Swine Evaluation Station but found no breed
or area predisposing factors. In his experimental studies at the Purdue
Agricultural Experiment Station, stomach ulcers were found in more than
50% of the swine, with the highest incidence occurring in swine used to
test the value of cooked or gelatinized corn as a component of growing
and finishing rations. In contrast, no ulcers were found in the stomachs
of pigs not fed the heat-treated corn. Therefore, he concluded that heat
treatment of corn, as in pelleting, was a contributory factor in the
development of gastric ulcers. In a later report, Perry 23.31. (1963)
discussed their previous work and described a subsequent eXperiment using
gelatinized rations, and they reported a 53% incidence of gastric ulcers.
Although the pigs fed raw corn had no stomach ulcers, supplemental
thiamine, vitamin 812, l-lysine, or fish meal failed to alter the inci-

dence in pigs fed gelatinized rations.

Clinical and Pathologic Findings

Bullard (1951) published a brief clinical description of eSOphago-
gastric ulceration in a large boar in which the ulcer was described as a
round lesion. 3% inches in diameter, surrouhding the esophageal orifice
which resulted in a secondary anemia and, finally, death.

KOwalczyk (1958), at the University of Wisconsin, described the
clinical symptoms and gross pathology of 6 cases of fatal gastrorrhagia
in swine during a Z-year period. Although some were indoors and others
on pasture, all were fed well-balanced rations. Two to 3 weeks before
death the animals appeared to lose weight. Later, vomition of blood and
bloody stools were observed. At necrOpsy the stomachs were filled with

fresh and clotted blood.

63

In a more elaborate report, Kowalczyk gt,§1. (1960) described the
stomach ulcers found in swine at the University of Wisconsin farms during
1957 to 1959. During the summer of 1957, several cases of fatal gastro-
rrhagia occurred in 3- to 5-month-old Chester White and Duroc swine.
These animals died suddenly and at necropsy had distended stomachs
filled with free and clotted blood and ulcerations in the glandless
region of the cardie and in the fundus. They also described a case
in an 8-month-old Hampshire boar in which the stomach was filled with
blood clots and tarry ingesta was present in the intestine. Besides 1
large ulcer, found in the glandless region of the stomach, others were
present in the fundic region and the torus pyloricus.

During this 1957 to 1959 period, a series of experiments *was con-
ducted to determine factors in the development of parakeratosis. Twelve
of the 161 swine died of acute stomach hemorrhage and 15 additional pigs
had stomach ulcers when the swine were slaughtered. In a subsequent
eXperiment using 35 swine, 3 died of acute stomach hemorrhages; and at
slaughter 15 of the remaining 27 had stomach ulcers. However, in a
later experiment with 56 weaned pigs fed similar rations, none died of
gastrorrhagia. At slaughter, 5 of the animals had slight erosions in
the glandless area of the cardisl region. These workers also reported
unsuccessful attempts to reproduce stomach ulcers by intravenous injec-
tion of microorganisms isolated from the base and edges of naturally
occurring ulcers. Supplements of zinc. c0pper, and cadmium neither caused
nor prevented the ulcers. A seasonal factor was suggested to have an

etiologic role.

6h

The pathOgeneses of the lesions were described in detail. In the
early stages, the stratified squamous epithelium proliferated with sub-
sequent necrosis of the mucosa. The acute inflammation was manifested
by congestion of the adjacent mucosa and submucosa, and the capillaries
and small blood vessels were prominent. A cellular reaction of neutro-
phils and a few lymphocytes formed the zone of demarcation. As the
necrotic process continued, the ulcer deepened, the walls of the capil-
laries and small vessels were destroyed, and blood oozed into the lumen.
Small thrombi developed in the adjacent vessels, and a more pronounced
_ leukocytic infiltration occurred. As the process of necrosis subsided,
lymphocytes and eosinophils infiltrated the lesion and the fibroblasts
became more prominent. If fatal hemorrhage did not occur, fibrosis at
the base of the ulcer was evident. The lesions in the glandular and
nonglandular portions of the stomach were described simultaneously,
indicating their apparent close relationship.

Rothenbacher gt 31. (1963) described the stomach ulcer-gastrorrhagia
syndrome in 70 Michigan pigs which died of stomach hemorrhages. This
report distinguished between the lesion found in the cardial portion of
the stomach and the peptic type ulcer found in the glandular region.

In the early or subclinical stage, the cardial lesion was characterized
as a parakeratotic proliferation of the squamous epithelium. This was
followed by progressive surface necrosis, erosion, ulceration and hemor-
rhage. Histologic changes in the malpighian and basal layers included
edema of the intercellular Spaces with microvesiculation, hydropic
degeneration, and a lack of keratinization. Although the fibrinonecrotic

membranes were invaded by microbial organisms, including cocci, bacilli,

65
and fungi, they were considered secondary invaders, since these micro-
organisms were not found in the submucosa. These authors compared this
ulcer-gastrorrhagia syndrome to various toxicoses or deficiency syndromes,
such as parakeratosis or a vitamin A deficiency. Since many of the pigs
were fed an all-plant ration, the possibilities of nitrite or other toxic
agents as etiologic factors were postulated. A seasonal variance in
incidence was also observed.

Curtin gt El. (1963) characterized the clinical and pathologic
aspects of the asaphagogastric ulcers in swine at the Indiana Swine
Evaluation Station. Of the 443 pigs examined, 383 (86.45%) had abnormal
keratinization of the stratified squamous epithelium of the stomach.
Although only 4 died as a result of hemorrhage, ulcerations were observed
in 87 (19.64%). Candida albigans was isolated from many of the lesions,
but these authors also considered it as a secondary invader. They cited
stresses and increased demands for necessary metabolic requirements as
a primary etiologic factor. Since more deaths occurred during late
spring and early winter months, they also observed a seasonal variance
in the incidence. ‘Introduction of diseases into a herd, changes in
management, environmental variation, and immunization were incriminated
as other stresses. Although sufficient vitamin A was present in the
diet to promote rapid growth, they postulated a subclinical deficiency
resulting frtm inhibitory substances or a lack of enzyme systems to
function at an accelerated growth rate. Since specific etiologic factors
‘were unknown. they indicated that rapid growth appeared important to

the formation of this asaphagogastric lesion.

66
Cgpdidiasis and Ulcers

Kovalev (1947) was one of the first to report clinical candidiasis
in pigs. Although the causative organism was Q_i_d_i_u_g albicans, it was
identical to the Monilia albigans, or Candida albicans, as named today.
The disease was characterized by lesions on the nose, gums and buccal
mucosa, which later resulted in emaciation and death. In addition to
local necrosis, he postulated that the organism produced a systemic toxin.

