ABSTRACT
PATHOLOGIC EFFECTS OF
ESCHERICHIA COLI 083:K.:NM IN
GNOTOBIOTIC PIGS

By Alfred Lenneth Britt

In a series of 6 experiments, 58 gnotobiotic pigs from 6 litters
were utilized in investigation of the pathologic effects of oral exposure
to Escherichia coli serotype 083:K.:NM in gnotobiotic pigs. The pigs
were germfree or monocontaminated with Staphylococcus _2, or Bacillus _2,
at 69 to 148 hours of age, when they were fed g, 22;; inoculums in volumes
varying from 1.25 ml. to 5.0 ml. of brain-heart infusion or thioglycol-
late liquid mediums. The inoculums were incubated at 37 C. for 24 or 36
hours just prior to their being fed to the pigs.

In the first 4 experiments there was 100% morbidity and 84% mortality
among 25 pigs between 10 and 162 hours after exposure to g, ggli_at
ages ranging from 69 to 148 hours. The most prominent lesions consisted
of serofibrinous to fibrinOpurulent polyserositis and/or polyarthritis
in 100% of the 25 principals.

Invasiveness of the organism was indicated by the septicemic nature
of the infection. Four principals were severely ill but not moribund
when they were euthanatized between 99 and 236 hours after exposure to
E, 991;. Septicemia was confirmed by post-mortem microbiological recovery

of E. coli from the exudates in serous and synovial cavities of the

 

euthanatized principals as well as from the principals that had died.

Alfred Lenneth Britt

Macroscopic and microscopic morphologic evidence of gastroenteritis
was usually mild or absent, and results of clinic0pathologic examinations
did not reveal evidence of anemia, hemoconcentration, or serum electro-
lyte (sodium and potassium) derangement in the pigs.

Clinical signs were nonspecific, rather than pathognomonic, and
included fever, hypothermia, diarrhea, partial anorexia, leukOpenia, and
leukocytosis. The occurrence of fever and leukocytosis indicated that
colostrum-deprived gnotobiotic pigs are capable of responding defensively
to bacterial stimuli in much the same manner as other animals.

In Experiments 5 and 6, anti-hog cholera serum was fed to one-half
of each group of principals at 6 hours of age or earlier as a substitute
for colostrum. Some biological protection was manifested by the absence
of mortality and reduced incidence of gross lesions in the pigs fed
anti-hog cholera serum as compared with the principals not fed anti-
hog cholera serum. Infection following exposure to a contaminated
fomite resulted in the only death among the principals in Experiment 6.
Evaluation of the degree of protection against E, ggli infection pro-
vided by feeding anti-hog cholera serum was complicated by an unexplained
reduction of mortality in the group of principals not fed anti—hog
cholera serum. Also, some pigs in both groups were able to eliminate
E, ggli_from the serous cavities after the acute septicemic phase of
infection.

Serums collected from the pigs at 5 to 12 days of age, before and
after exposure to E, ggli_in Experiment 6, were negative for 0 agglutinins
to whole cell E, ggli_083 somatic antigen, alone or combined with latex
particles, in tube agglutination tests in final dilutions of 1:10 and

1:20.

Alfred Lenneth Britt
Results of electrophoresis indicated a range of 0 to 13% gamma
globulin with similar ranges in pigs irrespective of the feeding of anti-
hog cholera serum. Also, there was no difference between groups in
(l) hemoglobin values and packed cell volumes, which increased to a peak

at 12 days of age, (2) declining alpha globulin levels with increasing

1

age, and (3) serum potassium values, which increased with increasing age

of the pigs.

PATHOLOGIC EFFECTS OF
ESCHERICHIA COLI 083:K.:NM IN

GNOTOBIOTIC PIGS

By

Alfred Lenneth Britt

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Pathology

1967

To my wife Jean
and our children

Nancy Jean
Rebecca Ann
Mary Lynn
Daniel Lenneth
David Henry
Kevin Victor

ii

ACKNOWLEDGEMENTS

The author wishes to express his gratitude to his major professor,
Dr. Glenn L. Waxler, for continued invaluable assistance, advice, and
encouragement in the conduct of the research and the writing of this
manuscript.

Appreciation is expressed to Dr. C. C. Morrill, Chairman of the
Department of Pathology, and Dr. R. F. Langham for assistance and
instruction during the period in which this research was conducted and
the manuscript written. Drs. C. K. Whitehair and C. K. Smith have
been especially helpful with counsel and assistance in the area of
gnotobiotic research in addition to serving on the author's guidance
committee.

Dr. S. D. Sleight and other members of the Department of Pathology
have earned the author's humble appreciation of their professional
and technical assistance.

Miss Janice Fuller has been most helpful in performing the essen—
tial service of typing the manuscript. The patience and encouragment
of the author's wife and children have inspired the completion of this

work.

iii

TABLE OF CONTENTS
Page
INTRODUCT I ON C O O O O O O O O O O O O O O O I O O O O I 0 O O O O 1

REVIEW OF LITERATURE. . . . . . . . . . . . . . . . . . . . . . . 3

 

General Characteristics of Escherichia coli. . . . . . . . . .3
Pathogenicity of E, coli . . . . . . . . . . . . . . . . . . 4
Some Factors in Susceptibility to Enteric Disease. . . . . . 8

Clinical Signs, Clinicopathologic Findings and
NecrOpsy Findings. . . . . . . . . . . . . . . . .*. . . . . 11

Prevention of Disease. . . . . . . . . . . . . . . . . . . . 15
Experimental Reproduction of Disease in Pigs . . . . . . . . 18
Artificial Rearing of Pigs . . . . . . . . . . . . . . . . . 21
Germfree (Gnotobiotic) Research. . . . . . . . . . . . . . . 24
Chronology of Development of Germfree Research. . . . . 25
Gnotobiotic Pigs as Laboratory Animals. . . . . . . . . 27

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . 31
Experimental Animals . . . . . . . . . . . . . . . . . . . . 31

Equipment and Technique for Procuring and Rearing
Gnotobiotic Pigs . . . . . . . . . . . . . . . . . . . . . . 31

Clinical Observations, Clinicopathologic Examinations
and NecrOpsy Procedures. . . . . . . . . . . . . . . . . . . 39

Preparation of Infective Agent and Exposure of Pigs. . . . . 41
RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Experiment 1 . . . . . . . . . . . . . . . . . . . . . . . . 44
Experiment 2 . . . . . . . . . . . . . . . . . . . . . . . . 53

Experiment 3 . . . . . . . . . . . . . . . . . . . . . . . . 58

iv

Page

Experiment 4 . . . . . . . . . . . . . . . . . . . . . . . . 65
Experiment 5 . . . . . . . . . . . . . . . . . . . . . . . . 82
Experiment 6 . . . . . . . . . . . . . . . . . . . . . . . . 87
DISCUSSION. . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Bacterial Status . . . . . . . . . . . . . . . . . . . . . . 96
Experiments 1, 2, 3, and 4 . . . . . . . ; . . . . . . . . . 96
Experiments 5 and 6. . . . . . . . . . . . . . . . . . . . . 102
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
BIBLIOGRAPHY. . . . . . . . . . . . . . . . . . . . . . . . . . . 110

VITA. . . . . .'. . . . . . . . . . . . . . . . . . . . . . . . . 119

Table

Distribution of pigs, conditions of oral exposure to

LIST OF TABLES

Escherichia coli and clinic0pathologic examinations
employed in each experiment. . . . . . . . . . .

Distribution of pigs and clinical signs observed in
Experiment 1 . .

Body temperatures (°F.) of pigs in Experiment 1.

Hemograms of surviving pigs and body temperatures of

pigs at the time blood samples were collected in

Experiment 1

Distribution
Experiment 2

Distribution
Experiment 3

Distribution
Experiment 4

Distribution

Experiment 5 .

Distribution
Experiment 6

of pigs

of pigs

of pigs

0 O O O

of pigs

of pigs

clinical signs observed

clinical signs observed

clinical signs observed

clinical signs observed

clinical signs observed

vi

Page

43

45

46

48

S4

59

66

83

88

Figure

10

ll

12

LIST OF FIGURES

Side view of flexible plastic film isolator used to
house gnOtObIOtiC P188. 0 o o o o o o o o o o o o o o 'o 0

Surgical isolator floor after modification by instal-
lation of rigid fiberglass ring . . . . . . . . . . . . .

Fiberglass ring being secured to the sow's flank by
means of cotton cords . . . . . . . . . . . . . .

Allis tissue forceps being held out of the surgical
field after they were applied to the incised plastic
and the sow's skin. . . . . . . . . . . . . . . . . . . .

Pig before removal from uterus. Uterine wall being
inCised With SCiSSOI‘S o o o o o '0 o o o o o o o o o o o

Pig G4480, 58 hours after exposure. Fibrinous to
fibrinopurulent exudates cover the surfaces of
abdominal viscera . . . . . . . . . . . . . . . . . . . .

Pig G4482, 75 hours after exposure. Polyserositis
with inflammatory exudates on peritoneal, pleural
and pericardial surfaces. . . . . . . . . . . . . . . . .

Pig c4481, 72 hours after exposure. Gastric serositis
with fibrin, subserosal edema and inflammatory cells.
extending to involve outer muscularis (M). Hematoxylin
and eosin. x 190. . . . . . . . . . . . . . . . . . . .

Pig G4480, 58 hours after exposure. Subserosal edema,
hyperemia, and leukocytic infiltration of spiral colon.
Hematoxylin and eosin. x 190. . . . . . . . . . . . . .

Pig G5228. Germfree control pig which weighed 2100
Gm. when it was euthanatized at 15 days of age
(Experiment 4). . . . . . . . . . . . . . . . . . . . . .

Pig G5226. Emaciated carcass of pig euthanatized
162 hours after oral exposure to Escherichia coli . . . .

Pig G5226. Polyserositis in the pig carcass illustrated

in Figure 11. Note exudates on peritoneal, pleural,
and epicardial surfaces . . . . . . . . . . . . . . . . .

vii

Page

32

32

34

36

36

62

62

64

64

69

69

70

Figure

13

14

15

16

17

18

19

20

21

22

23

24

Page

Pig G5224, 120 hours after exposure. Mesenteric lymph
node. Note absence of lymphoid follicles. Hematoxylin
and eosin. x 75 . . . . . . . . . . . . . . . . . . . . . 72

Higher magnification of mesenteric lymph node in Figure
13. Note small number of lymphocytes, edema (a), and
hyperemia (b). Hematoxylin and eosin. x 190. . . . . . . 72

Pig 65224, 120 hours after eXposure. Superficial inflam-
mation at hilus of spleen with serositis. Note mass of
neutrophils (N). Hematoxylin and eosin. x 75 . . . . . . 73

Pig G5224, 120 hours after exposure. Pancreas with
focal parenchymal necrosis, interlobular edema and ex-
tension of serositis. Hematoxylin and eosin. x 75. . . . 73

Pig G5224, 120 hours after exposure. Hepatic serositis

in proximity to gallbladder. Note hepatic parenchyma

(a), edematous Glisson's capsule (b), and subserosal
leukocytic infiltration (c). Hematoxylin and eosin.

x 75. . . . . . . . . . . . . . . . . . . . . . . . . . . 74

Pig G5224, 120 hours after exposure. Duodenal sero—
sitis. Note subserosal edema and leukocytic infil-
tration O O O O O O O O O O C O O O O O O I O O O I O O O 74

Normal duodenal mucosa from section in Figure 18.
Hematoxylin and eosin. x 190. . . . . . . . . . . . . . . 75

Cuboidal mucosal epithelium with subepithelial edema
also from section in Figure 18. Hematoxylin and
eosin. x 470. . . . . . . . . . . . . . . . . . . . . . . 75

Pig 65224, 120 hours after exposure. Fibrinopurulent
serositis of the mid-jejunum. Hematoxylin and eosin.
x 75. O O O O O I O O O O C I O O O O O O O O O O O O O O 77

Pig G5227. Mid-jejunal section. Note single layer of

serosal cells and the hydrOpic appearance of the mucosal
epithelium in a germfree control pig 15 days of age.
Hematoxylin and eosin. x 75 . . . . . . . . . . . . . . . 77

Pig G5224, 120 hours after exposure. Subserosal edema
and serositis of the ileum. Leukocytic infiltration of
the lamina propria is only slightly greater than in the
controls. Hematoxylin and eosin. x 75. . . . . . . . . . 78

Pig G5224, 120 hours after exposure. FibrinOpurulent
pericarditis and epicarditis with adhesions between

the pericardium (p) and epicardium (e). Hematoxylin

and eosin. x 75 . . . . . . . . . . . . . . . . . . . . . 79

viii

Figure

25

26

27

28

Page

Pig 65226, 162 hours after exposure. Suppurative myo-
carditis. Note neutrophils infiltrating the myocardium.
Hematoxylin and eosin. x 470. . . . . . . . . . . . . . . 79

Pig 65224, 120 hours after exposure. Interstitial pneu-
monia with interlobular edema and neutrOphilic infiltra-
tion. Alveolar septa are thickened but alveoli and
bronchioles are free of cellular exudates. Hematoxylin

and eosin. x 190. . . . . . . . . . . . . . . . . . . . . 80

Pig 65222, 94 hours after exposure. Synovitis of the

elbow joint. Note fibrinous and cellular exudates on

the synovial intima accompanied by intimal hyperplasia

and subintimal edema with leukocytic infiltration.
Hematoxylin and eosin. x 190. . . . . . . . . . . . . . . 81

Pig 65227. Histologic features of the synovial membrane

from the elbow of a germfree pig. Hematoxylin and
eOSin. x 190. I O O I O O I O O O O O O O O I O O O O O O 81

ix

INTRODUCTION

Enteric infectious diseases of swine constitute a major problem in

swine production. The role of Escherichia coli in enteric infections

 

of swine has been the subject of considerable discussion and has been
investigated by several groups and individuals. Among the causes for
the lack of certainty regarding the significance of E,‘ggl;.in enteric
diseases has been the cosmOpolitan distribution of E, gglg'as a member
of the intestinal flora in apparently healthy pigs and other species of
warmrblooded animals. Another cause of confusion has been the diffi-
culty encountered in attempts to determine which bacterial species
constitute the "normal intestinal flora" of pigs. Serological grouping
of E, gg;l_strains isolated from cases of enteric disease has shown
that certain serological groups or serotypes are more frequently iso-
lated than other serotypes.

The development of techniques and equipment for rearing pigs germs
free, or with specifically known associates as gnotobiotes, has made it
possible to investigate the effect of suspected enteropathogenic organisms
in pigs in the absence of other bacteria or in association with certain
known bacterial species.

The main objective of this study was to determine the effects of
.E,.ggli 083 on gnotobiotic pigs by exposing the animals orally to cul-
tures of the organism and observing the clinical signs, the results of

clinico-pathologic laboratory tests, and the gross and microscopic

2
lesions. Another objective was to determine the effect of anti-hog

cholera serum on the course of the disease produced by this organism.

REVIEW OF LITERATURE

General Characteristics of Escherichia coli

Definition of Escherichia coli. Escherichia coli is a Species of
straight, Gram-negative, nonsporulating, rod-shaped bacteria of the genus

Eschierichia, tribe Escherichieae and family Enterobacteriaceae according

 

to Breed, Murray and Smith (1957) in the 7th edition of Bergey's Manual.
Many species of the family Enterobacteriaceae live in the intestines
of man and other animals, frequently causing intestinal disturbances.
Escherichia coli is usually a short, plump rod, but its form is variable
under varying conditions and sometimes rather long filaments may be seen
(Hagan, 1947). Hagan also wrote that Bacterium col;.(E, gglg) is

"...strictly parasitic in its habits and, therefore,

is not found abundantly anywhere in nature except

in intestinal tracts, in feces, and in materials

which have been subjected to fecal pollution."
Classification of E. coli. Recommended procedures for biochemical dif-
ferentiation and classification of E. 3211. were presented by Breed ¢_e_t_:_ 2;.
(1957) and Edwards and Ewing (1962). Edwards and Ewing (1962) said
that the E, 22;; group is composed of bacteria that give similar bio-
chemical reactions but which may be separated into many different sero—
types by means of serological methods.