Quin (1952) described moniliasis in swine droves in Canada. Though
the clinical picture Varied, some droves had persistent diarrhea, encepha-
litic symptoms, excessive thirst, and a variable death loss. In 1 group
of pigs the esophageal-gastric junction was occluded by inflammatory
fungal lesions which yielded a pure culture of Monilia. Since death
losses stopped when antibiotics were removed from the ration, he concluded
that antibiotic residues in the feed were related to this syndrome.

McCrea and Osborne (1957) reported a case of "thrush" caused by
Q. albicans in an artificially raised pig. After 14 days on a semi-
synthetic diet the pig was vomiting and emaciated; and it was later
euthanatized. At necrOpsy, a pseudomembrane was nearly occluding the
terminal portion of the esophagus. Although g. albicans was identified
from the colon. an organism belonging to the genus Candida was isolated
from the esophagus. Histopathologic examination revealed degenerative
and necrotic cellular material, neutrophils, and the yeast hyphae. These
authors indicated that candidiasis is rarely diagnosed in swine but
that it was frequently diagnosed in man and poultry.

Gitter and Austwick (1959) reported mucormycosis and moniliasis in

a litter of pigs. In some of the lesions. Candida albicans occurred

67
concurrently with the mucormycotic organisms. But C. albicans were
present only in the stratified squamous epithelium in the stomach; the
mucoraceous hyphae were present only in the glandular portions of the
stomach. In the lesions attributed to Q. albicans, histopathologic
examination revealed necrotic and degenerative processes in the epithelium
and yeast cells and pseudohyphae in the cellular debris. These were
accompanied by numerous mononuclear cells and eosinOphils in the lamina
propria, marked thickening of the muscularis mucosa, and a slight
edema of the submucosa. Similar lesions were described in the esophagus,
lips and tongue. C. albicans was not only isolated from the lesions
but also from the bedding, drinking water, and air in the pigs' pens.
' These isolations indicated the possibility of exogenous transmission, a
concept of great importance in the study of both human and animal
candidiasis.

Osborne gt 21. (1960) reported this disease in 41 artificially
raised pigs, 25 of which died of the infection. The clinical signs
included dullness, anorexia, and diarrhea; and the mouth, tongue, hard
palate and stomach contained lesions. Although the stomach lesions were
in general less severe, occasionally the superficial mucosa was eroded.
Degeneration and necrosis of the stratified squamous epithelium and
infiltration of neutrophils, plasma cells, lymphocytes and eosinOphils
in the submucosa were noted microscopically. Though the epithelial
layer contained numerous yeast cells and pseudohyphae, the submucosa was
not penetrated. These authors treated the disease successfully with
Nystatin, an antimycotic agent. They also discussed the predisposing

factor of antibiotic-containing feeds and concluded that the small amount

68
of antibiotics used in the diet did not alter the normal gut flora
sufficiently to predispose to infection by Q. albicans.

In Wisconsin, Baker and Cadman (1963) isolated C, albicans from
unthrifty pigs, 2 to 8 weeks old, in 8 different herds. One case history
involved 160 pigs, 80 of which died of the infection and 30 of which were
severely stunted. Despite antibiotic and furazolidone therapy and excel-
lent management and sanitation, losses persisted. The clinical signs
included restlessness, inappetence, and vomition. At necropsy, the
stomachs were distended with gas and contained a dark green, slimy
liquid. The mucosa of the asephageal region, which was thickened and
had a rough, scaly, and corrugated appearance, was easily removed and
had shallow ulcerations and inflammation. The desquamated epithelium
contained numerous mycelial fragments and yeast cells of C, albicans.
Although the mortality and morbidity rates were not as high in the other
7 case histories described, the clinical signs and necropsy findings were
similar. These workers concluded that g, albicans had a predilection
for the esOphageal region of the stomach and for the lower portion of the
asephagus. Since it was not known whether Q, glbigans was capable of
infecting normal pigs, they also concluded that this problem may have
been secondary or incidental to other existing conditions.

An anonymous author (1963) reported a high incidence of fungi in
the stomachs of swine in which gastric ulcers were observed. 0f more
than 600 animals examined, 13.5% had gastric ulcers and 97% of these
‘were located in the esophageal region. Cgpdidg albicans was isolated
from 47.3% of the ulcers and Qandida.§pp. from 33.3%. In stomachs with
no ulcers, but which were not completely normal, 9, albicans was isolated

.from 13.8% and Candida gpp. from 21.6%. Moreover, 87% of the stomachs

69
from which 9. albicans was isolated had keratinization of the eSOphageal

region.

Prednisologg—Inggced Ulcers in flats
Nature of Prednisolone

Beckman (1958) described prednisolone as a synthetic dehydrOgenated
analogue of hydrocortisone. In addition to being one of the most power-
ful glucocorticosteroids, prednisolone is used to correct adrenal
insufficiencies, to inhibit inflammatory reactions, and to counteract

allergies.

Prednisolone-Ulcer Relationship

Robert and Nezamis (1958) referred to the develOpment or reactiva-
tion of gastroduodenal ulcers in man as a serious side effect of cortico-
steroid therapy. Thus, a method of producing similar ulcers in experi-
mental animals was available. They produced ulcers in both fasted and
nonfasted rats with steroids: however, in the nonfasted group it required
10 to 15 days of treatment with high doses. The steroid-induced ulcers
were always found in the glandular portion of the stomach in rats and
resembled the human peptic ulcers. These were characterized by hemorrhages,
necrosis of a segment of the mucosa and formation of a crater that was
composed of necrotic debris and leukocytes. Although these ulcers were
usually multiple in number, they never perforated the stomach wall.

These authors also reported that steroids prevented the appearance
of ulcers in the forestomachs of fasted rats or rats with a ligated
pylorus. The usual ulcerated lesion in flhe forestomach was an inflamma-

tony reaction initially caused by cOncentrated gastric juice. This

7O
lesion was characterized first by a tremendous edema of the forestomach
wall and an infiltration of inflammatory cells. Since the blood vessels
were compressed by the edema, a state of malnutrition of the surface
epithelium occurred. Necrosis resulted because the malnourished tissue
was not resistant to gastric juices. Steroids prevented the initial
inflammatory reaction: there was no edema or ischemia, and the mucosa
retained its integrity.

In a subsequent paper, Robert and Nezamis (1963) reported the
effects of prednisolone on mucus formation. Using fasted adult Sprague-
Dawley rats. 5 mg. of prednisolone was given daily subcutaneously for periods
from 1 to 6 days. From their findings of decreased gastric juice acidity,
they concluded that steroid ulcers were not related to hyperacidity.
They reported, however, a significant decrease in gastric mucus in the
steroid-treated rats. From these findings of decreased mucus, the
appearance of steroid-induced ulcers was attributed to the cortico-
steroids, cendering the mucosa more sensitive to the digestive and irri-
tating prOperties of gastric juices by weakening the mucous barrier.
This latter explanation of the pathogenesis of steroid-induced ulcers
overshadowed the widely accepted theory that the ulcers were related to
hyperacidit .