Serological typing is based upon the varied and complex antigenic

structure which is similar to that of Salmonella and Shigella. Kauffmann

(1947) deveIOped an antigenic scheme for serological differentiation or

4

serotyping of E, ggli_based upon his own previous work and that of
Knipschildt and Vahlne. Since 1947, many investigators have studied

E, ggEE_serotypes (Edwards and Ewing, 1962), especially those associ-
ated with cases of infantile diarrhea, and as a result of their efforts,
the E,.ggli_scheme has been extended to include 145 0 antigen groups,

86 K antigens, and 49 H antigens. There are 3 classes of antigens that
are important in E, ggli.serology. Edwards and Ewing (1962) listed

them as: (1) the 0 antigens, which are somatic antigens not inactivated
by heat at 100 C. or 121 6.; (2) the K antigens, which are somatic anti-
gens that occur as sheaths, envelopes or capsules and which act as
masking antigens that inhibit 0 agglutination. The K antigens are
inactivated by heat at 100 C. or 121 6.; (3) the H antigens or flagellar
antigens which are inactivated by heat at 100 C. Typing of an unknown 8
E. 52;; serotype is dependent upon the determination of its 0, K, and

H antigens. The K antigens are divisible into at least 3 varieties, L,
A, and B antigens, which are tabulated by Kauffmann (1947, 1966) and

Edwards and Ewing (1962). All of the K antigens inhibit agglutination

of living enterobacteria in 0 antisera.
Pathogenicity of E. coli

The problem ofgpathggenicity of E. coli. Because of the cosmopolitan
distribution of'E,'gg;i in the intestines of healthy animals (Hagan,
1947), it has been difficult to evaluate the significance of E, 32;;
isolations from diseased animals. Hagan (1956) cited the work of Jensen
(1903) who elucidated many of the facts regarding overwhelming E,'gg;;
septicemia, which he termed "colibacillosis" in colostrumrdeprived

calves. Many others have confirmed Jensen's findings and have studied

5
the coliform organism, endeavoring to learn whether they differed in any
way from the same Species found in normal animals. The earlier workers
were unable to find any distinguishing characteristics of these "patho-
genic" strains. McBryde (1934) reported that E, 32E;_was recovered
quite consistently in cultures taken from the mesenteric lymph nodes,
kidney, spleen, and heart blood from 31 selected cases on a large
garbage-feeding ranch in southern California where there was high mor-
tality in pigs 2 to 5 days of age. From the bacteriological and histo-
logical studies, it appeared that the disease was the result of invasion
by Bacterium coli (E, EQEE) which had assumed pathogenic and invasive
properties.

Kauffmann (1947) indicated that there are certain serological E,
gg;;_types which possess a particular pathogenicity. Ewing (1956) said
investigations in the past have made it clear that the diarrheal diseases
of infants may be caused by a variety of microorganisms. In a large
number of otherwise unexplained epidemics of diarrheal disease of infants,
however, certain'E,lggE£_serotypes are recovered. The designation
enteropathogenic E, ggl$_ has been suggested for these particular sero-
types.

Strains of the organism, which have been freshly isolated from a
diseased animal (Merchant and Packer, 1958), will produce death in guinea
pigs, mice, rats, and rabbits within 48 hours. The animals die with a
fibrinopurulent peritonitis and, in some cases, a septicemia. In the
larger animals, the organism has been isolated from numerous tissues
affected with disease. In some cases, it appears to be the only organism
present, but, in many, it occurs as a secondary invader. Colibacillosis

or diarrhea of baby pigs was described by Dunne (1958). In an analysis

6
of enteric conditions encountered in routine diagnosis, Roberts and
Vallely (1959) found that hemolytic E. 25$: was a common inhabitant
of the intestines of pigs of all ages. High concentrations of these
organisms, usually found throughout the intestines, were specifically
correlated with gastroenteritis, which occurred chiefly in 2- to 4-
month-old pigs. The hemolytic E, ggEE_serotypes in such cases were
found to belong principally to 5 serotypes, whereas strains isolated
in small numbers from the intestines in other diseases had much greater
serological variation. Clinical and epidemiologic data implicated a
disease syndrome resembling gut edema in many ways, but with edema as
an infrequent post-mortem feature. An analysis of enteric conditions
encountered in routine diagnosis underlined the importance of hemolytic
E,‘ggEE in 2- to 4-month-old feeder-pig mortality, but indicated that
"baby piglet enteritis" was more typically associated with nonhemolytic
E, EElic Sojka, Lloyd and Sweeney (1960) also found that in very young
piglets the strains isolated from enteritis were often nonhemolytic and
that the commonest serotypes were E68 and G7.

Escherichia coli was considered significant by Saunders £5.3E,
(1960) in 58 outbreaks of disease causing high mortality in baby pigs.
The syndrome was consistently associated with certain serotypes of E,
ggli_and evidence was presented to suggest that they were of primary
etiologic importance. They reported that lesions of acute hemorrhagic
enteritis were not present, and they were unable to demonstrate the
presence of clostridial toxin. Blood tests did not indicate anemia in
the piglets, and vitamin A deficiency seemed very unlikely as most sows
had grazed during pregnancy. There was no sign of hemolytic disease, and

very few of the dams had been vaccinated against hog cholera with crystal

violet vaccine.

7

Stevens (1963a) estimated that less than one-fourth of the porcine
enteritis outbreaks are associated with known specific causes (such as
swine fever, salmonellosis, transmissible gastroenteritis, inorganic
toxicoses, or deficiency of vitamin A or iron) and that many of the
remaining cases are likely to be associated with pathogenic E, ggli}
serotypes. Lecce and Reep (1962) identified E,.ggEE 08 as a pathogenic
serotype in pigs which were kept under excellent environmental conditions.

In studies on the pathogenicity of E, EQEE, Namioka and Murata
(1962) observed the effect on the organism of a substance obtained from
pseudoeosinophils of rabbits. Obvious differences of sensitivity of
various E, gglg_cultures to phagocytin's bactericidal effect allowed
them to be divided into 2 groups. The behavior of the Salmonella or-
ganisms was quite similar to that of the resistant E,.ggli.groups. Their
data indicated that the organisms isolated from pathological materials
had a tendency to resist the bactericidal action of phagocytin, whereas
the cultures derived from apparently healthy animals did not.

Moon, Sorensen, and Sautter (1966a) reported that 4 E, 22;; sero-
types isolated from newborn pigs with diarrhea were enterOpathogenic as
determined by their ability to cause ligated loops of the small intestine
in the newborn pig to become distended with fluid. Gossling and Rhoades
(1966) identified 2 serotypes "as possible specific etiologic agents of

enteritis in baby pigs".

Host specificity of E. coli. Saunders ggngl. (1960) stated that it is
now evident that E, 22;; strains are host—specific. Ingram and Lovell
(1960) listed 3 serotypes originally isolated from scouring calves and
subsequently from cases of piglet enteritis. Taylor (1961) reported

that serotypes isolated from calf scours have not been found to affect

8
man or other animals; similarly, serotypes related to pig diseases
appear to be concerned solely with this animal. Human pathogenic types
have been isolated from domestic pets. Taylor listed serotypes 026,
0114, and 0126 as having been isolated from babies with infantile
diarrhea and from calves with calf scours, but only those serotypes
isolated from infants had the ability to cause dilatation of ligated

rabbit gut loops.

Some Factors in Susceptibility to Enteric Disease

Age of host. Pigs are highly susceptible to E, £2E$_infections only
during the first few days of life (Dunne, 1958). Dillard (1964) stated
that colibacillosis is the greatest disease hazard during the pig's
first week, with the incidence being determined mainly by age of the
host. However, Saunders ££Hél° (1960) reported that the disease some-

times started in 3-week-old pigs and progressed to the younger ones.

Colostrum deprivation. Lecce, Matrone and Morgan (1961) stated that
colostrum fed to piglets is rich in antibodies (gamma globulin) to coli-
form organisms. Lecce and Reep (1962) found that specific pathogen-
free (SPF) pigs, deprived of colostrum and reared in isolation, often
develOped diarrhea when 3 days old and sometimes half of the litter was
lost within 2 weeks. Eighty-five per cent of the bacteremia and death
correlated with E,‘ggEE isolations from blood or liver occurred in pigs
fed diets lacking bovine and porcine gamma globulin. Agglutinins for

E. c_o_l._i_ 08 and other E. 39;; serotypes were demonstrated in bovine and
porcine colostrum by tube agglutination tests. These agglutinins were

practically absent from porcine and bovine gamma globulin.

9
Egmunolqgip behavior of babyppig_, In studies on the electrophoretic
mobility of serum protein of colostrum-deprived baby pigs, Bauriedel 3;
.21° (1954) showed that there was no measurable gamma globulin at birth
and that none was present before 6 weeks of age.

Baby pigs under 8 weeks of age, deprived of colostrum, did not pro-
duce a measurable serologic response to the Brucella abortus bacteria
and ovine RBC antigens used by Hoerlein (1957). Baby pigs that received
normal (nonimmune) colostrum responded to antigens when inoculated at
3 weeks of age. This response increased markedly to 6 weeks of age.
Passive transfer of antibodies through "immune" colostrum interfered with
active antibody production between 3 and 6 weeks of age. Segre and
Kaeberle (1962) reported that colostrum-deprived pigs could form tetanus,
but not diphtheria antibodies at birth. Colostrum-fed pigs formed
diphtheria antibodies after intraperitoneal inoculation of a toxoid at
birth, but their antibody response to tetanus toxoid was depressed. The
results were interpreted as evidence that a degree of transplacental
transfer of maternal antibody took place and that the sow's blood con-
tained anti-tetanus antibodies, but not antidiphtheria antibodies.

Myers and Segre (1963) used a double-diffusion agar precipitation system
against anti-swine gamma globulin serum of rabbit origin to demonstrate
the presence of gamma globulin in the blood of newborn, colostrumr
deprived pigs.

Miller ££_§E, (1962) found that Salmonella pullorum antibodies
acquired by newborn pigs fed serum from hyperimmunized swine were re-
tained in measurable concentration for as long as 7 days, but not for 17
days. Colostrum-derived E, pullorum antibodies reached a peak in the

pigs' serum at 24 hours of age and dimdnished logarithmically thereafter

10

with a half life of 4.7 days. Kaeberle and Segre (1964) administered
porcine, ovine, and equine globulin concentrate by oral or intraperitoneal
routes to colostrum-deprived pigs within 12 hours of birth. With the
exception of the intraperitoneally administered bovine globulin, the
homologous and heterologous globulins were eliminated at logarithmic
rates. Elimination of heterologous globulin at a logarithmic rate and

the absence of detectable antiglobulin were accepted as evidence that

immunologic tolerance had been induced.

Concurrent disease. McBryde (1934) suggested that inbreeding and the
presence of brucellosis in the sows had contributed to the birth of weak
pigs highly susceptible to colibacillosis. However, one of the criteria
for selection of the 58 outbreaks of disease investigated by Saunders
££_§l, (1960) was the absence of any identifiable disease other than

E, coli infection.

 

Environmental factors. Gordon and Luke (1960) found that gastroenteritis
was not always associated with poor sanitation. Lamont, Luke and Gordon
(1950) discussed the effects of chilling and the relationship of housing
to the health of the pig in general. Dunne (1958) recommended ample

dry bedding, elimination of drafts, provision of heat lamps in chilly
weather, clean dry pens, and a good sow ration, in control of colibacil-
losis. Sojka (1965) assumed that predisposing factors must play an
important role in the development of enteritis. These predisposing
factors are largely unknown and may be of infectious or noninfectious

nature, but they may be of prime importance in initiating disease.

11
Clinical Signs, Clinicopatholpgic Findings, and Necropsy Findings

Fever, depression of appetite, and dullness. Saunders EEHEL° (1963a)
reported that fever did not occur in their "pathogen-free" piglets experi-
mentally infected with E, 52E$_E68 Type I, but the body temperatures were
often subnormal. Carpenter (1965) and Smith, Conant, and Overman (1964)
reported that the response of experimental animals to endotoxin (lipo-
polysaccharide from Gram-negative bacteria) is complex. Among the
reactions to a single injection listed by Carpenter were: fever, hypo—
thermia in some species; leukopenia, followed by leukocytosis; thrombo-
cytOpenia; hyperglycemia followed by hypoglycemia; impeded phagocytosis
by reticuloendothelial cells; disturbances of the reactivity of terminal
blood vessels; peripheral vasoconstriction and vasomotor shock. Depres-
sion of appetite might be expected as a part of the listlessness and

dullness described by Dunne (1958) in pigs with colibacillosis.

Diarrhea. Colibacillosis, white scours, diarrhea neonatorum, or diarrhea
of baby pigs, is an acute, sometimes highly fatal disease of suckling
pigs characterized by diarrhea with yellowish-white, watery feces and
often accompanied by septicemia (Dunne, 1958). Within the first few

days after birth, pigs become listless and develOp diarrhea with watery,
yellowish—white feces having a fetid odor. Infected animals lose weight
rapidly and become very weak. The death rate of infected untreated pigs
varies but may be as high as 100%. Saunders'ggngE. (1960) accepted a
high incidence of diarrhea and/or mortality in the herds as one of their
criteria for investigation of E, 22E;_infection in piglets. Smith and

Jones (1963) reported a 102 to 104 increase over the usual number of

12
E, 22;; in the small intestines of newborn pigs with E, 32;; infection,
and bacteremia was absent in the diarrheal disease.

Moon EEHEL- (1966b) concluded that E, 92;; infection of the newborn
pig was most frequently a diarrheal disease without bacteremia or mor-
phologic evidence of enteritis. The §:.£Eli associated with the diarrheal
syndrome were of different serotypes than those associated with the

generalized infections encountered in their study.

Hematologic and serologic alterations. Saunders g£_gl, (1960) reported
that blood tests did not indicate anemia in the piglets with E, ggli.
infection, and there was no sign of hemolytic disease. Roy g£_gl,

(1959) investigated the effect of white scours on the sodium and potas-
sium concentration in the serum of newborn calves. Sodium and potassium
values from those calves that died from E, ggll_septicemia were similar
to those of calves that scoured, but recovered. Serum sodium values

of calves that died with localized intestinal E, 221$ infection were
similar to the sodium values of calves that died from‘E,'ggEE septicemia.
However, serum potassium values of calves dying with localized intestinal
E, 22;; infection increased until death. The authors discussed the

possibility of cardiac arrest being caused by increased serum potassium.

Enteritis and gastroenteritis. Enteritis in pigs 2 to 5 days of age was
reported by McBryde (1934) and in very young piglets by Sojka _E._E.
(1960). Gordon and Luke (1960) described their investigation of an
acute gastroenteritis affecting piglets as young as 6 hours to a few
days of age in which the same serotype was involved in 5 of 7 farms.

The sows remained normal, the pigs scoured badly, mortality was heavy,

and sometimes whole litters died. On some farms the disease started

13

in 3- to 4-week-old pigs and progressed to the younger ones. E, 22;;
was recovered from all levels of the gastrointestinal tract but rarely
from the viscera. Baby piglet enteritis, associated with E, 22;;
infection, was included in a report of investigations by Roberts and
Vallely (1959). Kernkamp (1950) noted that gastrointestinal disorders
in which coliform organisms are an etiologic factor are not manifested
at necropsy by diphtheritic exudates and necrosis that reach deeper
than the epithelium. In acute E, 32;; infection in suckling pigs,
post-mortem examination revealed catarrhal inflammation of the stomach
and intestine and reddening, with swelling, of the intestinal mucous

membrane (Glasser, Hupka and Wetzel, 1950, cited by Sojka, 1965).