In summary, the steroid-induced ulcers in rats were limited to the

glandular region of the stomach and were related directly to the decreased

gastric mucus rather than hyperacidity.

OBJECTIVES

The specific objectives of this research were:

1. To determixma the nature of the stomach ulcer problem with
respect to -fi) the incidence, (B) the role of vitamin A deficiency,

(C) gastric hyperacidity, ~D) the eosinOphilic hepatitis complex, and
(E) the role of Candidé glbigans.

2. To observe and record gross and microscopic lesions associated
with the gastric ulcer syndrome, candidiasis, and steroid-induced ulcers
in pigs.

3. To determine the pathogenicity of Candida albicans when given
by various routes of administration to week-old normal pigs.

4. To attempt to produce ulcers by giving high levels of predniso-

lone intramuscularly to pigs.

71

+
a

u“.

g was used as X.e mental ar dmal bec cause it was (1) the

(D

snoozes in which the stomac F ulc s were observed naturally and from

Ina alcicans was isolated, and (2 ) availaole for this research.

From December 1. 1952. to June 30, 1953, the data related to the

CllnlC€L study of stomach ulcers were accumulated from lu7l pigs slaughtered

- 4-
'54
(D
n
'1)

at ”ichig an State University Meats Laboratory. The average age of

pigs was 5 to 6 months, the aveflage weight was 200 pounds, and all an eared

'5)

Q

1 ' o

clinically normal on antemortem inspection.

’5

Experiment I consisted of an epizsological study of 3 groups 0:

pigs: (1) from the Hichigan State University Swine Farm and their experi-

‘

rom the Swine Evaluation Station at fiicnigan State University;

and (3) from all over Xichigan that were entered in various Lefi, FFA, and

at

other shows which required carcass data. In addition to these 3(1 pigs,

N!

’( pigs were fed diets var;w ing in vitamin a and carotene content, and
at slaughter the stomachs were examined.

Exneriment II consisted of 12 pi gs used in a study of candidiasis.

‘ J

‘1—

These were ra‘ntained in rooms 57 and 53, Giltner hall, richigan Ftate

University. Assignment to various treatrw nts was made V “Sin? F95a3*

p

numbers (Dixon and La

"\
4

ssey,1;571.

‘

F.xperim?nt III, Trials 1 and 7, consisted of 24 pigs used in the

study of the eprednisolone-ulcer syndrome. In Trial 1, 4 pigs were

“Q
"J

73
purchased from a nearby farm and maintained at the Veterinary Research
Farm. In Trial 2,:he 20 rigs that were used were raised at the Michigan

«.50

State University Swine Farm.

Agglytiggl_frocedures
Hematology. Blood samples were collected from the anterior vena cava,
using ethylene diaminotetraacetic acid (EDTA) or heparin as an anti-
coagulant. The samples were collected at selected intervals. depending
on the experiment. HcmOglobin was determined by the cyanmethemoglobin
method, packed cell volume Values by the micro method (capillary tube),
and total white blood cell counts were made using Turk's diluting fluid‘
and the hemacytometer. At the time blood samples were collected, blood

smears were made, then stained with Nright's stain and a differential

white blood cell count was determined.

Histopathology. Tissue sections were collected, preserved in buffered
10% formalin, and were stained with hematoxylin and eosin. Selected
sections from Experiments I and II were stained by the Gomori's silver
methenamine method to demonstrate fungi, and selected sections from
Experiment III were preserved in Carnoy's fluid and stained by the
Best's carmine method to demonstrate glyc0gen. These procedures were

according to the United States Armed Forces Institute of Pathology

14

Manual of Histoloric an: Special Staining Technics (1957).

L

Mycologio Examinations
Sabouraud's glucose agar with 20 units of penicillin and #0 units

of streptomycin added per ml. was used for the isolation f C. albicans

 

in Experiments I and II. For identification of the typical chlamydospores,

"Difco" cornmeal agar was used (Beneke, 1962).

7#

Specific Procedures for Each ”garment
EXperiment I (Nature of Ulcer Problem)

. These pigs were fasted approximately 18 hours prior to slaughter.
Using an ice tong type electric immobilizer (Valley Forge Research, Inc.),
the pigs were stunned by a humane method. Then they were exsanguinated
by severing the large vessels anterior to the heart. After death, the
carcasses were scalded at 1&2 F. and dehaired. The average time lapse
from stunning to evisceration was 30 minutes. After evisceration, the
viscera were examined according to routine post-mortem procedures. Then
the stomach was opened by incising along the greater curvature, which
allowed a careful gross examination of the mucosal surface and the wall.
The stomachs were classified as (1) normal, (2) keratinization of the
esophageal region. (3) ulceration of the eSOphageal region, and (h)
ulceration of the fundic region.

The pH values of 167 stomachs were determined with a Beckman Glass
Electrode pH Meter - Model H-Z using a Beckman 39142 Combination Electrode
(Beckman Instruments, Inc., Fullerton, Calif.). These values were
determined in the eSOphageal region and were recorded after inserting
the electrode 1 cm. into the esophageal orifice. The pyloric values were
obtained with the electrode Opposite the torus pyloricus.

Samples for mycologic examination were collected from 10 stomachs
‘with esophageal ulcers and 10 with keratinized lesions. The lesions were
swabbed, streaked on Sabouraud's pen-strep agar, and incubated at 22 C.
for 72 hours. The characteristic colonies were removed, streaked on
cornmeal agar and incubated. .Q. glbigggg was identified by means of its

typical chlamydospores.

75

Experiment II (Candidiasis)
Twelve Yorkshire crossbred baby pigs were born April 17, 19€3.
At 5 days of age, the pigs were given 2 ml. of an iron preparation
(Armidcxtran, Armour & Co.) intramuscularly. At 6 days of age, the

pigs were weaned, ear notched and weighed, and blood samples and rectal

swabs were collected.

.-

”he 12 pigs were randomly selected and put into 5 groups, 3 groups

,s

of 2 each and 2 groups of 1 each. The ration consisted of homogenized,

J

pasteurized, whole milk and eggs (1 egg per quart of milk), and mixed

by a Haring Flendor. The pigs were fed 3 times a day ad libitum.