Bacteremia and septicemia. McBryde (1934), in examination of 31 selected
cases, reported that in every case in which enteritis had developed in

2- to 5-day-old pigs, cultures showed invasion by E, 22;; (now §:.Egl$)°
Dunne (1958) noted that E, 22;; can usually be isolated from the paren-
chymal organs of pigs which have died following severe diarrhea or which
have been killed in a moribund condition. Jubb and Kennedy (1963)
mentioned that the infection is septicemic and the organisms can be
isolated from all tissues. The intestinal tract seems to be the constant
site of infection, but other viscera may be invaded in the terminal

stages of disease (Stevens, 1963b).

Polyserositis and arthritis. The term "polyserositis" was used by

Roberts, Switzer, and Ramsey (1963a) to refer to peritonitis, pleuritis,
pericarditis and arthritis which are frequently observed in young swine
at necropsy. Lecce (1960) reported on porcine polyserositis with arth-

ritis in SPF pigs which were transferred at 8 weeks of age from isolation

 

l4
cages to a pig farm where they became infected with a pleuropneumonia-
like organism and Hemophilus influenzae suis. Polyserositis was caused
by Pasteurella multocida, Hemophilus suis, and/or Mycoplasma hyprhinis,
according to Roberts 25 El- (1963a, 1963b).

Pneumonia, pleuritis and peritonitis may be present as complica-
tions in colibacillosis (Dunne, 1958) and abscessed joints sometimes
occur as a secondary infection after the disease appears to have abated.
Jubb and Kennedy (1963) stated that E, ggEE_infection is septicemic
and localization can occur in meninges, joints and lungs. Localizations
may accompany the enteric phase, but their manifestations are usually
delayed. Also, the bacteria may enter through the umbilicus to localize

in these sites.

Edema disease and anaphylaxis. Edema disease usually occurs in wean-
lings or weaned pigs, but occasionally may be seen in any pigs (Schofield
and Davis, 1955). According to Lamont g£.§E, (1950), so-called "gut
oedema" in the pig was first recognized in Northern Ireland in 1932.
Shanks and Lamont (1938) described the condition as "an unusual condi-
tion affecting the digestive organs of the pig". The symptomatology

and post-mortem findings of gut edema were described by Lamont g; 3;.
(1950). They indicated that edema of the stomach wall between the
muscular coat and the mucosa was the most consistent post-mortem finding.
The folds of the mesentery supporting the spiral coil of the colon was
another common site of edema which was noted as having occurred in 1

pig only 1 week old and was restricted to the mesentery without involve-
ment of the wall of the colon. Subcutaneous edema, extending along the

belly, has been noted in many cases. Edema of the eyelids was also

15
reported as a frequent lesion and the ears, throat region, and elbow
and hock joints were sometimes edematous.

Sojka, Erskine and Lloyd (1957) stated that there was evidence of
specific serotypes of E, ggli_associated with edema disease, thus
suggesting a toxin is produced by E, 22;}, The serotypes appear to
be noninvasive and are not common in pigs except with edema disease.

Lemcke, Bellis and Hirsch (1957) gave antiserum to pigs affected
with gut edema and encountered a peculiar hypersensitivity which required
that the pigs be desensitized to prevent anaphylaxis. The sensitivity
of the pigs was shown to be specific to the E, 32;; antibodies. Thomlinson
and Buxton (1963) reported the production of active anaphylactic shock
using egg albumin, and reversed passive anaphylaxis using E, 32;; (0138)
in pigs. The signs and lesions closely resembled those of edema disease
and hemorrhagic gastroenteritis. Catarrhal enteritis was also observed.
There was a relationship between the character of the lesions which were
produced and the severity and duration of the anaphylactic symptoms.
Chopra, Blackwood, and Dale (1964) injected cell-free extracts of E, 22;;
0139:K92(B) and produced edema disease in pigs raised in disease-free
and conventional environments. The severity of clinical signs was pro-
portional to the amount of extract administered. Two injections, ad-
ministered 48 hours apart, produced signs of the same magnitude on both
occasions, which is in contrast to what would be expected if edema were

caused by a hypersensitivity reaction.

Prevention of Disease
In a treatise on coliform infections in the young pig and a practical
approach to the control of enteritis, Stevens (1963b) gave a general

description of a coliform enteritis syndrome in baby pigs and suggested

16
that the disease arises when there is a lack of balance between passive
immunity and certain strains of'E,‘ggEE. His recommendations for con-
trol of enteritis in piglets a few days old encompassed 3 general areas:
(1) reducing the numbers of E, ggli; (2) increasing the immunity; and
(3) improving the management. Thorough disinfection of farrowing houses
will reduce the numbers of E, ggli'present. Hysterectomy techniques
have offered the promise of total elimination of some pathogens. These
could include "pathogenic" serotypes of E, EQEE, but the ability of the
organisms to survive for long periods outside the host and in the soil
makes continuous freedom unlikely. The use of antibiotics is the most
frequent method attempted for reducing the number of E, ggii, but it may
cause other procedures to be ignored. Dosing by mouth is preferable
to injection (Stevens, 1963b; Kohler and Bohl, 1964), because the
action of the E, 22;; is believed to be largely in the intestines, and
some drugs, given by injection, fail to produce high concentration in
the intestines. Stevens considered intramuscular injection of one gram
of oxytetracycline into the sow just prior to farrowing as wasteful and
unreliable because a comparatively small preportion of the antibiotic
finds its way into the milk. Also, it is not always easy to time the
injection to be just prior to farrowing.

For increasing the immunity of the dam and the antibody content of
the sow's colostrum, there should be advantage in keeping a closed herd
(Stevens, 1963b) and allowing new gilts or sows to mix with the older
sows for several weeks before farrowing. Gordon and Luke (1958) reported
that an autogenous bacterin, administered to the dams during gestation,
protected the pigs. But Jones, Sellers and Smith (1962) failed to reduce
the incidence of scours in litters born subsequent to vaccination of the

sows with living and dead autogenous vaccines.

17

The use of specific immune serum (Dunne, 1959) has proved effective
in the treatment of diarrhea caused by E, 22;; infection in baby pigs.
Lecce and Reep (1962) found that gamma globulin in the diet was apparently
important in preventing disease only in the first 2 days when the neo-
natal pig was still absorbing large molecules from the gut. They sug-
gested that gamma globulin might function by promoting the localized
inhibition of the bacteria in the gut, by inhibiting the stray bacteria
that passed the first line of defense and by influencing the rapid
maturation of the piglets' serum protein profile. Switzer, Preston
and Morrissey (1962) administered 10 ml. of commercial swine serum con-
centrate orally by stomach tube and another 5 m1. subcutaneously, plus

2 ml. of a polyvalent El, perfringens antitoxin to SPF pigs to substi-

 

tue for colostrum in biological protection of the pigs.

Management factors contributing to the apparent increase of the
baby piglet enteritis syndrome during the last decade were listed by
Stevens (1963b) as the increase in intensive pig breeding, the use of
large farrowing houses, and the births of a rapid succession of litters
providing a regular supply of susceptible hosts. McBryde (1934) recommended
improved sow rations and elimination of disease in the sows to prevent
the birth of weak pigs. Dunne (1958) recommended treatment of the umbili-
cus with iodine at the time of birth in controlling joint infection.
Stevens (1963b) recommended good creeps with warmth and protection for
the pigs, control of agalactia and "farrowing fever" in the sows and good pre-
farrowing rations to include vitamin A and iron. Stevens advocated a
water supply for the baby pigs since a pig with diarrhea is adversely
affected by dehydration. Parenteral injection of glucose and electrolyte

solutions may save some sick pigs.

18

Experimental Reproduction of Disease in Pigs

In naturally reared pig_. Saunders g£.§E, (1963b) were successful in
experimentally reproducing a naturally occurring syndrome which was
described by them in 1960 as being associated with certain serotypes

of E, 29;; and characterized by diarrhea and death in piglets under a
week of age. They found it necessary in view of their results to modify
their methods with each succeeding experiment and to devise means of
limiting cross infection of the controls. Large numbers of freshly
isolated organisms of the 2 most common E, 33;; serotypes from typical
outbreaks were given orally to newborn colostrum—deprived piglets which
were the offspring of gilts. There was positive correlation between
the time of appearance of the test serotype in rectal swabs and the
onset of diarrhea which usually followed exposure by 12 to 24 hours.
Body temperatures were never elevated, but often subnormal. All 21
newborn piglets exposed to the 2 different serotypes, designated 67

and E68 Type I, scoured and 11 of them died. In the fatal infections
the pigs continued to eat until near death in spite of obvious illness,
and death frequently occurred within 3 days of exposure. In those pigs
which survived, disappearance of the test serotype from rectal swabs
was usually followed by clinical recovery. The clinical course and
gross lesions at necropsy in all 5 experiments closely resembled those
of field cases. Post-mortem bacteriological recovery of the test
organisms in profuse pure culture, not only from the intestines, but
frequently from other viscera, revealed the septicemic nature of the

infection with a propensity for infection of the brain.

l9

Saunders ££_El, (1963b) undertook an additional eXperiment to
assess the effect of giving a "non-pathogenic" strain designated P50
to 7 piglets. At 12 to 24 hours after the pigs were orally exposed,
serotype P50 appeared in profuse pure culture in the rectal swabs of
the 7 inoculated pigs, but there was no diarrhea. Post—mortem examina-
tion of one piglet which died suddenly at 34 hours after exposure with-
out having scoured revealed fibrinous peritonitis, congestion of the
lungs and gastroenteritis. The results of bacteriologic examination
suggested a fatal septicemic infection. No other evidence of pathogeni-
city was displayed by serotype P50, and the gross lesions were not
typical of cases of E, 22EE_infection observed in the field. Serotype
P50 had not spread to the control group of piglets and was rapidly
displaced in the exposed group following accidental infection by E68
Type I. The controls also became infected with E68 Type I and all pigs
died within 2 days of first excreting that organism and the onset of
diarrhea. E68 Type I was recovered from most visceral and intestinal
sites of 5 piglets. Two piglets in each of the experiments 5 and 6
(Saunders g£_§E,, 1963b) were allowed to take colostrum before dosing
but did not exhibit increased resistance to infection. All piglets
which survived to the end of each experiment at 7 to 15 days were killed
and blood was collected for serological examination. The 26 serum
samples were all negative on the rapid slide test for bacterial agglu-

tinating antibodies to the respective serotype used.

En pathogen-freeppigs. .The term "pathogen-free" or specific pathogen-
free (SPF) pigs is used to designate pigs which have been obtained by

hysterectomy or cesarotomy and prevented from nursing the sow. These

20
colostrumrdeprived pigs are then reared in an environment designed to
prevent the infection of the pig with specifically known pathogenic
viruses or bacteria such as the agents causing atrOphic rhinitis and
virus pig pneumonia.

In reporting their investigations of E, 29;; associated with
colostrum-free neonatal pigs raised in isolation, Lecce and Reep (1962)
indicated that E, EQEE strain 08 was a part of the "normal" fecal flora
in their piglet population and was also the strain most frequently
isolated in bacterial cultures of blood or liver from moribund or dead
pigs. In a litter of healthy colostrum—deprived, gamma globulin-free
pigs raised in isolation, E, £2EE_serotype 08 was identified by Lecce
and Reep in 17% of the fecal cultures taken from the 9 pigs at 4 and
8 days of age. When the same pigs were 2 weeks of age, they were fed
an inoculum of 109 E, 22;; 08, but did not develOp signs of disease.
Agar immunoelectrophoresis of 2 of the piglets' sera taken at the time
of feeding the E, 22;; indicated that gamma globulin was not present at
2 weeks but was present by 3 weeks of age. At serum dilutions of 1:10
and 1:40, blood serum samples taken at 2, 3 and 6 weeks of age were
negative for agglutinins to the 08 isolate fed. Lecce and Reep sug-
gested that resistance to infection was not dependent upon antibody
(gamma globulin) except during the first 2 days of a pig's life when it
is capable of absorbing large molecules from the gut.

Saunders §£_§E, (1963a) experimented with pigs obtained by hys-
terectomy, deprived of colostrum and artificially reared in individual
incubators in strict isolation. They were able to avoid outside contami-
nation in their study of the effects of feeding a pathogenic serotype

(E68 Type I) of E, coli at varied dosage rates to pigs of different ages.

21
Also, they studied the effect of giving a pathogenic serotype 6 days
after feeding a supposedly nonpathogenic serotype. The experimental
pathogen-free pigs of Saunders g£_§E, were equally susceptible to
disease whether the pigs were 2 or 12 days of age at the time of oral
inoculation with E, 22E1_E68 Type I in doses ranging from 103 to 108
bacteria. In their experiments disease production by invasion of a
pathogenic serotype was not prevented by the previous introduction of
a nonpathogenic E, EQEE, Two experiments in which colicinogenic E, £2EE_
(618) were established in pigs weaned at 10 and 11 days of age showed
that the predominant hemolytic strains (E4 and E57) were not suppressed
by the colicinogenic strain (Tadd and Hurst, 1961).

Saunders g£_gl, (1963a) found that the disease experimentally pro-
duced in their artificially reared animals did not differ in any essential
respect from disease produced by the same E, 23;; serotypes in naturally
reared animals. Saunders and his associates considered that their
experiments clearly indicated the value of pathogen-free piglets for

this type of research.
Artificial Rearing of Pigs

Dietapy need for colostrum and hexose sugars. Early workers attempting
to rear pigs artificially from birth (Bustad, l948)on a synthetic milk
diet containing all known vitamins found that colostrum, or plasma or
serum as a substitute for colostrum, in the diet was essential for the
pigs' survival. Without the colostrum or the colostrum substitute in
their synthetic milk diet for the first day of life, the pigs deve10ped
severe diarrhea and died shortly after birth. A diet of casein, lard,

sucrose, minerals and vitamins was used successfully by Wintrobe (1939)

22
for rearing pigs weaned at 2 to 23 days of age. Pigs allowed access
to colostrum for 24 hours were unable (Becker 2; 21°: 1954) to utilize
sucrose as a source of carbohydrates. When sucrose was replaced with
glucose as the source of carbohydrate in the synthetic diet, satisfac-
tory results were obtained. Pigs weaned at a minimum of 24 hours (Johnson,
James, and Krider, 1948) were reared on a diet containing casein, cere-

1ose, lard, salt and vitamins.

Function of colostrum. Young and Underdahl (1949) used a hemagglutination-

 

inhibition test to demonstrate the absence of antibodies for swine
influenza in the serum of newborn pigs prior to ingesting colostrum and
the appearance of those antibodies in the pigs' serum 30 minutes after
obtaining colostrum by nursing. In addition to proving the colostral
transfer of hemagglutinin inhibitors (antibodies) for swine influenza
virus from dam to offspring, they also showed that the HI titer in pig
serum continued to increase to 6 hours of age, but there was very little
increase after that time, although the colostral content of antibody
remained high. Tiselius and Kabat (1939) demonstrated by electrophoresis
that bacterial antibody in pig serum was contained in the gamma globulin
fraction. Hoerlein (1957), Rutqvist (1958), Lecce g£_§E, (1961), Miller
._§._E. (1962) and Segre and Kaeberle (1962) are among the investigators
who have confirmed the function of colostrum in the transfer of antibody
or gamma globulin to the newborn pig. Evidence has accumulated (Young
and Underdahl, 1949; Speer.g£H§E., 1959; Lecce g£_§E,, 1961; Miller 3E

.21-: 1962) to indicate that there is a cessation of gamma globulin absorp-

tion from the pig's intestines after the first 24 hours.

23

Diets and techniques for rearing colostrum-deprived pig_, Young and
Underdahl (1951) described a diet and technique for starting pigs
without colostrum. Young and Underdahl (1951) and Hoerlein (1957)
recommended protective isolation of the colostrum-deprived pigs to pre-
vent their exposure to infections which may be fatal to them although
they may be enzootic or inapparent in pigs that have had colostrum.
Morrill (1952) showed that fatal hypoglycemia in fasting newborn pigs
was delayed by elevating the environmental temperature from 60 to 90 F.
Young and Underdahl (1951) collected pigs in sterile bags at birth and
fed them a diet based on cow's milk in an isolated room where the
temperature was maintained at 80 to 90 P. Young, Underdahl and Hinz‘
(1955) reported a technique for procurement of baby pigs by hysterectomy
from full-term sows. Improvements in apparatus, technique and diet were
described by Underdahl and Young (1957a, 1957b). Among other changes,
they kept the isolation units at 90 to 100 F. during the first few days
and then gradually lowered the temperature. The diet for starting the
young pigs consisted of 1 quart of homogenized milk, 1 whole egg and 5
m1. of a mineral mixture containing ferrous sulfate, copper sulfate,
manganese chloride and potassium iodide. At one week of age, the pigs
were transferred to an isolation brooder large enough to hold 12 pigs.
The starting diet was continued and gradually changed to a commercial
pig starter. The pigs were transferred at 4 weeks of age to a clean
area. These "disease-free" pigs were used to repopulate herds where
atrophic rhinitis and virus pneumonia of pigs had been problems (Young

35 g” 1959).

24

Whitehair and Thompson (1956) took pigs by hysterotomy rather than
hysterectomy and reared the cesarean-derived pigs in individual metabo-
lism cages on a diet composed of casein, fat, lactose, minerals and
vitamins. More recently, commercially prepared diets for rearing SPF
pigs have become available (Waxler, Schmidt, and Whitehair, 1966;
Betts, Lamont, and Littleworth, 1960).

Saunders EEHEA- (1963a) obtained pathogen-free pigs by hysterectomy
after the methods reported by Betts g£_§E, (1960). Betts and his
associates had procured pathogen-free, colostrum-deprived pigs by
hysterectomy using the techniques which had been developed by Young and
Underdahl (1953) and Young g£_gl, (1955). The sows were stunned elec-
trically and then anesthetized with carbon dioxide gas at 110 to 113
days Of gestation. The entire uterus was removed aseptically and trans-
ferred through an antiseptic lock into a sterile Operating hood. The
piglets were removed from the uterus by 2 Operators working through
long rubber gloves. The operators tore Open the uterus, removed the
pigs, ligated the umbilical cords and dried the pigs. The pigs were
reared in sterile incubators supplied with a source Of heated, filtered
air. The diet was a commercially available milk substitute dissolved
in water and sterilized by heat. Betts g£_gl, (1960) reported a sur-
vival rate Of 82.3% of the pathogen-free colostrumrdeprived pigs they

Obtained in 39 litters.

6ermfreep(6notobiotic) Research

Terminology. The problem Of terminology for the expanding discipline
of research with germfree life forms was discussed'by Reyniers g£_gE,

(1949) and their recommendations were published in Lobund Reports NO.

25

2, "The Need for a Unified Terminology in Germfree Life Studies". They
suggested the term "gnotobiotics", which was derived from two Greek words
(gagg, known and Eigg, life), as a designation for the field Of investi-
gation concerned with growing living organisms by themselves or in
association with other known organisms. The term "gnotobiote" may then
be used to refer to an animal whose bacterial flora is known, and gnoto-
biotes may be designated as germfree, monocontaminated, dicontaminated,
or in some other classification based upon the number of species Of

bacteria with which the gnotobiote shares its environment.

Chronology of develppment Of germfree research

Theoretical,phase. According to Reyniers (1959), a definite date
for the first pure culture concept is not established but must have pre-
ceded the recognition that microbes might be involved in the health and
disease of man and animals. The first reported direct experimentation
on germfree life was done by Duclaux, who reported in 1885 on his attempts
tO grow peas and beans as pure cultures in a sterile medium. Pasteur
commented in the same journal in his introduction to Duclaux's work
that he thought microbes were necessary to the life Of animals and that
animal life without microbes would probably be impossible. Reyniers
speculated that Duclaux's work might have been forgotten except for the
controversy stirred by Pasteur's remarks. Pasteur's negative philosophy
was supported by the experiments Of Schottelius (Gordon, 1959) at the
turn of the century.

The school of supporters of the feasibility Of germfree life was
headed by Nencki who, in 1886, attacked Pasteur on theoretical grounds

(Gordon, 1959). Nuttall and Thierfelder in 1895 and 1896 sought to

26
prove that Nencki was right by means Of the first experiments on germ—
free mammals. Metchnikoff (1903) supported the philosophy that the host
may be unfavorably affected by substances produced by various normal
intestinal flora, and Cohendy (1912) successfully reared germfree

chickens of remarkable quality.

Basic research phase. Gordon (1959) stated that Schottelius fed
inadequate diets to his germfree chickens as did Nuttall and Thierfelder
tO their guinea pigs, and little information was gained in terms Of the
effects on the body of the missing bacterial flora. However, each in-
vestigator contributed much to the methodology of rearing germfree
animals. Raster reared germfree goats in 1914 and proved that the
weight gain Of the germfree animals was not due to retention Of water
only, but represented true growth. Glimstedt (1936) found moderate
inhibition of development in various organs and reduced amount of lym-
phoid tissue in approximately 30- and 60-day-Old germfree guinea pigs.
The difference from organs and tissues Of normally reared guinea pigs
was greatest in organs that are normally in close association with the

intestinal flora.

Instrumentation of basic and applied.research. With the exception
Of the Lobund group at Notre Dame (Reyniers, 1959), there were only 8
investigative attempts to use higher animals in germfree research between
1895 and 1950. Early research in the field of gnotobiotics was directed
toward solving the academic question Of whether life and growth were
possible for animals in the germfree condition. To progress from the
theoretical phase to one of basic and applied research in gnotobiotics

required the development of dependable apparatus (Reyniers, 1959) and

27
techniques for procuring germfree animals and maintaining them germfree
from birth to old age to death. The apparatus developed by KHster is
the basis for most Of that now used for rearing and studying of germfree
higher animals (Trexler, 1959). Gustaffsson (1959) described the light-
weight germfree rearing systems and instrumentation for rearing germfree
vertebrates used at the University Of Lund, Lund, Sweden, and Miyakawa
(1959a) described the remote controlled germfree rearing unit develOped
by Miyakawa and associates in Japan. Reyniers (1959) reported on the
design and Operationcflfgermfree systems develOped and standardized in
use over a period Of 25 years at the Lobund Institute, University Of
Notre Dame, Notre Dame, Indiana. The adaptation of plastic flexible
film apparatus for the rearing and use Of germfree animals was described
by Trexler and Reynolds in 1957. Waxler (1961) reviewed the development
of techniques and equipment and the use Of germfree animals in gnoto-
biotic investigations from 1885 through 1960. In most instances (Waxler
ggngl., 1966), efforts to rear mammals free Of demonstrable microor-
ganisms have been limited to the smaller laboratory animals, although
Smith (1961) reared germfree lambs and goats and Reyniers (1942) reported

the rearing of a germfree monkey.
Gnotobiotic pigg as laboratory animals

Technique for rearinngpotobiot c pigs. The swine industry derived
much benefit from the SPF pig programs which provided a means Of inter-
rupting the disease cycle for some specific diseases and supplying pigs
for herd repopulation (Waxler g£_gl,, 1966). While the SPF and "disease—
free" pigs were satisfactory for disease control through repOpulatiOn and‘

for some reSearch purposes, Other phases Of research required the rearing

28
of pigs under even more carefully controlled conditions to reduce the
number Of variables encountered in such things as studying the action Of
specific infectious agents. Landy 2; El: (1961) discussed the use of
large germfree animals in medical research. Meyer, Bohl and Kohler
(1964) published an account of procurement and maintenance Of germfree
swine for microbiological investigations. The technique, equipment
and diet developed by Waxler (1961) made it possible for him to rear
gnotobiotic pigs to ages Of 17 to 52 days in the presence of not more
than 2 isolates of bacteria. His results indicate that the technique
can be used in producing gnotobiotic pigs for use in research. Two
problems which persisted at the end of the experiments were: (1) de-
termining the prOper time to perform the hysterotomy to Obtain strong,
healthy pigs and (2) development of a diet adequate not only for sup—
porting life and slow growth rates, but one which will produce weight

gains comparable to those of pigs reared by the sow.

Characteristics ofpgnotobiotic pigs. The need for characteriza-
tion of germfree animals exists and it can be justified in at least 3
ways according to Gordon (1959). First, there is a need to determine
whether the available germfree animals are truly the long-anticipated
standardized biological tOOl. Secondly, parameters should be established
to supply some standards for comparison Of morphological and physiologi-
cal differences between conventional and germfree stock which occur as
a result of absent bacterial stimuli. Thirdly, the effects on the host
caused by the environmental presence of the normal bacterial flora should

be clarified.

29

Wostmann (1959) reported decreased gamma globulin in the electro-
phoretic serum protein profile Of rats, mice, chickens and rabbits, while
gamma globulin was completely lacking in germfree guinea pigs. The data
Of Wostmann indicated the presence of circulating antibodies to proto-
zoan and helminthic parasites in the alpha and beta fractions Of serum
protein in rats and mice. These data seemed to be paralleled in the
case Of bacterial antibodies and the gamma fraction also showed an in-
crease with a lag time Of at least 2 weeks following bacterial invasion.

Wagner (1959) reported that antibacterial agglutinins were absent
from the serum of rats and young germfree chickens long after conventional
rats and chickens had developed positive titers. Glimstedt (1936) and
Miyakawa (1959b) found the lymphoid tissues to be less develOped in
germfree guinea pigs than in conventional guinea pigs. Thorbecke (1959)
concluded that such differences are only quantitative and not qualita-
tive. Thorbecke and Benacerraf (1959) found that blood clearance of
intravenously injected E, ggll_was slower in germfree mice than in
immunized germfree, conventional control and immunized control mice.
Further, they concluded that qualitative differences regarding develop-
ment Of lymphoid tissue, serum gamma globulin levels and phagocytic
indices for bacteria may become apparent in germfree animals only after
it becomes possible to rear the animals not only free of demonstrable
living bacteria, but also free Of antigenic stimuli from dead bacteria
and other antigenic substances.

After reviewing the literature concerning the histology of the
lymphatic system and the liver Of the pig, Waxler (1961) found only quan-
titative histologic differences of the lymph nodes in germfree pigs as

compared with those of pigs raised under farm conditions. There were

30

no significant differences in the histology of the spleen Of various
groups Of pigs reared artificially or naturally, but artificially reared
pigs appeared to have more iron-containing pigment in the spleen. The
hepatic interlobular septa, which are prominent fibrous bands in the
farm-raised pigs, were Often absent or reduced to a few reticular fibers
in the germfree pigs.

Schmidt (1961) investigated the hematologic characteristics and the
morphology of the intestinal tract in germfree pigs. Again, only quan-
titative differences were found between the germfree pigs and naturally

raised pigs.

MATERIALS AND METHODS

Experimental Animals
The pigs utilized in this series of 6 experiments were all sired
by the same purebred Yorkshire boar and were collected by cesarotomy on
the 112th day of gestation, or later, from related purebred Yorkshire
sows and gilts. Medroxyprogesterone acetate* was added to the feed at
the rate Of 500 mg. per day for the sows in Experiments 2 through 5 for
periods Of time ranging from 9 to 26 days immediately prior to the

scheduled date for cesarotomy procedures.

Equipment and Technique for Procuring and Rearing Gnotobiotic Pigs

Equipment. The isolator systems used in this work consisted Of 60 x 60
x 180 cm. flexiblepflastic film isolators, each equipped with 1 pair Of
shoulder-length gloves on each side; an air lock at 1 end to permit ex-
change Of supplies, pigs, and accessory items; and a filtering system for
the exchange Of bacteria-free forced air under slight positive pressure
(Figure 1). The preparation and sterilization of the equipment and
techniques for rearing gnotobiotic pigs were described by Waxler _E _E.
(1966).

The surgical isolator was modified by installing a rigid fiberglass

ring 30 cm. in diameter by 10 cm. in length in the center Of the plastic

 

* Repromix, The Upjohn Company, Kalamazoo, Mich.

31

32

4 .

 

Figure 1. Side view of flexible plastic film isolator

used to house gnotobiotic pigs.

 

Figure 2.

Surgical isolator floor after modification
by installation Of rigid fiberglass ring.

33
film forming the floor of the isolator (Figure 2). Holes Of 3 mm.
diameter were drilled at 5 cm. intervals around the periphery of the
fiberglass ring 5 mm. ventral to its uppermost edge. Sterilized rubber
bands were looped through the holes to anchor Allis tissue forceps
during the surgical procedures. Three complete turns Of plastic film
tape served to fasten the fiberglass ring in the Opening provided for
it in the center of the floor Of the isolator, as described by Waxler
_£__E, (1966). The lower end Of the ring was covered with a cap Of 20
mil vinyl plastic which formed the only area Of contact between the
sow's skin and the plastic isolator when it was sealed to her flank in
preparation for surgery. The ring was further secured in place by means

of 3 cotton cords passed beneath the sow and anchored to 3 eyelets pro-

vided in each side Of the flange (Figure 3).

Supgical procedures. 0n approximately the 112th day of gestation the
sows or gilts were scrubbed with water and detergent. For preOperative
sedation promazine hydrochloride solution* was injected intravenously

at the rate Of 1 ml. per 100 pounds estimated body weight and 20 to 25
m1. of 2.5% procaine hydrochloride solution** were injected epidurally

at the lumbosacral articulation. When the epidural anesthesia had become
effective, the sow was placed on the Operating table surface with her

left side uppermost and elevated to working height for completion of

preoperative preparation.

 

* Sparine, Wyeth Laboratories, Cleveland, Ohio.

** Epidural, Haver-Lockhart, Kansas City, MO.

34

 

Figure 3. Fiberglass ring being secured to the sow's
flank by means of cotton cords.

35
The hair was shaved from the surgical site and adjacent surrounding
skin. The shaved area was scrubbed 3 times with a detergent solution* and
rinsed with water, dried with sterile towels after the 3rd rinse, and
subjected to 3 alternating floodings Of 70% alcohol and phenylmercuric
nitrate solution. In Experiments 5 and 6, chloroform was added as a
final cleansing and defatting rinse before the surgical site was sprayed

* The surgical procedure was continued

with a sterile adhesive compound.*
according to the method of WaxlerlggugE. (1966). When the bottom Of the
isolator and the sow's skin were incised with electric cautery, Allis
tissue forceps were applied at approximately 2 cm. intervals tO hold the
incised edges of the plastic and the skin in close apposition (Figure 4).
The handles Of the Allis tissue forceps were secured in the rubber bands
at the edge of the fiberglass ring to prevent their obstructing the field
Of surgery. Only one man of the team working from Opposite sides of the
surgical isolator actually performed the laparotomy and lifted the pigs
individually but still within the uterus into the surgical isolator
(Figure 5). The second member of the veterinary surgical team refrained
from touching the gloves of the other Operator and anything else that
might have come into contact with the skin of the sow. Scissors were
employed to incise the uterus and placental membrane over the elevated

pig from its head tO midway Of its back. As the incised uterus and pla—

cental membranes retracted ventrally along the sides Of the pig, the

 

* Liquid Germicidal Detergent, Parke, Davis and Company, Detroit,
Mich.