 

Candida albicans, type B, were grown on Sabouraud's medium. The
colonies were then removed and placed in physiological saline. With a
hemacytometer, the yeast cells were counted and the average per ml. was
calculated as 357,000 organisms. Except for the controls, each pig was
given 1 ml. of this stock culture. The oral administration was in 20 ml.
of the milk-egg mixture at feeding time, the intravenous injection was
made into the anterior vena cava, and the intraperitoneal injection was

made into tne rirht lower abdominal region. In the remaining 2 pigs the

D

o H

foramen magnum was located, and with a 2-inch, 23-gauge needle, the 1 ml.
was inoculated into the subarachnoid space. Blood samples were collected
at selected intervals using EDTA as an anticoagulant, rectal swabs were
taken, and weights were recorded twice per week. The eXperiment lasted
21 days. The pigs were then euthanatized with electricity and carefully
observed for gross changes at necroosy. Specimens from several vital

rgans were collected for histopathologic examination.

76

Experiment III (Prednisolone)
Trial 1. Four Yorkshire crossbred pigs, 8 weeks of age, were divided
into 2 groups of 2 each. Using 1 group as the control, the other 2 pigs
were given 100 mg. of prednisolone‘ intramuscularly daily for 7 days.
Feed was withheld the last 72 hours for both groups, and the pigs were
necrOpsied 12 hours after the last injection. Gross observations were
recorded and selected specimens were preserved for histOpathologic

examination.

Trial 2. Twenty.crossbred pigs were divided into 2 groups of 10 each.
One group of l0 was used as controls. The experimental group of 10 was
subdivided into 2 groups of 5 each. One group was given 100 mg. of
prednisolone intramuscularly per pig per day for 7 days, and the other
group was given 200 mg. in a similar manner. Feed was withheld 27 hours
before slaughter, and the pigs were slaughtered 12 hours after the

last injection. Heights were recorded at selected intervals. At
slaughter, the tissues were examined for gross lesions, selected sec-
tions were preserved for histopathologic examination, and the weights

of the livers, hearts, stomachs, and adrenal glands were recorded.

 

*Delta-Cortef, Sterile Aqueous Suspension, the Upjohn Company, Inc.,
Kalamazoo, Michigan.

RESULTS
WW
Incidence and Epizoology

The incidence of stomach lesions was determined in 1071 slaughter-
age pigs which included 673 pigs from the Michigan State University
Swine Farm, 170 from the san.‘ valuation Station, and 228 from outside
sources. The data regarding the monthly incidence of lesions are given
in TABLE 2-1. Data.summarizing the source of the pigs and incidence of
stomach lesions are presented in TABLE 2-2. Although all the pigs
appeared clinically normal on antemortem inspection, 39 of the 115 with
esophageal ulcers had free blood in the stomachs on examination at
slaughter. The highest incidence of all types of stomach lesions
occurred in the pigs from the Swine Evaluation Station, and the highest

incidence of ulcers occurred during the months from December to April.

Role of Vitamin A

Stomachs of 97 pigs from a vitamin A experiment were examined at
slaughter to determine the role of vitamin A. These pigs were fed a basal
ration low in vitamin A and the same ration with varying levels of caro-
tene and vitamin A supplemented. The information regarding the incidence
of stomach lesions in pigs fed various amounts of vitamin A and carotene
is given in TABLE 2-3. No major differences were noted in the incidence
of stomach lesions among the different groups: however, only 9 of the

97 stomachs examined were grossly normal.

77

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AELB 2-2.--Incidence of Stomach Lesions in slaughtered Pigs from
Various Origins.

 

 

 

No. of Stomach Lesions
Origin Pigs Keratinization Esophageal Ulcer Fundic Ulcer
Swine
farm 673 222 63 23
7e 33.0 9.6 3.4
Swine
Eval. 170 115 47 1h
.13 67.7 27.7 8.2
Other 223 #1 5 l
f‘é 13.0 2.2 o.u
Total 1071 376 115 38
:‘é 35.3 10.7 3.5

 

TABLE 2-3.--Incidence of Stomach Lesions in Figs on Basal Rations Low
in Vitamin A With Various Levels of Carotene or Jitamin A

 

 

 

 

 

Supplemented.
No. Stomach Lesions
Group of Esoohageal Fundic
No. Pig: Ration Normal Keratinization Ulcer Ulcer
1 9 Basal 2 7 O O
2 44 Carotene b 3h 3 3
added
. 3 “4 Vitamin A 3 38 l 2
added

Total 97 9 79 u 5

 

80

The Role of pH

The pH values were determined in stomachs from the 97 pigs of the
vitamin A experiment and 70 pigs fed a standard swine ration. The
average pH values of the esophageal and pyloric regions of the stomachs
and the relationship to the gross lesions are given in TABLE 2-4. The
average pH value in the esOphageal region of normal stomachs was 2.5
and in the pyloric region. 3.5. Ln contrast, the average pH Value was
3.8 in the keratinized esophageal regions, 5.2 in the esophageal region
of the stomachs with fundic ulcers and 6.2 in the ulcerated esophageal
regions. Although free blood was present in 3 of 9 stomachs, the pH
range for all stomachs with ulcers in the eSOphageal region was 6.2 to
6.6. In general, the highest pH values were recorded in stomachs con-

taining free blood.

Relation to Eosinophilic Hepatitis Complex
The data on liver condemnations from 1305 pigs slaughtered between
August 1, 1962, and June l, 196“, are given in TABLES 2-5 and 2-6. The
highest incidence of liver condemnations for eosinOphilic hepatitis
occurred from September through January of each year and occurred from
swine raised at the Swine EValuation Station. The incidence of stomach
ulcers and eosinophilic hepatitis in the same animal was lower than the

naturally occurring incidence of either (TABLES 2-2 and 2-5).

Gross and MicroscOpic Lesions
Keratinization. Keratinization was characterized grossly by a prolifera-
tion of the squamous epithelium of the esophageal region of the stomach.

This proliferation appeared as bright yellow to brownish-yellow plaques.

81

TABLE 2-4.--Average pH Values of Stomachs from (A) Pigs Fed Rations
Varying in Carotene and Vitamin A. and (B) Pigs fed
Standard Rations.

W
Stomach Average pH
Lesions ESOphageal Orifice Pyloric Orifice

A. Pigs fed rations varying in carotene and vitamin A

Normal (9)* 2.6 3.5
Slight keratinization (23) 3.4 4.3
Severe keratinization (56) 4.3 4.9
Total keratinization (79) 3.7 4.5
ESOphageal ulcer (4) 6.1 6.2
Fundic ulcer (5) 5.0 5.8
Total (97) 3.4 4.3
B. Pigs fed standard rations
Normal (31») 2.5 3.5
Keratinization (30) 3.9 4.7
Esophageal ulcer (5) 6.3 6.5
Fundic ulcer (l) 5.7 5.8
Total (70) 2.8 3.8
C. Composite of A and B
Normal (#3) 2.5 3.5
Keratinization (109) 3.8 4.6
ESOphageal ulcer (9) 6.2 6.3
Fundic ulcer (6) 5.0 5.8
Total (167) 3.0 4.0

 

*( ) Number of stomachs examined.