** Vi-Hesive, AerOplast Corp., Dayton, Ohio.

36

‘ n

r i (01:15:; .
I} ’,1
\I.’ '

:1"
.m

 

 

 

Figure 4. Allis tissue forcepsibeinggheId out Of the“—
surgical field after they were applied to the incised plastic
and the sow's skin.

 

 

 

Figure 5. Pig before removal from uterus. Uterine
wall being incised with scissors.

37
assisting surgeon was able to grasp the pig without touching the uterus,
lift the pig from the uterus, and apply 2 hemostatic forceps to the
umbilical cord. He then severed the cord between the forceps and passed
the pig into the attached receiving isolator where umbilical ligatures
were applied and the hemostatic forceps removed by 2 additional assis-
tants working at the receiving isolator. All other surgical procedures
were according to the method described by Waxler ££H21° (1966). As soon
as the surgical procedures and identification by ear notching of the
pigs was completed, the receiving isolator was separated from the surgi-

cal isolator and used to transport the pigs to the rearing room.

Rearing technique and diet. In the rearing room, the pigs were distri—
buted in approximately equal groups among 3 isolators where they were
kept individually in stainless steel cages and reared according to
established methods (Waxler §£_§E,, 1966). The pigs in one isolator
served as controls and the pigs in the other isolators served as princi—
pals in these experiments. The pigs were weighed at 24 hours of age.
The diet consisted of a liquid milk substitute* which was supplemented
with 5 ml. of sterilized 50% dextrose solution at each feeding during
the first 3 days. The pigs were fed 3 ounces SPF Lac 5 times daily
until the 4th day, when the feedings were reduced to 4 times daily and
the volume increased to 4 ounces per feeding.

In Experiments 5 and 6 a commercial anti-hog cholera serum was
added to the first feeding for 6 pigs in each experiment. In Experiment

5 the first feeding at 5-1/2 hours after the birth by hysterotomy

 

* SPF Lac, Borden CO., New York, N.Y.

38
consisted of 50 m1. anti—hog cholera serum and 2 ounces Of the liquid
diet with 5 m1. Of 50% dextrose solution for each of 6 pigs. On the third
day, the liquid diet was increased to 5 ounces per feeding. In Experi-
ment 6, the first feeding to 6 pigs at 4 hours of age was 25 ml. of
anti-hog cholera serum plus 2 ounces of liquid diet and 5 ml. of 50%
dextrose solution, while the other 6 pigs received 3 ounces liquid diet

and 5 ml. 50% dextrose solution but no anti-hog cholera serum.

Bacteriologic procedures. Rectal swabs for bacteriologic cultural deter-

 

minations were collected from the pigs at birth and 3 times weekly
thereafter. Three tubes of thioglycollate medium* were inoculated for
culture at 25, 37, and 55 6. Then the swabs were streaked directly

onto 5% bovine blood agar* plates and eosin-methylene blue* (EMB) plates
for incubation aerobically at 37 C. The culture mediums were examined
twice daily for evidence of bacterial growth. When growth occurred in
the liquid mediums they were subinoculated onto blood agar and EMB plates,
then final cultural examination and determinations were made. In Experi-
ments 5 and 6, the use of thioglycollate liquid medium was discontinued
in favor of brain-heart infusion (BHI) broth,* and all cultures of rec-
tal swabs were incubated only at 37 C. The blood agar and EMB plates
were incubated for 72 hours and brain-heart infusion tubes for 1 week.
Slides were prepared according to standard methods from thioglycollate
liquid medium or BHI broth and from typical discrete colonies on the
blood agar and EMB plates and were stained by Gram's method and examined

at high dry and oil immersion magnifications by transmitted light microsc0py.

 

* A Bacto dehydrated base for each of the mediums was obtained
from Difco Laboratories, Detroit 1, Mich.

39

Clinical Observations, Clinicopathologic Examinations and NecrOpsy Procedures

Clinical observations. Body temperatures were recorded daily from rectal
thermometer readings from all pigs. Observations of appetite, physical
activity and general appearance of the pigs, and the character of the

feces were recorded daily or at each feeding when changes were observed.

Clinicopatholggic examinations. Blood samples were collected 3 times
weekly in volumes Of 3 to 5 ml. from the anterior vena cava according

to the method of Carle and Dewhirst (1942). One milliliter of blood from
each pig was put into a vial containing ethylenediaminetetraacetic acid
(EDTA) as an anticoagulant and the remaining volume of blood was put into
a vial without anticoagulant. Hematologic examinations of specimens

from the nonclotted blood were according to the methods of Benjamin
(1961) for micro-hematocrit determination of packed cell volume of
erythrocytes (PCV), cyanmethemoglobin estimation of hemoglobin (Hb.) and
total and differential leukocyte counts. Examinations of serum samples
were according to methods described by Frankel and Reitman (1963) in
Gradwohl's Clinical Laboratory Methods and Diagnosis and included the
biuret method for determination of total serum proteins, paper electro-
phoresis* for quantitative determination of serum albumin and globulins,

and sodium and potassium determinations by flame spectrophotometry.**

 

* Beckman-Spinco paper electrophoresis, Duostat Power Supply,
and Analytrol Automatic Densitometer, Beckman Instruments, Inc., Spinco
Div., Palo Alto, Calif.

** Coleman Model 21 Flame Photometer, Coleman Instruments, Inc.,
Maywood, Ill.

40
The serums from pigs in Experiment 6 were examined in tube aggluti-
nation tests for 0 agglutinins to E, £2EE_083 somatic antigens. Porcine
antiserum to E. 2211.083 was obtained from the blood of 2 conventionally
reared and weaned pigs immunized with 0 antigens prepared according to
the method of Campbell 2; 31° (1963). Polystyrene latex particles* were
used experimentally as indirect indicators to improve the sensitivity

and visibility of the tube agglutination test as described by Singer

and Plotz (1956) and Muraschi (1958).

Necropsy procedures. NecrOpsy of each pig was done as soon as possible

 

after death whether death occurred as the result of experimental infec-
tion or euthanasia. External appearance Of the carcass was noted before
a midventral skin incision was made from the mandibular symphysis to

the posterior pelvic region. The skin and limbs on each side were dis-
sected from the trunk and reflected laterally. In all experiments
except Experiment 1, a flame-heated spatula was used to sear the exposed
subcutaneous tissues covering the elbow and stifle joints preparatory

to incising the joint capsules with a flamed scalpel. Sterile inocula-
ting loops were inserted deeply into the Opened joints to contact the
synovial surfaces and then withdrawn to streak identified areas on pre-
pared blood agar and EMB plates. The pleural and peritoneal cavities
were similarly sampled for bacteriologic examination, then the cavities
were fully Opened and the pericardial cavity exposed for bacteriologic

examination.

 

* Bacto-Latex 0.81 (Code 3102), Difco Laboratories, Detroit,
Mich.

41

The viscera were Observed for lesions and, in some cases, were
photographed. The organs were then removed, and blocks of tissue no more
than 0.5 cm. thick were collected from the mandibular, superficial in-
guinal, prefemoral and mesenteric lymph nodes; adrenal glands; pancreas;
spleen; liver; midportion of the kidneys; urocyst; cardiac, fundic and
pyloric regions of the stomach; 5 equidistant levels of the small in—
testine; cecum; spiral colon; terminal colon; heart; lungs. Rarely,
sections were taken from the elbow and/or stifle joints. The tissues
were fixed in 10% neutral buffered formalin, retrimmed and processed
for histOpathologic examination according to procedures outlined in the
Manual of Histologic and Special Staining Technics of the Armed Forces
Institute of Pathology, Washington, D.C. (1957). Paraffin-embedded
tissues were sectioned at 7 microns thickness, mounted on glass slides,
stained with hematoxylin and eosin, and coverslipped for histOpathologic
examination. In the 6th experiment, blocks of liver tissue were fixed
in Carnoy's solution for subsequent staining with Best's carmine and

examination for glycogen.

Preparation of Infective Agent and Exposure of Pigs
The original culture of E, 23;; 083 was isolated from a pig which
develOped pericarditis and died at the Veterinary Research Farm at
Michigan State University. Serially transferred cultures were maintained
on nutrient agar* slants stored at 25 C. and were serotyped in 1963 at

The University of Minnesota** and again in 1965 at The Pennsylvania State

 

* Nutrient Agar 1.5% (B69), Difco Laboratories, Detroit, Mich.

** Bacterial cultures serotyped as 083 (Minnesota #344) by Dr.
Harley Moon, The University of Minnesota, St. Paul, Minn.

42
University.* Inoculums for the pigs were prepared by inoculating screw-
capped vials containing 5 ml. quantities of thioglycollate liquid medium
or 5 ml. of brain-heart infusion broth with organisms picked from at
least 3 typical colonies on 5% bovine blood agar which had been inocu-
lated from the storage tubes and incubated for 24 hours aerobically at
37 C. The inoculated thioglycollate and BHI liquid mediums were incu-
bated at 37 C. for 24 hours in Experiments 2, 5 and 6 and 36 hours in
Experiments 1, 3 and 4. The inoculums were transferred after incubation
into the isolators containing the principals.

Gnotobiotic pigs Of different ages in separate experiments were fed
varied volumes of the cultures of E, ggll_mixed in their liquid diet.
The sex distribution and mean 24-hour body weights of the controls and
principals, age of the pigs at exposure to E, 221;, volume Of inoculum
fed, culture medium and length Of time the inoculum was incubated, and
the types of clinic0pathologic examinations conducted in each experiment

are given in Table l.

 

* Bacterial cultures serotyped as 0 group 83 (083:K.:HNM) by
courtesy of Dr. Paul J. Glantz, The Pennsylvania State University, Uni-
versity Park, Pa.

43

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Hem ««
assume sassHH mumHHoousoHne . cane *

 

 

 

 

 

 

 

 

umm a N m H
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o.m
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m m m N
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o.m
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amm m m a H
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amm a N a N
mo» Oz 02 02 wow *OHnH om o.m Nu .nmmum Oz o.H 2 q o.H E H H
humouoma umou mHmm mmu%H OH 5: A.M£V A.HEV A.H£V wudmoaxo mom A.wxv xmm .oz A.wxv xom .Oz .02
um OOHO OOHu luona Iouu IwOH IHvoa mouse mom musmoaxm um ucoa Bayou .uB .u3 .mxm
ImaHsmxo ImOHu Iouu IOOHO loo musu IOOOH .Ho> um mem IaouH>am IHOO< mHmmHOaHum - wadMueQm
OHwOHO Iuswm IOOHO asumm Imaom IHso mo Ow< :H mHumu
IHuouomm NoumH asuom BOHOOOOH loom
chHumOHamxo OHNOHOSOOQOOHOHHU
.uaoaHummxo sumo aH OO%OHQBO chHumaHamxm
OHmOHOcOOQOOHOHHO pom .HHOO mHnuHumsomm Ou musmoaxo Hmuo mo chHuHOOOO .mea mo OOHusoHuumHn .H OHan

RESULTS

Experiment 1

The details of the pigs' identification numbers, sex and body weight
Of the principals and controls with clinical signs of illness and mor-
tality are tabulated (Table 2). Bacteriologic examination of the pigs
at birth revealed no bacterial contamination, but monocontamination of
pigs in all 3 isolators with Staphylococcus g2, was detected in culture
of rectal swabs collected when the pigs were 72 hours Of age and immediately
prior to their exposure to E, EQEE. Escherichia coli was recovered from
the principals in culture of rectal swabs collected at 20 hours post-
exposure.

One of the controls, Pig 5, had been weakened by loss of blood when
the umbilical ligature loosened. The pig remained weak and maintained
a subnormal body temperature until it died at approximately 84 hours of
age. Hypothermia was recorded for principal Pigs 1, 2, and 3 at 24 hours
of age and body temperatures became more depressed after exposure to
§:.Efll$: Pig 3, in Isolator l, and Pigs 7, 8, and 9, in Isolator 3, all
developed fevers following exposure to E, 22;}, The body temperatures
are given in Table 3.

Clinical signs of illness in the principals included listlessness,

fever, rough hair, partial to complete anorexia, and moderate diarrhea.

44

45

uuomoum mmz OOHOHOOOU
Osmond wm3 OOHOHOOOU
uaoawummxm mo GOHumOHBumu um OONHumamzusm mem .ausm

zuuHo mo musos «N OHnuHB poanos mums mem «a
meo Houuaoo «

II
+

 

 

«w + + + + + + + oo.H z NNoNo m m

mm H.535 + I + I + + + «04 2 268 w m

«m + + .+ + + + + mo.H m omONU N m

cm + + + + + + + «o.H z wooNu m H

o« + + I + I + I Nw.o m Nmew N H

m« + + I + I + I «o.H z mmeu H H

as H.533 I I I I I I I no.0 a 330 is N

E H + I + I I I .54 s «was In N

as éfismv I I H I I I I so; a .268 .3 N
A.unv mEHu Ammouoma summm moss mewuos< mHmoumO chmm um>mm A.wxv wow .02 .02 .Oz
Hm>H>u=m um mOOHm IumHQ IOHOOH onamH ««.u3. unmouomz me uOumH
musmooxmumom IOH mmouu kwom IOmH

 

 

.H uamaHHmaxm CH vm>ummno mawHw HmOHGHHO mam mem mo GOHuanuumHQ .N OHan

Table 3. Body temperatures (°F.) of pigs in Experiment 1.

 

 

 

 

 

1::Or Pig Pre-exposure (hr,)r Postexposure ,

No. No. 52 24 44 72 _ 93
2 4* 100. 99.4 101.0 101.6 101.4
2 5* 97. ** --- -—- ---
2 6* 101. 102.0 101.2 100.8 102.0
1 1 98. 97.8 96.6 Dead ---
1 2 96. 95.2 94.0 Dead ---
l 3 98. 99.0 105.0 Dead ---
3 7 100. 102.6 106.4 106.0 98.8
3 8 100. 100.2 102.5 104.0 106.0
3 9 99. 100.2 100.6 105.2 Dead

 

* Control pigs

** Died at 84 hours of age after being weakened by blood loss

47

Hematology. The hemograms are shown in Table 4 together with the pigs'
body temperatures at the time the blood samples were collected. The

arithmetic means Of blood values for all 9 pigs at 24 hours of age were:

Hemoglobin (6m./100 ml.) 7.9
Packed cell volume (%) 27.7
Corrected total leukocytes
(x 103/cu.mm.) 5.49
Differential leukocyte count (%)
Segmented neutrophils 46.5
Nonsegmented neutrophils 7.8
Lymphocytes 41.0
Monocytes 4.4
Basophils , 0.2
Eosinophils 0.0
Nucleated erythrocytes
(/100 leukocytes) 1.7

At 40 hours postexposure the 2 hypothermic principals had leukopenia,
which was essentially absolute neutropenia, and the other 4 principals
had leukocytosis, which represented both relative and absolute neutro-
philia and lymphocytosis. The lymphocytosis persisted in the 2 surviving
principals at 93 hours postexposure, but neutrOphilia was changed to
severe absolute neutrOpenia (Table 4).

ElectrOphoretic examination of serum samples collected terminally
from pigs 4, 6, and 8, prior to euthanasia, resulted in poor separation
of the beta and gamma globulin fractions, but the albumin and globulins
were well separated and were calculated as being in the ratios of 0.56:1,

0.74:1, and 0.19:1, respectively.

Necropsy findings, There were dried brownish feces on the tail and peri-
neal area of one of the controls, but the other control was entirely

free of signs of diarrhea. There were no grossly visible inflammatory
exudates in the serous cavities. The Spleens were smaller and much

brighter red than in the principals. Each of the stomachs contained

48

.mmuhuounuxum woummHuac u ummz .mHHna
IOchOO u m .mHHLQOmmn u m .mmukoosoe u z .mmuNO03OENH u H .mmuomawmmcoc n mz .OOOOOEwmm u wow {e
mem Houucoo «

 

 

 

 

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N H O H NO O H NNNN O.NN N.N N.NO N
N O O H NN OH NN ONNN O.NN O.HH O.NOH «N mnsmoaxm
H O O H NN NH HN NNNN O.HN N.N O.HOH «N umumm NO
O O O H NN NH ON ONONH O.NN H.OH N.OOH O
N O O H NN NN NN NNNNH O.NN O.N N.NOH N
O O O O NN NN HN OONNH N.NN O.N N.NOH N
H O O O NO NN sN NONOH O.ON N.N O.NOH N
O O O N NN O NH HNNN N.NN H.OH O.OO N
N H O HH ON H N HNNN O.NN N.N N.NO H
O H O N NN N NN ONNN N.NN N.OH N.HOH IN
III III III III III III III III III III Odom em ousmoaxw
O H H O NN NH ON NHNN N.ON N.N O.HOH NO Hmumm «O
O O O N «N N ON ONNN N.NN O.OH O.OO O
H O N N NO NH ON NHHN N.NN N.N O.OOH N
N O O O NN NH NO NOON O.NN N.N N.OOH N
N O O N NN N NN NHHO N.NN N.N O.NO N
N O O O NN N ON NNON O.ON O.N O.NO N
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H O O OH NN OH NN NNNN O.HN N.N N.HOH «N
N O O O ON N NN OONN N.NN N.N O.NO «N musmoaxm
N O O N NN N NO NHNO O.ON N.N 0.00H «O ONONON ON
*«ONNz m m 2 H Nz .NON Om; ANN H.Ha OOH N.N.N when .02 musmoaxm on
I mHHSgHHDmZ Hmuou >Um \.EUV Imummamu me M>HumHmH
ufiDOU muNAUOxDMH HNHUGOHOMMHHO UQUUQHHOU UOHUUHHOU . n—m Nnfiom wHSOS

OH maHa

 

 

4‘ ‘1

.H unmaHuomxm OH OOOOOHHOO
mum3 mOHaEmm OOOHH OEHO mnu ad mea mo mOHSOOHOQEOO hoop cam mem wcH>H>u=m mo mamumoamm .« mHan

49
approximately 2 ounces of milk curd. The livers were slightly yellow,
the gallbladders were not edematous, and all other organs were without
visible lesions.

Pig 2 died 45 hours after exposure to §:.£Qli' Grossly, the skele-
tal muscles were pale, and the prefemoral lymph nodes were edematous.
There were several milliliters of serosanguineous fluid in the pleural
cavity and serofibrinous fluid in the peritoneal cavity. The liver was
faintly yellow, and the spleen was enlarged and dull red. The stomach
was distended with gas but contained no food. There was hyperemia of
the mesenteric lymph nodes and the small intestine, especially in the
upper jejunum 2 to 5 feet posterior to the pylorus. The kidneys were
pale, and the urinary bladder was faintly petechiated on the serous and
mucous surfaces.

Pig 1 died 46 hours after exposure, and the lesions were similar
to those in Pig 2. However, there was a small amount of milk curd in
the stomach and a small amount of fluid in the small intestines, which
were more edematous than hyperemic. There was marked subserosal edema
of the spiral colon.

There were dried feces on the tail and rear legs of Pig 9, which
died 84 hours postexposure. Edema or serous inflammation involved the
muscles and subcutis Of the posterior ventral abdominal wall and the
associated lymph nodes. There was severe serofibrinous inflammation Of
the peritoneal, pleural, and pericardial membranes. Fibrin covered the
epicardium, and the myocardium was pale. There were scattered focal
areas of emphysema in the lungs with some congestion and with consolida-
tion Of the ventral parts of the apical lobes. Fibrinous adhesions

occurred between the visceral and parietal layers of the pleura. The

50

spleen was enlarged and dark. The liver was enlarged, dark red and
faintly mottled, but the margins of the lobes were thin. The gallbladder
was edematous. There was excess mucus but no food in the stomach. The
abdominal viscera were firmly adherent to each other due to a heavy
coating of fibrinopurulent exudate. The mesenteric lymph nodes were
edematous. The small intestine was hyperemic and the contents were more
fluid than in the 3 principals previously examined. The colon and rectum
contained yellow, mucinous, viscous, nongaseous fluid. There was apparent
diffuse petechiation of the cortical surfaces of the pale kidneys. The
urinary bladder was empty and slightly thickened.

Lesions Observed in principals 3, 7, and 8 were similar to those
described for Pig 9, with the exceptions that the gallbladders were not
edematous and one stomach contained some milk curd. Experiment 1 was

terminated at 99 hours postexposure with euthanasia of Pigs 8, 4, and 6.

Bacteriologic findings. Post-mortem bacteriologic examination Of the
controls revealed no growth in mediums inoculated from the peritoneal and

pericardial cavities, but Staphylococcus _p, grew on the blood agar plate

 

subinoculated from the primary culture of a rectal swab from Pig 6.

Escherichia coli was recovered from the principals from all sites sampled,

 

which included the peritoneal cavity and rectal swab from Pig 7 and peri-

toneal and pericardial cavities and rectal swab from Pig 8.

Histopatholggic findings. The tissues from Pigs 4 and 6 appeared to be
normal, but the other control (Pig 5), which died at 3 days of age, had
edema in all lymph nodes. Lymphoid tissue in the controls was not dense,
and secondary nodules were absent. The lymphoid tissue was composed of

plasma cells, lymphocytes and medium-sized to large reticuloendothelial

51

cells with some mitoses. There were a few foci of medium—sized dark
lymphocytes, but small dark lymphocytes were infrequent. Myeloid elements,
which were mostly polymorphonuclear neutrophils (PMN) with some eosino-
phils, were found in clusters or diffusely scattered between the lymphoid
nodules and, sometimes, mixed with cells in the lymphoid nodules.

The mandibular lymph nodes from the principals were characterized
by mild edema, faint follicular pattern, infrequency of small dark lympho—
cytes, and increased granulocytes, which were primarily neutrOphils with
a few prominent eosinOphils. There was a fibrinoid substance in the
mandibular lymph node sinuses of Pig 9.

The lesions in the mesenteric lymph nodes were edema, hyperemia
and depletion of lymphocytes, with increased prominence of eosinophils
and neutrophilic granulocytes, some of which were necrotic. The super-
ficial inguinal and prefemoral lymph nodes were similar to the mesenteric
lymph nodes.

The adrenal glands were not hyperemic, except in Pig 3, which had
fibrinous to fibrinopurulent serositis of the adrenal capsule, and in
Pig 8, which was similarly affected and had focal hemorrhages in the
capsule. The pancreatic parenchyma was essentially normal, although
there were lesions of serositis which were most severe at the hilus.
There were no significant lesions in any of the thyroid gland sections.

Lymphoid tissue of the spleens resembled that of the lymph nodes.
The Malpighian corpuscles were poorly developed and usually consisted of
2 or 3 concentric layers Of lymphocytes. Serositis of varying severity
was found in all the splenic sections but did not appear to penetrate

into the parenchyma.

52

Hepatic lobular outlines were more distinct than in the controls
and perilobular connective tissue was not demonstrated by the hematoxylin-
eosin stain. There were scattered foci of hematOpoiesis in the hepatic
sinusoids. Serositis of Glisson's capsule was of varying severity, and
the relative composition of fibrinous and leukocytic elements in the
exudate also varied. The cytOplasm of the hepatocytes was usually lightly
stained and the cellular outline was often indistinct. Lesions less
frequently seen in the liver sections were: scattered focal necrosis,
hemorrhage, and some fatty metamorphosis. In one liver section there
was moderate edema with neutrophils surrounding the cholecyst.

The most common renal lesion was cloudy swelling of the epithelium
of the convoluted tubules. FibrinOpurulent serositis was seen in 4 pigs
and there was subserosal extension of the inflammation at the renal
hilus in one section, but the pelvis was free of cellular exudates.

There was fibrinOpurulent serositis of the urinary bladder in 5 princi-
pals and one pig had a few discrete submucosal hemorrhages. There were
no lesions of urocystitis in the bladder lumens.

There were lesions of serositis of varying severity and character
in some sections from the stomach of each of the principals, but there
were no lesions of severe mucosal hyperemia, hemorrhage, necrosis, or
leukocytic infiltration in the gastric sections. There was an apparent
increase of mucous secretion.

Duodenal serositis was seen in 5 principals. The inflammation pene-
trated subserOsally in one pig and through the muscularis into the sub—
mucosa in another pig. FibrinOpurulent serositis was seen in most of
the sections from 3 levels of the jejunum, and hyperemia of the mucosa

and submucosa was seen in approximately 50% of the sections. There was

53
fibrinopurulent serositis of the ileum in sections from 3 pigs. Ileal
enteritis was manifested in one pig by leukocytic infiltration of the
lamina propria, neutrophilic granulocytes in the lacteals of some villi,
and increased eosinophils in the lymphoid tissue. Lesions of fibrino—
purulent serositis were features of the cecum in 4 pigs, of the spiral
colon of 3 pigs, and of the terminal colon of 5 pigs. Subserosal edema
of the spiral colon was severe in 2 pigs.

Pericarditis and epicarditis were fibrinous in one pig and fibrino-
purulent in 2 pigs. Pleuritis varied from fibrinous to fibrinopurulent
in 3 pigs, and in one pig there were interlobular fibrinopurulent exudates
in the lung and interstitial pneumonia with septal thickening. One of
3 pigs with focal infiltration of neutrophils in the mesenchymal stroma
of the umbilicus had developed a microabscess with early peripheral

fibroplasia.

Experiment 2
Information concerning distribution of the controls and principals,
their identifying numbers, sex, and 24-hour body weights is included
with clinical signs in Table 5. Bacteriologic examination of rectal
swabs collected from the pigs at 48 hours of age revealed Staphylococcus
ER, in pure culture from 7 of the 8 pigs and no bacterial growth from
the remaining pig. Rectal swabs collected 24 hours after exposure

yielded E, coli from all the principals.

Hematology. No pattern was established in the hemograms of blood samples
collected 1, 4, and 6 days after the pigs were exposed to E, coli.
Severe neutrOpenia (neutrophils — 3 and 13%, lymphocytes._ 97 and 87%,

corrected total leukocyte counts - 24,260 and 22,850) was coexistent

54

ucmmmua mm3 GOHOHOOOU u +
powwow mmz OOHOHOOOU u I

uooaHHmaxo mo OOHumcHauwu um OONHumamnusm wme .nuam
mem Houucoo «

 

 

Nu + + + + I I I ao.H z Nmmmo w m

NNN H.535 + I + I I I + 3H m NONNO N N

NoH + + + + + I + MN.H z Nmmmw m m

NN + + + + I I + NN.H Z Hmmmu m H

«NH + + + + + I + mo.H m «mmmw N H

om + + + + I I + m«.H m mmmmu H H

NNN éfismv I I I I I I I OH ,N NONNO «N N

NNN H.695 I I I I I I I NH.H z NONNO 3 N
A.unv msHu unmouooo spoon moss meOHOG4 mHmOuho mHama um>mm A.wxv xmm .Oz .02 .Oz
Hw>H>uam um maOHm IHOHQ onde onsmH .u3 homouumz me HoumH
ounmooxoumom IOH mmouu moon IOmH
1|“

 

.N unmaHumaxm OH Om>ummno mcme HOOHOHHO mam mem mo sowuanuumHn .m OHamH

55
with lymphocytosis in samples from Pigs 2 and 5, respectively, approxi-
mately 100 hours after exposure. The controls and principals had very
similar hemoglobin and erythrocyte PCV values in each day's blood

samples.

Fever and other clinical signs. The maximum body temperature recorded
for either of the controls was 103.0 F. Five of the 6 principals had at
least one temperature reading of 104.0 F. or above, with 106.8 F. being
the maximum. The feces were noticeably more fluid in the principals
than in the controls from the first postexposure day onward. Decreased
appetite seemed to be associated with pain or stiffness and reluctance
to move rather than with decreased alertness or fever, as some of the
principals would not arise when fed and would protest when lifted but
would then eat a part or all of the feeding.

Serum electrolytes in 28 samples were found to be essentially the
same in both groups of pigs. Sodium values in flame photometry ranged
from 131 to 152.5 milliequivalents/liter for the principals and in the
controls the range was from 135 to 151 mEq./liter. Serum potassium
increased with age of the pigs. The extremes were 4.35 and 8.45 mEq./
liter, with the highest concentration being from one of the controls.
Separation of the serum protein fractions in electrOphoresis was not

sufficient to permit interpretation of the results.

Necropsy findingp, There were no gross lesions in the controls. Signs
of diarrhea, cachexia, enlarged external lymph nodes, and fibrinous to
fibrinopurulent polyserositis were constant findings in the principals.
Inflammatory edema of the ventrolateral abdominal areas was extensive

in 3 pigs and a fourth pig had subcutaneous edema of the shoulder, neck

56
and head regions. It also had swollen leg joints with excess fluid in
the synovial sacs. Splenic enlargement was present in all principals.
Hepatic enlargement or congestion and mesenteric lymph node edema and/or
hyperemia were associated lesions in 4 pigs. There was mild catarrhal
gastritis, and 3 pigs had milk or curd filling their stomachs. Hyperemia
of the small intestine was seen in only 1 pig. The small intestinal con-
tents were scanty yellow fluid and similar material of greater quantity
filled the cecum and colon. The cecum of one pig was distended with gas,
but the feces were much alike in all the principals. Petechiation of the
renal cortical surfaces was apparent in the principals but not in the
controls. One principal pig had polycystic kidneys and the urinary
bladder contained about 10 ml. of viscous urine. In the male pigs there

was extension of the peritonitis into the scrotal sac. Escherichia coli

 

was recovered in culture of the 3 serous cavities and the humeroradial
synovial sacs from all the principals, including one that was euthana-

tized with electricity when it was seriously ill but not yet moribund.

HistOpathologic findings. Lymphoid tissue from the controls was more
dense than in the pigs from Experiment 1, but secondary nodules were
still not seen. Granulocytes were prominent in the lymph nodes and in
the spleens. There were some foci of hematOpoiesis in the spleen and
liver sections. No lesions were seen in the gastric tissues. The duo-
denum had a small number of granulocytes in the lamina prOpria and
mitoses in the mucosal epithelium. There was hyperemia Of the mucosa
in the upper, middle and lower levels of the jejunum. The epithelial

cells of villi in the lower section of the jejunum appeared hydropic

57
but were not desquamating, and there were very few leukocytes in the
lamina propria. Peyer's patches in the ileum were composed of nondense
lymphoid tissue and there were small numbers of granulocytes in the
ileal stroma. The cecum, spiral colon, and terminal colon presented no
unusual features. Other organs were also without lesions, except there
appeared to be some mild hyperemia of the adrenal cortex.

The principals had a tendency toward suppurative lymphadenitis in
addition to the lesions of edema, hyperemia, and depletion of lymphocytes
viewed in tissues in Experiment 1. Fibrinous to fibrinopurulent sero-
sitis was found in most of the sections of the viscera. Congestion of
the hepatic sinusoids was found in sections from 5 pigs and 2 sections
had edema around the gallbladders. Mild leukocytic infiltration was
found in one pancreatic section.