TABLE 2-5.--Incidence of Liver Condemnations in Slaughtered Pigs from
Various Origins.

 

 

Eosinophilic
No. of EosinOphilic Ascarid Hepatitis
Origin Pigs Hepatitis Sears and Ulcers
Swine Farm 856 137 26 13
3% 16.0 3.0 1.5
Swine EVal. 221 57 8 7
6 25.6 3.6 3.2
Other 228 33 38 2
6 14.5 16.7 1.0
Total 1305 227 72 22

% 170“ 5'5 1'7

 

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83

and the extent of keratinization varied from small areas to the entire
esophageal region. Fig. 2-1 illustrates moderate to heavy keratinization.

Histopathologic examination of these keratinized lesions indicated
a parakeratotic proliferation of the squamous epithelium. Below the
outer layers of nucleated epithelial cells, a 5- to 8-ce11 layer had
ballooning degeneration. The malpighian layer was hyperplastic. similar
to acanthosis. EVen though fungal hyphae were often seen in the super-
ficial layers of these lesions, little to no inflammatory reaction was
noted. Fig. 2-2 illustrates uniform parakeratosis and acanthosis. and

Fig. 2-3 illustrates parakeratotic plaque formation.

Esoghageal ulcers. Nearly all eSOphageal ulcers were accompanied by
moderate to extensive keratinization of the squamous epithelium. Grossly,
these ulcers varied from (1) a single break in the continuity of the
mucosa, to (2) numerous bleeding ulcers with clots attached. or to (3)
complete loss of epithelium and replacement with granulation tissue.
The first type of ulcer was characterized by a separation in the mucosal
surface at or near the squamous-glandular epithelial junction (Fig. 2-4).
The second type consisted of the first plus hemorrhage (Fig. 2-5). Only
a few stomachs contained attached clots. and usually the stomachs were
filled with free blood clots and unclotted blood. Although a few of the
stomachs contained a small amount of feed, usually no food material was
detected. The third type of ulceration consisted of a chronic fibrino-
necrotic gastritis of the esOphageal region in which no mucosal surface
was evident (Fig. 2-6).

Microscopically. the first type of ulcer had parakeratotic prolifera-

tion in some areas and complete loss of epithelium in others. The

,..‘
‘ (.y H ~ , I ‘
‘ 7 . 1 . mlmummmlmlmumnmm

 

 

 

iFig. 241.;A stoma—ch of pig with a keratinized esophageal
region and adhering mucus in the fundic region.

  

Fig. 2421JPhO—{omifirhgr-Vaph—ofT‘ig. 211. Fake—rattan pro-
liferation of the squamous epithelium of the esophageal region.
B a E stain; x 75. '

 
    

I. - '- ‘ ' - -a

 

Fig. 2L3.--Parakeratotic proliferation of squamous epithelium
on the esophageal region of the stomach. Note the plaque formation.
H & E stain; x 75.

 

 

 

. . ‘ f. ..
n:"\"“' "‘ 1 "V I H.‘ , u r v
I . .2 Q n n m u n n+1.

 

 

Fig. 2-4.--The stomach of a pig with a keratinized esophageal
region. At the squamous-glandular junction. small ulcers are
present.

 

 

Fig. 2-5.--E50phagea1 region of a pig's stomach with ulcers
and attached blood clots.

 

 

Fig. 2-6.--The stomach of a pig with a fibrinonecrotic lesion
in the esophageal region and adhering mucus in the fundic region.

phils. l"mphocvtes, and varying degrees of edema (
Colonies of bacteria and fungal hyphae were occasionally present on the
surface. but rarely invaded the submucosa. The second. or bleeding-type,
ulcer contained inflammatory exudates. thrombosis. erythrocytes. and
necrotic debris. The third type of ulceration was characterized by
numerous lvmphccytes. neutrOphils and fibroblasts infiltrating the

filrinonecrctic area. Jo epithelium was present. and usually bacteria

invaded the surface of the lesion.

Fundic ulcers. Grossly, these lesions appeared as dark reddish to

black ulcers in the fundic region and were surrounded by an area of

‘3

hyperemia (Aig. 2-9}. In different stomachs. these lesions varied in

I

number from 1 to 6 and in average diameter from 1.5 to 70 mm. hiero—
scopicclly, coagulation necrosis of the glandular epithelium, occasional
thrombosis of olood vessels. and erythrocytes on the ulcerated surface
he -nflammatcry reSponse included infiltrations of
lymmhocytes and neutronhils and proliferation of fibroblasts (Fig. 2-10).

so fungal or bacterial o~ganisms were noted in these fundic lesions.

Eosinophilic hepatitis and ascarid-scarred livers. The livers seen on
routine post-mortem examination classed as eosinophilic hepatitis con-
tained varying numbers and sizes of lesions which were visible not only
on the surface but also in the parenchyma of the liver. Some 0 the
lesions were raised from the surface. causing the capsule to bulge.

The affected livers varied from normal in size to greatly enlarged.

88

 

FEET 2-7.:;Submucosa 6E esophageal §3gioh of stomaoh'shown
in Fig. 2-4. Note the neutrophils and lymphocytes. H & E stain;
x

 

a ’ ' A

 

fiéjgée8.--Higher power of Fig. 2-7. Note the numerous neutro-
phils and proliferating fibroblasts. H & E stain; x 750.

 

 

 

 

 

"Fۤ. 2-9.--Stomach of a pig with 2 ulcers in the fundic
region.

    

3.: ”3%;Mmflz'whv'wfiw

fig. é:10.--Photomicrogra£h_of Fig. 2-9. Ulcerative gastritis
of the fundic region of stomach. Note coagulation necrosis of the
mucosa with infiltration: of inflammatory cells. H & E stain; x 75.

 

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,

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0
cf
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3.1.
F J
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Palpation of the all dicated that the most severely
affected were friaole. The less extensively affected livers were normal

idual lesions were from 1 to h cm. in

ow 4
‘1
(D
Ho
:3
> L:
«c:

to firm on palpation.
diameter. and the color varied from gray to yellow. Since no tendency
toward encapsulation by fibrosis was seen. the lesions appeared prolifera-
tive and disseminated into the normal parenchyma. The cut surface of

the lesion abreared as yellowish to gray perilobular discoloration with

‘- . ‘5. -‘ u‘ ' \\'- -- 1‘ u a 0 fl ‘ 1 ‘J _‘ v " "1
,eCLial hemorrhages. the nenatic lvmon nodes were normal

(J

F-J

4 4L
9.4, \a-‘e

atter oeing congested and edematous.