Sections from the stomachs of the principals had lesions of mild
to severe serositis but only one section had signs of inflammation of the
muscularis. The mucus formed a thicker layer on the mucosa in the prin-
cipals than in the controls. There was mucosal necrosis in one duodenal
section and inflammation of the muscularis in another, but the remainder
appeared to have only serositis. Two sections of the upper jejunum
were hyperemic and a third section had edema and leukocytic infiltration
of the muscularis. Pig 8 had lesions of enteritis consisting of hyperemia,
edema, mixed leukocytic infiltration of the lamina prOpria, and necrosis
of the mucosal epithelium, especially at the tips of the villi in the
middle and lower jejunum and ileum. Leukocytic infiltration of the
lamina prOpria in the middle and lower jejunum in the other pigs was mild
or absent, but catarrhal changes of increased mucous secretion and mucosal

hyperemia were found in some of the sections. Two sections of the ileum

58
had necrosis of the mucosal epithelium with leukocytic infiltration of
the lamina prOpria and some necrosis of Peyer's patches. The outer mus-
cularis of the ileum was affected by serocellular inflammation which
appeared to be an extension of the serositis. Mitoses were frequent in
the ileocecal lymphoid tissue in one section and necrosis was evident
in another section. Submucosal and subserosal edema was mild, but
fibrinOpurulent serositis was severe in 2 sections. Severe fibrinopuru-
lent serositis with subserosal edema and some leukocytic infiltration
were common lesions of the spiral colon and the terminal colon. Heart
and lung lesions resembled those in Experiment 1. Pig 7 had acute

exudative synovitis of the elbow and stifle joints.

Experiment 3

Bacteriologic examination of rectal swabs from 11 pigs at birth
revealed no bacterial growth after 72 hours' incubation but the original
germfree status of the pigs was converted to monocontaminated when
Staphylocgccus _p, grew in cultures of rectal swabs taken from the pigs
at 48 hours of age. The pigs in Isolator 1 (Table 6) were fed 5 ml. of
the E. 39}; inoculum at 72 hours of age, and the pigs in Isolator 3
were fed only 1.25 ml. of the inoculum. Both groups of principals were
positive for E, £2Ei_in culture of the rectal swabs collected the next
day.

Although 11 pigs were transferred into the receiving isolator, only
10 pigs were distributed among the isolators in the rearing room.’ Pig
2 had escaped from the uterus into the peritoneal cavity of the sow
during the surgical proceedings and it died about one hour afterward.
Pigs 7 and 9 were weak and had poor appetites; one of them had lost a

large volume of blood from loosening Of the umbilical ligature.

59

ucommua mmz OOHOHOOOU u +
uammnm mm3 OOHOHOOOU n I
own mo whom m um OONHumcmsusm mem .suom
.Lummp HHua: nuan Soum mmuHuomam noon own new Hmm3 muo3 mem mmOSH e«
mea Houuaoo «

 

 

mm + + + + I I I mo.o z ow««u HH m

««H H.5usmv + I + I I I I Hm.o m mw««0 OH m

OH + ««+ + «N I I I mm.o m nm««o m m

mm + + + + I I I Hm.o m Nw««o w m

«m + + + + I I + Nw.o z wn««u « H

NN + + + + I I + Hm.o m Hw««u m H

mm + + I + I I + mm.o m mn««o H H

ocoz I + I «x I I I NN.o m om««o «N N

««H H.3usmv I I I I I I I NN.o m mw««o «o N

««H A.nu=mV I I I I I I I Hm.c m «m««o «m N
A.unv OaHu hmmouomc nummo moan memuoq< mHmOuho «Homo um>mm A.wxv xmm .02 .Oz .02
HO>H>H=m um m:OHm IHOHQ IOHOOH IOHOOH .u3 mmmouomz me HoumH
muzmoaxoumom IOH mmouu zoom IowH

 

 

.m unmaHuomxm OH po>ummno mame HOOHOHHO pom meo «O :OHuonHuumHm .o mHan

60

Fever and other clinical sigp_, The clinical signs are tabulated with
the details of distribution of the pigs in Experiment 3 (Table 6). Body
temperatures of 104.0, 105.6, and 106.3 F. were observed in Pigs 4, l,
and 3, respectively, approximately 12 hours before they died. Blood
samples from all 10 pigs 24 hours before exposure of 7 pigs and from
all 8 remaining pigs 21 hours after the principals were exposed had only
minor changes in individual and group values. The mean hemogram values
for the 10 pigs at 48 hours and 8 pigs at 93 hours of age were:

Age

48 hours 93 hours

Hemoglobin (Gm./100 ml.) 8.18 6.79
Packed cell volume (%) 26.50 24.25
Total leukocytes (x 103/cu.mm.) 7.68 7.30

Differential leukocyte counts (%)

Segmented neutrOphils 65.10 45.75
Nonsegmented neutrophils 11.2 26.0
Lymphocytes 22.3 26.6
MOnocytes 1.5 1.4
Basophils 0.0 0.0
Eosinophils 0.0 0.25
Nucleated erythrocytes

(/100 leukocytes) 1.6 2.62

The hemogram values for the one surviving principal at the next blood
sampling and the mean values for the 2 remaining controls 6 days after
exposure were:

Controls Principal

Hemoglobin (6m./100 m1.) 10.2 9.2
Packed cell volume (%) 33.0 28.0
Total corrected leukocytes
(x 103/cu.mm.) 6.65 9.80
Differential leukocyte counts (%)
Segmented neutrophils 46.5 36.0
Nonsegmented neutrophils 5.5 15.0
Lymphocytes 46.0 49.0
Monocytes 0.5 0.0
Basophils 0.5 0.0
EosinOphils 0.5 0.0

Nucleated erythrocytes
(/100 leukocytes) 3.0 0.0

61
Serum electrolytes were not determined in this experiment. Lack of
separation of the serum proteins in electrophoresis produced highly

erratic results which are not presented here.

Necropsy findings. No gross lesions were found in the controls and all
their body cavities were negative for bacterial growth on culture. Gross
lesions in the principals were similar to those in pigs on Experiment 2.
There were external signs of diarrhea in 5 pigs and 3 carcasses were
dehydrated. Subcutaneous ventral abdominal edema was severe in 3 pigs,
and the edema extended to the neck in one of them. Hyperemia and edema
were seen in the external and internal lymph nodes. Polyserositis in

6 pigs varied from serofibrinous to fibrinOpurulent. The extensive
peritoneal exudates in Fig 11 (64480) are illustrated in Figure 6.

That pig also had 20 m1. pleural serous exudate. Peritoneal exudates,
pleural adhesions, and pericardial exudates (Figure 7) are shown in

the photograph Of Pig 8 (64482). Pig 9 was cyanotic at the time of
exposure and died some 10 hours later with signs of diarrhea, intestinal
hyperemia, and rounded diastolic heart. Cultures of the pericardial and
pleural cavities were negative for bacterial growth. Three pigs had

30 to 60 cc. of milk curd in the stomach, but others were empty. There
‘was mild hyperemia of the small intestine in 5 pigs and in approximately
the first 125 cm. of the jejunum in Pig 10, which was euthanatized.

The latter pig also had lesions of polyarthritis and mild fibrinous
peritonitis. The liver and spleen were enlarged and dark in 5 pigs.

The kidneys were usually pale and the urinary bladder was adherent to
the ventral parietal peritoneum. Lesions of acute pneumonia were patchy
or' confined to the apical and cardiac lobes. Septicemic infection was

indicated by the recovery of E, coli from the 3 major serous cavities

62

 

I '”

 

Figure 6. Pig 64480, 58 hours after exposure. Fibrinous
to fibrinopurulent exudates cover the surfaces of abdominal
viscera.

 

 

Figure 7. Fig 64482, 75 hours after exposure. Poly-
serositis with inflammatory exudates on peritoneal, pleural
and perciardial surfaces.

63
and the left elbow synovial surface in 5 pigs, peritoneal cavity and
elbow in one pig (Pig 9) and from the hock and elbow but not from the

serous cavities in the 9-day-old Pig 10.

Histopathologig findings. In most of the lymph node sections there were
lesions of edema, hyperemia, and/or depletion of lymphocytes, as in the
previous experiments. Diffuse suppurative lymphadenitis occurred in

the superficial inguinal and mesenteric lymph node sections of 2 pigs.
Four pigs had serositis of the adrenal capsule, and one of the 4 had
hyperemia of the zona glomerulosa accompanied by superficial necrosis.
Two pancreatic sections had interlobular edema and some leukocytic in—
filtration deeper than the serosa. There was congestion in some splenic
sections and some lymphocytic depletion. There was some cloudy swelling
of the hepatic parenchyma and congestion of the sinusoids in most sections.
In one liver there was edema of the gallbladder, and fatty vacuolation

was seen in another section. Gastric serositis did not usually pene-
trate deeper than the serosa, but 2 sections were found to have edema

and leukocytic infiltration of the outer muscularis (Figure 8). Mucosal
hyperemia was found in one or more levels of the small intestine in 5

pigs and 1 had mild hemorrhagic enteritis in the middle and lower jejunum.
Serositis was the most prominent lesion of the cecum. Subserosal edema
of the spiral colon with hyperemia and leukocytic infiltration accompanied
inflammation of the colonic muscularis in one pig (Figure 9). Fibrino-
purulent pericarditis and epicarditis were severe in 3 pigs. Pig 9 had
the most severe epicarditis and myocarditis of this group. Fibrino-
purulent pleuritis,in some of the lung sections,was accompanied by septal
thickening and atelectasis which resembled incompletely expanded fetal

lung tissue.

I- ll‘t il’uvu

‘I I.

64

 

 

 

Gastric
Hematoxylin

Pig 64481, 72 hours after exposure.

Figure 8.
serositis with fibrin, subserosal edema and inflammatory

cells extending to involve outer muscularis (M).

and eosin.

x 190.

4"
I'. t
\ :a.‘
.. '1:

92%;}. 9"?"
4;; g --

H

 

 

Sub-

Pig 64480, 58 hours after exposure.

hFigure 9.
serosal edema, hyperemia, and leukocytic infiltration of

spiral colon.

Hematoxylin and eosin. x 190.

65

Experiment 4

 

Bacterial,status and clinical observations. The distribution of the pigs
and the clinical signs observed are shown in Table 7. The pigs in Experi-
ment 4 were gremfree at birth and remained free of any demonstrable bac-
teria throughout the experiment, except for the inoculum which was fed

to the principals when they were 6 days (148 hours) of age. Viability

of the inoculum.was confirmed by the recovery of typical E, ggl1_from
rectal swabs from the principals at 40 hours after exposure.

At 18 hours after exposure the body temperatures ranged from 102.4
to 103.0 F. The pigs were all eating normally at 21 hours after exposure,
but at 24 hours 4 of the 6 principals did not eat all their feeding at
once, and Pigs 6 and 7 were shivering. By 40 hours after exposure Pigs
6, 7, and 3 had body temperatures of 107.0, 104.5, and 107.0 F., respec-
tively. Twenty-four hours later, Pigs 1, 2, and 4 had temperatures of
105.6, 106.0, and 104.6 F., respectively. At that time Pig 6 was dead
and none of the principals ate their complete feeding within 30 minutes.
Pig 4 maintained body temperatures above 105.0 F. for another 4 days
until it was euthanatized. Lameness or soreness and reluctance to move
about was manifested by 3 pigs. ‘Diarrhea was severe in only 3 of the
6 principals, although the feces were more fluid in all the principals
than in the controls.

Blood samples collected at 40 hours after exposure were calculated
to have the following mean values with total leukocyte counts corrected

for nucleated erythrocytes in the controls and principals:

66

Oommmua mus OOHOHOOOO n +
unmmnm mos OOHOHOOOO u I
.umwm OOOHH co ausouw HmHumuOmn
OOOOOHO uo: OHO OOHOO onu aouw mucmuaoo .HHOO am Ou musmoaxm muomon OONHumsmnusm was m me *«
.mem Houucou «

 

 

 

NI N.Nusmv I *«III III III III III III HO.O N ONNNO N N

«m + + + + + I + H0.0 m NNNmO N m

«m + + + + + I + Nm.O m HNNmO O m

NNH H.535 + I + I + + + NH.H z NNNNO « N

ONH + + I + + I + Nw.O m «NNmO m H

NNH + + I .+ + I + NO.O N NNNNO N H

«OH + + I + + I + H0.0 m MNNmo H H

OON H.5usmv I I I I I I I N0.0 m ONNmO «O N

OON H.3usmv I I I I I I I m0.0 m NNNmu «m N
A.H£v maHu honouomc gamma moss meouoc< mHmoumo OHOOO Ho>om A.wxv xom .02 .oz .02
HO>H>H=m um OOOHm IumHO IOHOOH onaoH .uB NOOOHOOZ me HoumH
ousmomxoumom IOH mmouu Hmom IomH

 

 

.« uOmBHummxm OH mo>ummno mstm HmOHOHHO mam mmHO mo OOHOOOHuumHm .N OHomH

67

Controls Principals
Hemoglobin (Gm./100 ml.) 8.75 8.03
Packed cell volume (%) 26.50 26.17
Total corrected leukocytes
(x 103/cu.mm.) 5.11 9. 73
Differential leukocyte counts (%)
Segmented neutrophils 44.0 41.67
Nonsegmented neutrophils 2.0 9.33
Lymphocytes 53.0 49.0
Monocytes 0.5 0.0
BaSOphils 0.0 0.17
EosinOphils 0.5 0.0
Nucleated erythrocytes
(/100 leukocytes) 14.5 14.3

While the other principals had leukocytosis, Pig 4 had leukopenia with
relative and absolute lymphopenia. At the next blood sampling Pig 4
was the sole surviving principal and the leukOpenia had been converted

to leukocytosis. The hemogram was:

Hemoglobin (Gm./100 ml.) 9.0
Packed cell volume (%) 28.0
Total leukocyte count (/cu.mm.) 17,100
Total corrected leukocyte count

(/cu.mm.) 11,958
Absolute differential counts

(/cu.mm.)

Segmented neutrOphils 2,870

Nonsegmented neutrophils 1,315

Lymphocytes 7,773

The results of determinations for serum electrolytes in 19 samples
indicated similar ranges of values for the controls and the principals.
The results of electrOphoresis of 23 serum samples from 7 pigs at 5 days
of age (1 day before exposure) and at 8 days of age (2 days after
exposure) indicated that albumin remained nearly constant and the alpha
globulin component increased slightly in percent of total protein in the
5 principals, while albumin increased sharply and the alpha globulin
component decreased almost prOportionately in the 2 controls. Change in

the beta globulin component was variable in the principals and controls.

68
The gamma globulin component remained unchanged in the principals but
decreased in both controls. The lack of total protein determinations

for these samples prevented quantitative evaluation.

Necropsy fipding_. Pig 5 was found in Opisthotonic convulsions and was
removed from the isolator for euthanasia when it was 3 days of age.
There was no ligature on the umbilical stump. Subcutaneous edema of the
limbs and ventral abdomen was the only grossly visible lesion. Tissues
were not saved for histOpathologic examination, but contents from the
colon were streaked on a blood agar plate and no bacterial growth occurred
during 72 hours of aerobic incubation at 37 C.

The controls had no visible gross lesions and had approximately
doubled their birth weights when they were euthanatized at 15 days of
age (Figure 10). The cecum and colon in each of the controls were larger
than in the principals. The carcasses of the principals were emaciated
(Figure 11) in contrast to the controls. Diarrheal feces covered the
rear quarters of Pigs 4 and 6. Elbows, hocks and points of jaw contact
with the cages were denuded of hair in some pigs. Hyperemia and edema
of the external lymph nodes varied from none to moderate. Polyserositis
varied from serofibrinous in the pigs which died earlier to fibrinous
or fibrinopurulent in the pigs which survived longer (Figure 12) but
was the most prominent lesion in each of the pigs. Hepatic congestion
and splenic enlargement were consistent lesions. Each of 5 principals
had about 1 ounce of dry milk curd in the stomach; Pig 4 had about 2
ounces. Mesenteric lymph node hyperemia or edema was found in 4 pigs
but was mild in 3 of them. Intestinal hyperemia was absent or mild.