L‘1

O ‘ I O ‘ .0} .—
\_A

i;. T-ll illustrates a liver witn eosinonhi ic hepa‘; .. In contrast.
grossly. ascarid-scarred livers had whitish foci. crima‘ily on the
surface of the liv.r. These foci were 2 to 5 mm. in diameter and did
not extend deeclv into the carenchyma (Fig. e-lll. .ne he atic lymph

‘ V“ r ‘ ‘ A 'A. 1 .-- r‘ . ' r- c .- ‘ -: ,‘ -~ ' p O r" ' ‘ ‘1‘ .
n.des were normal. and in eacn Case ascarlos were found in -ne small

he microscotic exanination of the eosinophilic heoatiti: lesions

. 3"» n .‘ r -' ~ . 4’ 1‘ : ~ W n ' .‘ ‘ - .'
ind-catei acute. subacute. or chronic types. the acute tch was cnarac-

.~ \‘I - a la n .‘ e ‘ '. 94‘ u ' A e: : .1 ‘ ' W ' "‘
twtlceq by an eften1ive in.fltration ox eOslnoohils and occa31onal lymono-

cytes and neutrochils into the perinortal areas. Many of the sinusoids

" 1
:5
L1.
*4
O
O
t.
'4
’1!

' n
J
T
}_ J
(u
:5

ear the lesions were distended with blood. and some of
tee liver cells were undergoing atrochy and necrosis. The subacute

type of eosinoohilic hepatitis consisted of large numbers of eosino-
onils. proliferating fibroblasts. and a few lymphocytes in the ceri-
portal areas. Many of the lobules were reduced in size due to the atrophy
and necrosis of the hepatic cells in the periportal areas (Fig. 2-12).

’1'“

lne chronic type of eosinOphilic hepatitis was characterized by a

 

 

Fig. 2—ll.--Forcine liver. weighing 2740 Gm. Note the
yellowish nodules and the rounded edges.

\

:23

 

~\(?: (.':5 I. :‘E ' ,4. O?
. i‘xn.“ OW

 

‘ligj 2-l2.--Photonicrograhh of Fig. 2-ll. Sutacute perilobular
eosinophilic hepatitis in a pig liver. H & E stain: x 187.

92

marked thickening of the interlobular septa, fibroblastic proliferation,
and the production of collagen. Again, numerous eosinophils and a few
lymphocytes were seen in the septa. Microsc0pic examination of sections
through livers with lesions resembling typical ascarid scarring had a
focal hepatitis characterized by a marked increase in lymphocytes and
neutrOphils. Fibroblastic proliferation and parenchymal cell regenera-
tion were also seen. Some eosinOphils were noted in the examination of

these lesions (Fig. Z-lh).

Mycology
gandidg albicans was cultured and identified from 7 of the 10
esophageal ulcerative lesions and from 5 of the 10 keratinized lesions
(Fig. 2-15). Microscopic examination of some of the stomachs indicated

pseudohyphae in the superficial areas of the parakeratotic lesions

(Fig. 2'16).

Experiment II (Candidiasis)
Analyses
HematolOgy. Hemoglobin and packed cell volume values and total and dif-
ferential white blood cell counts remained within the normal ranges for
the entire experiment in all pigs in each group. Nucleated red blood
cells were seen. but in numbers within the normal range for pigs of this
age. During the first week of the experiment. a moderate macrocytosis

of the red blood cells was noted.

Culture. Rectal swabs were negative for Q. albicans by culture on

Sabouraud's pen-strep agar, except for 2 of the 3 pigs which were fed the

 

\
Fig. 2-13.--Liver of a pig which had numerous large ascarids

in the small intestine. Note the numerous whitish foci (ascarid
scars?) on the surface.

 

 

Fig. Z-lh.--A disseminating typemof lesion in a grossly
typical ascarid-scarred liver. Note the necrosis and the
infiltration of numerous inflammatory cells. H & E stain;
x 18”.

9a

 

 

iiiglré-15.-—Yellow keratinized and eroded esophageal region
of a stomach of a pig. Q. albicans was isolated from this lesion.

 

 

Fig: §:16.e-Q. albiéans in the mucosal surface of a pig stomach.
Gomori's silver methenamine stain: x 187.

95

organism. t necrOpsy. g, albicans was isolated from the cerebrOSpinal
fluid in both pigs given the organism in the subarachnoid space. In 1
of the 2 pigs given the organism intravenously. Q. albicans was isolated
from the lung and liver. In 2 of the 3 pigs which were fed the organism.
Q. albicans was cultured from the rectum and stomach. The typical

chlamydospores for identifying g, albicans are given in Fig. 2-17.

Signs

The daily rectal temperatures of all pigs were within the normal
range, except for pigs given 9, albicans intravenously. Their tempera-
ture increased slightly 24 hours after inoculation and remained elevated
for 3 days. Both pigs given the organism intravenously were dyspneic
‘ for h days after inoculation. On the nth day l of the pigs was weak.
unable to rise. and was then euthanatiZed and necropsied. The 2 pigs given
2.3}pigfgg in the subarachntid space had ataxia, depression and weakness
for 3 days following inoculation. All other pigs were clinically normal

throughout the experiment.

Gross lesions
The lungs of the pig that was necropsied on the 4th day after intra-
venous inoculation contained numerous areas of red hepatization scattered
throughout all lobes. These lesions were 1 cm. in diameter and gave a
speckledcn*ncttled appearance to the lung.The liver contained a few
white foci, which were 1 to 3 mm. in diameter and scattered throughout
the parenchyma. At necrOpsy. the rematning pigs had no gross lesions

attributable to Q. albicans.

 

l ,, a ,, _ ,__ ,aa,v _,,H- ,._u____,a,__aa-_
Fig. 2-17.--Typical chlamydospores of Q. albicans grown on
cornmeal agar. LactOphenol preparation; x 750.

\

Q
‘ "1'
33‘

0 1

  

 

 

~Fig.3-18quranuloma in the lung of a pig given Q. albicans
intravenously. H & E stain; x 187.

 

Microscopic Lesions

Microscopically, the tissues of the pig necrOpsied 4 days after the
intravenous injection had the following lesions:

(1). The lung had numerous interstitial granulomatous lesions
containing macrOphages. lymphocytes. neutrophils, and the fungal
organisms (Fig. 2-18).

(2). The liver contained numerous small granulomas made up of
macrOphages, an occasional giant cell, lymphocytes, neutrOphils. and
the fungal organisms (Figs. 2-19. 2-20, and 2-21).

(3). The myocardium had a few small granulomatous lesions composed
of macrophages. neutrOphils, lymphocytes, and the fungal organisms.

(b). The kidney had a few lymphocytes in the interstitial tissue
of the cortex, but no organisms.

Two of the 3 pigs given the organism orally had the following
lesions:

(1). Erosions of the buccal mucosa containing lymphocytes, neutro-
phils, bacteria. and a few fungal organisms. '

(2). The esophageal region of the stomach had an occasional para-
keratotic plaque. lymphocytic infiltration of the mucosa. and a few
Q. alhicans.

The 2 pigs in which Q. albicans was given in the subarachnoid
space had a mild meningitis. characterized by numerous lymphocytes and

the organisms (Fig. 2—22 .

 

 

fig)" 2493-5353103; with necrosis R the lime; of a pig
given Q. albicans intravenously. H & E stain; x 187.

  

.Q‘} Elfiv‘e: k; t‘, ;;f';:.‘l‘

Fig. 2-20. --Higher power of Fig. 2-19. Note the neutrophils,
lymphocytes and macrOphages. H & E stain; x 750.

99

 
 

Fig. 2-21:4. albicans iii the’g’ranulcfi’sflowniin Fig. 2-19.
Gomori's silver methenamine stain; x 750.

(fig. 2-22.--Q. aIBiéEBE'in the meninges—oan_pig given the
organism in the subarachnoid Space. Gomori's silver methenamine
stain; x 750.

 

Signs
The 2 pigs given 100 mg. of prednisolone daily for 7 days appeared
to consume excessive amounts of feed and water. From the 2nd through
the 4th day, they had extensive abdominal distention which was probably
due to fluid retention. Feed was withheld at the end of the hth day
and the abdominal distention was not apparent the last 2 days of the

experiment.

Gross Lesions
At necrOpsy, the eSOphageal region of the stomachs of the
prednisolone-treated pigs was keratinized and ulcerated. One of the
stomachs contained free blood and a clot attached at the ulcer (Figs.
2-23 and 2-2Q). The fundic region of l stomach had numerous small
ulcers (Figs. 2-25 and 2-26). The control pigs had keratinization of

the stomachs but no ulcers.

Microsc0pic Lesions
The stomach lesions of the prednisolone-treated pigs had parakera-
totic proliferation in some areas, necrosis and complete lack of squamous
epithelium in others. Lymphocytes, a few neutrophils, and erythrocytes
were present in the necrotic ulcerated area. Examination of the fundic
region indicated minute areas of necrosis of the glandular epithelium
and infiltration of the necrotic areas by lymphocytes and a few neutro-

phils.

 

 

Fig. 2-23.--Stomachs from prednisolone-treated and control
pigs. Note the hemorrhage in the eSOphageal region of the
stomach of the prednisolone-treated pig on the left.

 

 

 

Fig. 2-24.--Closeup of Fig. 2-23. Note the clot adhering to
the ulcerative lesion near the junction of the eSOphageal and
cardiac regions.

 

 

 

Fig. 2:25.--Stomachs from prednisolone-treated and control—
pigs. .Hemorrhages and ulcerations are present in the fundic
region of the stomach of the prednisolone-treated pig on the
left.

 

 

 

Fig. -26.--Closeup of Fig. 2-25. Hemorrhages and ulcerations
on the fundic region of the stomach of prednisolOne-treated pig.

103

Trial 2
Signs
No clinical signs of prednisolone toxicity were observed in the
2nd and more detailed study of prednisolone-induced ulcers in pigs.

These pigs were considerable larger than the pigs used in Trial 1.

Analyses

Since no major ‘ifferences were noted in the 2 groups given different
amounts of prednisolone, these 2 groups were combined and the analytical
data are given in TABLE 2-7. Although major differences were noted in
the average weight gains over a 6-day period for the control and treated
groups, after a 27-hour shrink prior to slaughter, these gains were lost
in the prednisolone-treated pigs. In the treated pigs the average
weights of the livers ahd hearts were greater but the average weights
of the stomachs and adrenal glands were lower than those of the controls.
No major differences were noted in the average hemoglobin and packed
cell volume values, but the average total white blood cell counts and

lymphocyte counts were lower, which constituted an absolute lymphOpenia

in the treated pigs.

Gross Lesions
The data on the incidence of stomach lesions are given in TABLE 2-8.'
Only 1 esophageal ulcer was noted in the control pigs, in contrast to

8 in the treated pigs. These ulcers were small, and no evidence of intra-

gastric bleeding was noted.

10%

TABLE 2-7.-—Experiment III. Trial 2. Average Live Weights, Orgar
Heights, and Blood Values of Figs Given Prednisolone

 

 

 

 

 

 

 

Intramuscularly.
Controls Prcdnisolone-Treated
(13 nigsl (lO pigs)
Heights (lbLI
Initial (3.23-5u, 2 p.m.) 219 208
Final (3-29-éu, 6 a.m.) ' 229 225
Gain (6 days) 10 17
At slaughter (3-30-6h, 9 a.m.) 217 207
(Shrink 27 hours) 12 18
Weightsngm.)
liver 137A 2058
Heart 307 368
Stomach 690 600
Left adrenal 2.0 1.5
Hb. (omij1oo mltl 15.u 16.1
Pcv Til A5 #6
was jpeizgjcmmgl 15,900 11.100
#3: ' 3h 59
Stab. O l I
lvmph. 62 35
Mono. 2 b
Baso. n O ‘ O
Eosin. 2 l

 

TABLF 2-3.--Experiment III. Trial 2. Incidence of Stomach Lesions in
Pigs Given Prednisolone Intramuscularly.

 

 

Controls Prednisolone-Treated

(10 pigS) (10 pigS)
Keratinization 8 8
Esophageal ulcers 1 8

Fundic ulcers 1 2

 

105

Microsc0pic Lesions
Microsc0pic examination of the prednisolone-induced ulcers indicated
necrosis of the surface epithelium, a slight infiltration of neutrOphils
and lymphocytes, and a slight edema. The inflammatory response in the
fundic ulcers was also minimal as compared to the control. Other
interesting findings accompanying the ulcer syndrome in the prednisolone-
treated pigs were extensive glchgen deposits in the liver and heart,

as demonstrated by the Best's carmine stain, and atrophy of the adrenal

cortex.

DISCUSSION
A high incidence of gastric ulcers in pigs was indicated by the

data from a total of 1071 pigs, of Which 378 (35.3%) had abnormal
keratinization of the stomachs, 115 (10.74) had eSOphageal ulcers, and
33 (3.5%) had fundic ulcers. The information on the pigs from the Swine
Evaluation Station that had the following incidence of lesions - 67.7%
with keratinization, 27.7% with eSOphageal ulcers, and 8.2% with fundic
ulcers - agrees closely with she Purdue University report of Perry
(1962), who recorded 2;}? incidence of eSOphageal ulcers, and of Olrtin

t al. (l963), who gave a 20% incidence. The data reported herein

ndicate a lower incidence of ulcers in the pigs from the Swine Farm

PI.

and outside sources than those from the Swine EValuation Station.
Although in many cases the rations were similar. the pigs from the
evaluation station were subjected to environmental changes when trans-
ported from the farms to the station.. This is suggested as a possible
stress factor for the pigs having the highest incidence of stomach
ulcers as compared to pigs raised to slaughter weight in a constant environ-
ment. Recently, Muggenburg gt 3;, (l96bb) reported a 15% incidence in
farm raised swine in Wisconsin and a 73 incidence in Iowa and Illinois.
The data in this paper, along with the report by Berg (1960) at the Iowa
Boar Testing Station, indicate that the highest incidence of ulcers
occurs in production pregrams that strive for tap feed efficiency and
growth rate. Regarding seasonal variance in incidence, the work here
indicated the highest incidence of ulcers was from December to April.
This is somewhat in contrast to the report of Curtin §£_al. (1963),

106

107
who reported more deaths due to ulcers in the early winter and late
spring months.

The pathOgenesis and gross and microscopic pathology of the stomach
lesions were similar tothose described by K0walczyk gt_gl, (1960),
Rothenbacher gt al. (1963). and more recently in a detailed report by
Muggenburg gt_al. (1965b). The stomach lesions appear to be initiated
with a parakeratotic proliferation of the squamous epithelium, followed
by necrosis and later ulcerations. The esophageal ulcer and‘fundic
ulcer appeared as separate and unrelated entities from both the epizo-
ologic and pathologic standpoints.

In the experiment on feeding rations containing varying levels of
vitamin A and carotene, there were no major differences in the incidence
of stomach lesions. This information would not support the suggestion
of Rothenbacher gt,gl. (1963) and Curtin gt,2l. (1963), who postulated
that a subclinical vitamin A deficiency was an underlying etiologic
factor in the gastric ulcer complex. The work described previously in
Part I of this thesis indicated that prolonged nitrite ingestion was
not associated with the incidence of stomach ulcers. This does not
confirm the suggestions offered by several workers.

Hyperacidity of the stomach was not incriminated as a possible
etiologic factor. Since the average pH value in the e50phageal region
of normal stomachs was 2.5 as compared to 3.8 in the keratinized
stomachs and 6.2 in the stomachs with esophageal ulcers, hyperacidity
was not associated with the gastric ulcer complex. In fact, these data
indicate that the reverse may be true. The average hydrogen—ion concen-

tration in eSOphageal region of normal stomachs was over 10 times

108
greater than the average hydrogen-ion concentration of stomachs with
keratinization.

A possible relationship between the eosinOphilic hepatitis complex
and the gastric ulcer syndrome was not confirmed. 0n the basis of the
epizoologic study, these 2 conditions appeared to be unrelated. In
addition, other workers have not noted the eosinophilic hepatitis prob-
lem as being associated with the gastric ulcer complex.

Candida albicans was cultured from a high percentage of the kerati-
nized lesions and ulcers in the asephageal region of the stomach.
However, microscOpically, the organisms were in the superficial layers
and not in the submucosa. g. albicans was probably a secondary invader,
but may have possibly interfered with healing. This is in agreement
with the reports of numerous other research workers. In Experiment II,
ulcers were not reproduced by oral administration of g. albicans. This
also indicates that this organism represents a secondary factor in the
gastric ulcer complex. Candida albicans, however, was capable of
establishing itself by intravenous, subarachnoidal, and oral routes of
administration. The information in this thesis indicates that C,
albicans is pathogenic to pigs and causes a granulomatous type of
inflammation.

In Experiment III. the use of the cortical steroid, prednisolone,
indicated that stress factors play an important role in the gastric
ulcer complex. Of the 12 pigs given prednisolone in 2 different trials
for 7 days, 10 had eSOphageal ulcers in their stomachs: in contrast,

1 of 12 controls had an eSOphageal ulcer. Since the steroid-related

stomach ulcers in pigs were not similar anatomically to the same type

109
in man and rats, this indicated a possible inherited weakness in the
eSOphageal region to stress factors. Sections of the livers and hearts
indicated extensive glyCOgen deposition which accounted for the increased
weights of these organs in pigs given prednisolone. The decreased
average weight of the stomachs of the prednisolone-treated pigs may
have been due to its anti-inflammatory prOperties. Pigs fasted prior
to slaughter usuallyinnnaedema of the stomach wall, and perhaps predniso-
lone inhibited the edema that usually accompanies fasting. The blood
values of the prednisolone-treated pigs showed a leukopenia with an

absolute lymphOpenia.

SEGRRY

The epizoology of the gastric ulcer problem was studied using 1071

(D

slaughter-age pigs from the Swine Evaluation Station, Mic igan Stat
University Swine Farm, and outside sources. The study indicated 373
(35.3%) had keratinization of the stomach, 115 (10.7%) had ulcers in
the eSOphageal region (hereinafter called eSOphageal ulcers), and 33
(3.5%) had fundic ulcers. The 170 pigs from the Swine Evaluation Station
had the highest incidence of stomach lesions at slaughter. Although all
the pigs were clinically normal, 39 of 115 with eSOphageal ulcers had
free and clotted blood in their stomachs. The pigs subjected to the
stresses associated with growth experiments and environmental and
management changes had the highest incidence of stomach lesions. Kersti-
nization of the asephageal region was characterized by a parakeratotic
proliferation of the squamous epithelium. This appeared to precede
necrosis and ulcer formation. The e30phageal ulcers and fundic ulcers
appeared as separate and unrelated entities.

No differences were noted in the incidence of stomach lesions in
9? pigs given rations varying in vitamin A and carotene content. The
research on stomach pH values indicated that the average pH value was
2.5 in the normal stomachs, 3.3 in those with keratinization, and 6.2
in those with esophageal ulcers. Thus, low acidity, rather than hyper-
acidity, appeared to be related to the stomach lesions.

Candida albicgns was frequently cultured from stomach lesions, but
this organism was considered a secondary factor because ulcers could not

be experimentally reproduced. Using 12 pigs, it was established that

110

Q. albicans was capable of producing lesions following intravenous,
subarachnoidal, and oral routes of administration.
Twenty-four pigs were used in the study of prednisolone-induced
ulcers. Daily injections of prednisolone caused a high incidence of
stomach ulcers, increased gluconeogenesis, inhibited inflammatory

responses, and caused a leuk0penia with an absolute lymphOpenia.

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‘.I“|’.* Il'rfib‘l ll