There was subserosal edema of the spiral colon in 2 pigs. The cecum

69

 

,7 ,HH—rv—r—vv—v—

Figure 10. Pig 65228. Germfree control pig which
weighed 2100 Gm. when it was euthanatized at 15 days of age
(Experiment 4).

 

_'—.v—.H .w

 

Figure 11. Pig 65226. Emaciated carcass of pig euthana-
tized 162 hours after oral exposure to Escherichia coli.

70

    

Figure 12. Pig 65226. Polyserositis in the pig carcass
illustrated in Figure 11. Note exudates on peritoneal,
pleural, and epicardial surfaces.

L . ./ '.‘ '

I , Ira , ,1? :PT~"-;

I 3155' ‘- 6“; 7:
.a-‘vs‘j'r r ,3. _.
‘.:.‘-;_<H‘--F~ ,J‘...

I tvluu" H, 1.1.“.

I -‘,-'O‘ I‘Iz . Satan,
u{}3:§f‘f‘ 7% Xi

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I ;._ f1 US», ‘5‘ i,
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4“ H-?\1‘ ‘Jé L ‘ a".

I 2.143; :,.ét.73v*‘. ‘ .
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. L

 

I “x ‘ , ‘ 1‘ ‘ .
.: “ } .i , R
l {f .a/ I? .132": 1‘? '3 .

Figure 13. Pig 65224, 120 hours after exposure. Mesen—
teric lymph node. Note absence of lymphoid follicles.
Hematoxylin and eosin. x 75.

 

 

Figure 14. Higher magnification of mesenteric lymph
node in Figure 13. Note small number of lymphocytes, edema
(a), and hyperemia (b). Hematoxylin and eosin. x 190.

 

 

Figure 15. Pig 65224, 120 hours after exposure. Super—
ficial inflammation at hilus of spleen with serositis. Note
mass of neutrophils (N). Hematoxylin and eosin. x 75.

 

 

 

Figure 165 Pig 65224, 120 hours after exposure. Pancreas
with focal parenchymal necrosis, interlobular edema and ex-
tension Of serositis. Hematoxylin and eosin. x 75.

 

Figure 17. Pig 65224, 120 hours after exposure. Hepatic
serositis in proximity to gallbladder. Note hepatic parenchyma
(a), edematous Glisson's capsule (b), and subserosal leuko—
cytic infiltration (c). Hematoxylin and eosin. x 75.

 

 

L

 

Figure 18. Pig 65224, 120 hours after exposure. Duodenal
serositis. Note subserosal edema and leukocytic infiltration.

 

75

 

Figure 19. Normal duodenal mucosa from section in
Figure 18. Hematoxylin and eosin. x 190.

 

 

 

Figure 20. Cuboidal mucosal epithelium with subepi-
thelial edema also from section in Figure-18. Hematoxylin
and eosin. x 470. . .

I

76
edema (Figure 20). One pig had lesions of catarrhal enteritis in the
upper jejunum with increased mucus, epithelial debris, and degenerating
neutrophils in the lumen; another pig had severe lesions of fibrinopurulent
'serositis with some extension to the mucosa. Similar lesions were found
in the middle and lower jejunal regions of the same 2 pigs (Figure 21).
A normal serosa and the hydropic appearance of the lower jejunal mucosal
epithelium in a control pig are illustrated in Figure 22. FibrinOpurulent
serositis of the ileum varied from mild to severe in 3 pigs (Figure 23),
which had only slightly greater leukocytic infiltration of the lamina
propria than in the controls. The same 3 principals had lesions of
similar nature and severity in sections of the cecum, spiral colon, and
terminal colon. All but one principal had lesions of fibrinous to
fibrinopurulent pericarditis (Figure 24), and Pig 4 had lesions of
suppurative myocarditis (Figure 25). All the principals had lesions of
pleuritis ranging from fibrinous to suppurative with mixed leukocytic
infiltration, and 2 of the pigs had lesions of interstitial pneumonia
with interlobular edema and neutrophilic infiltration (Figure 26)
accompanied by alveolar septal thickening but without alveolar and
bronchiolar cellular exudates. Two Of 3 elbow joints had lesions of
synovitis. The most severe synovitis (Figure 27) was represented by
fibrinocellular exudates on the intimal surface near the limbus, synovial
intimal hyperplasia, and subintimal edema with focal necrosis and leuko-
cytic infiltration. Figure 28 illustrates the histologic features of

the synovial membrane of the elbow from a similar site in a control pig.

77

 

 

Figure 21. Pig 65224, 120 hours after expdsure. Fibrino-
purulent serositis of the mid-jejunum. Hematoxylin and
eosin. x 75. 9

. ._.1..I...~-..

 

.-___ “mu-4...-.— A

 

 

Figure 22. Pig 65227. Mid-jejunal section. Note
single layer of serosal cells and the hydropic appearance of
the mudbsal epithelium in a germfree control pig 15 days Of
age. Hematoxylin and eosin. x 75.

78

 

 

Figure 23. Pig 65224, 120 hours after exposure. Sub—
serosal edema and serositis of the ileum. Leukocytic infil-
tration of the lamina propria is only slightly greater than
in the controls. Hematoxylin and eosin. x 75.

 

Figure 24.- Pig 65224, 120 hours after exposure. Fibrino-
purulent pericarditis and epicarditis with adhesions between
the pericardium (p) and epicardium (e). Hematoxylin and
eosin. x 75.

  

 

-—Figure 25. Pig 65226, 162 hours after exposure. Suppu-
rative myocarditis. Note neutrophils infiltrating the myo-
cardium. Hematoxylin and eosin. x 470.

80

 

 

 

 

Figure 26. Pig 65224, 120 hours after exposure. Inter—
stitial pneumonia with interlobular edema and neutrophilic ‘
infiltration. Alveolar septa are thickened but alveoli and
bronchioles are free of cellular exudates. Hematoxylin and
eosin. x 190.

81

 

 

 

Figure 27. Pig 65222, 94 hours after exposure. Syno-
vitis Of the elbow joint. Note fibrinous and cellular exudates
on the synovial intima accompanied by intimal hyperplasia
and subintimal edema with leukocytic infiltration. Hematoxylin
and eosin. x 190.

 

 

Figure 28. Pig 65227. Histologic features of the
synovial membrane from the elbow of a germfree control pig.
Hematoxylin and eosin. x 190.

82

Experiment 5

 

Anti-hog cholera serum was fed to each of 6 pigs in Experiment 5
to determine whether this would provide passive immunity against infec-
tion by‘E,‘ggll 083 in colostrum-deprived gnotobiotic pigs and to investi-
gate some Of the ways the course of the disease might be altered by the
anti-hog cholera serum.

Rectal swabs taken before the pigs' first feeding were negative on
culture for bacterial growth. Monocontamination with a Gram—positive,
spore-forming rod which grew in chains and was classified as Bacillus _p,
was disclosed in culture of rectal swabs collected from the pigs at 4

days of age. Escherichia coli was recovered from all the principals at

 

44 hours after oral exposure. Also, the principals and controls still
had the Gram-positive bacilli but they were reported as few or rare in
cultures of the principals' rectal swabs from 44 hours postexposure

onward, and none was recovered from Pig 6 at one week after exposure.

Clinical Observations. The controls' body temperatures of 103 to 104.6
F. were generally higher than in previous experiments, but fever was
Observed in only one principal (106.8 F. at 5 days postexposure).
Anorexia was of short duration in one pig and persistent but incomplete
until near death in another. Diarrhea or erythema of the perineum was
observed in 3 pigs. Details of the pigs' distribution and the clinical

observations are shown in Table 8.

Hematologic and serologic findings. In 5 successive samplings between
96 and 311 hours of age, the controls and principals had increasing
hemoglobin values -- 9.8 to 11.4 Gm./100 ml. —- and packed cell volumes

-- 30.7 to 34.1%. The maximum mean of nonsegmented neutrOphils Observed

83

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.BOHOOHOOO OO an: OOH hmm .Ha ONH OOH3 OOHOOHOO Omouuxov Non .Ha m
vow mums HH OOO OH .O .« .m mem .me mo muOon O OO wOHOOOH OmuHm OOO mm OOH mmm .Ha OO OOO OOHOOHOm
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Amhmpv OONHO hOmOHOOO numon mosh meO .mHmOONO OHOOO um>om mmOOHOOz nOuHm xmm .Oz .oz .02
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.m OOOEHHOONM OH Ow>ummno mOme HOOHOHHO OOO OMHO mo OOHOOHHHOmHn .w OHOOB

84
in the differential leukocyte counts for the controls was 1.0 and for
the principals was 2.2; therefore, they were included with the segmented
neutrophils in the data above. Corrected mean total leukocyte counts
and differential neutrOphil and lymphocyte counts for the controls and

principals in 5 successive samplings were:

 

 

Age of Differential (% of total)

Pigs Total.WBC/cu.mm. Neutrophils Lymphocytes
(hr.) Controls Principals Controls .Principals Controls Principals
96 6513 5772 43.6 39.5 55.7 58.7
140 6429 9541 28.7 26.2 71.0 73.1
192 7078 14469 25.0 18.6 74.3 81.0
264 6139 15784 26.0 24.9 72.3 74.5
311 4471 13075 19.6 18.4 79.3 81.1

Monocytes and basophils were generally absent, and eosinophils were less
than 1.0% except in the fourth sampling of the controls when the mean of
the eosinOphils was 1.67% of the leukocytes counted in the differential.

Among the principals, the pigs fed anti-hog cholera serum had the
lowest (7573) and the highest (28,300) corrected total leukocyte counts
after the day of exposure. However, the pigs not fed anti-hog cholera
serum (-) and those fed the antiserum (+) alternated in having the
higher mean total corrected leukocyte counts on successive days of
sampling: (-) 10,806, (+) 15,704, (-) 16,606, and (+) 13,256. Relative
neutrOpenia and moderate absolute neutrOpenia were observed only in
Pigs 6 and 10 at 146 hours after exposure.

The total serum protein tended to increase gradually from a mean
of 2.76 Gm./100 ml. (range — 2.3 to 3.3) in the samples from 11 pigs
96 hours of age to a mean of 4.38 6m./100 ml. (range - 4.2 to 5.2) in
samples from 10 pigs 311 hours of age. Ten samples collected termi-
nally, just prior to euthanasia, ranged from 3.6 to 4.4 6m./100 ml.

total serum protein.

85

Paper electrophoresis of 64 serum samples revealed that there was
not a corrleation between the distribution (percentage) of serum pro-
teins migrating in the albumin and globulin fractions when anti-hog
cholera serum was fed in the first feeding and compared to control serum
samples. The albumin fraction decreased from 32% before exposure to
20% terminally in the pig that died, but the total serum protein increased
from 2.6 to 4.6% to indicate an actual increase in the total amount of
albumin in the terminal serum sample. The results of electrophoretic
analysis of protein (expressed as percentage of total serum protein) in
the first sampling of 11 pigs at 4 days of age (96 hours) and in the

terminal samples from 10 pigs at 14 and 15 days of age ranged as follows:

 

I _ Globulins
Age of pigs Albumin Alpha1 Alpha2 Beta Gamma
4 days 23—35 4-11 23-40 13-36 6-10
14 and 15
days 45-63 ‘ 2-4 22-37 10-18 2-5

The albumin:globu1in ratio and total amount of serum albumin increased
with the age of the pigs. Gamma globulin was decreased in proportion
(ratio) to total proteins but was maintained nearly constant in total
amount in the samples in the 10 surviving pigs.

In 54 samplings representing 11 pigs in each of the first 4 groups,
and 10 pigs in thefflfth sample group, the flame photometer analysis indi-
cated a range from 139 to 155 mEq.Na/liter in the first group of serums,
and 140 to 145 mEq.Na/liter in the final group. Potassium ranges in
the same groups of serum samples were 4.6 to 7.0 mEq.K/liter in the
first group and 7.4 to 10.5 mEq.K/liter in the final group. The mean
sodium values in the 5 successive groups of samples were 147, 144, 150,

145, and 143 mEq.Na/liter. The means for serum potassium were 5.9, 6.1,

86
6.3, 8.1, and 8.8 mEq.K/liter. No differences were noted between the

experimental groups.

NecrOpsy finding_, Gross lesions were found in 2 pigs at necrOpsy. At
the ventral margin of the left central lobe of the liver in Pig 2 there
was a pale yellow, l-cm.-diameter, circumscribed lesion that resembled
inspissated fibrin but seemed to be an integral part of the liver. No
other lesions were seen in Pig 2, which had been fed anti-hog cholera
serum.

Pig 6 was similar to principals in previous experiments in having
severe fibrinous polyserositis. In all 3 cavities, the exudates appeared
dry and organizing. There was diffuse pulmonary congestion. There were
mild catarrhal gastritis and enteritis.

In bacteriologic examination, pure cultures of E, £2EE_were recovered
from the pleural, peritoneal and pericardial cavities of Pig 6. Peri-
toneal swabs from Pigs 3, 7, 8 and 11 produced no growth in culture.
Gram-positive rods with spores (Bacillus _p,) were isolated from the
peritoneal swabs of Pigs 5, 9, and 10. Similar Bacillus _p, and E,‘ggli

were isolated from Pigs 1, 2, and 4.

HistOpathologic finding_. The lesion from the liver of Pig 2 was com-
posed of a central zone of necrotic neutrophils with a peripheral zone
of lymphocytes in a mass of fibrin. The tissues of Pig 6 were similar
to those from principals in previous experiments with fibrinopurulent
polyserositis. There were signs of edema, hyperemia, and depletion

of lymphocytes in the mandibular and mesenteric lymph nodes. Mucous
secretion was increased in the stomach and lower intestinal sections.

There were adhesions between the visceral and parietal pleural surfaces

87
in the lung section which was taken to include the parietal pleura and
intimate subjacent tissues in the ventral thorax. No lesions were seen
in sections from the other principals and the controls, but the liver
sections presented the usual pale, glycogen-rich appearance of the hepato-
cytes. The middle and lower jejunal sections frequently had hydrOpic

appearing mucosal epithelium.

Experiment 6

Anti-hog cholera serum (25 ml./pig) was fed to each of 6 pigs in
their first feeding at 4 hours of age. The details of distribution of
the pigs and the clinical signs observed in Experiment 6 are shown in
Table 9, together with comparative body weights at birth (24 hours) and
at necropsy. Fifteen pigs were delivered by hysterotomy, but 3 of them
were discarded because of small size and weakness. Pig 9 died at 3
days of age following probable thermometer perforation of the rectum
which caused intraperitoneal retention of sterile feces.

No microorganisms were demonstrated in swabs from the liquid diet
and rectal swabs from 11 of the 12 pigs at 16 hours of age. However,
one pig's rectal swab yielded a Gram-positive Bacillus _p, and 8 of the
pigs were monocontaminated with the Bacillus by their fifth day.

Prior to exposure to E, £2Ei_at 5 days (115 hours) of age, Pigs 11
and 12 were each fed 2.5 ml. of a 36-hour BHI culture of the monocontami-
nant Bacillus which was already present in their environment. Pig 11
was never fed the usual inoculum of £3.2211 but was given the empty feed-
ing tray from the cage of Pig 13 some 20 hours and 2 feedings after the
.§3.22££ inoculum had been fed in it to Pig 13. In addition to the con-

taminant Bacillus gp,, E, coli was recovered from the rectal swabs Of all

88

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Ommsu mo :OOO OO OOH was EOuOm OHOHono wonIHOOm HOHOHOEEOO O HO .Ha ON «

.anuoumumhn Ono mo OOHOOHOEOO HOOHO muaon

 

 

 

 

 

NN + I + + + I + OO.N O0.0 z OO«Nm mH m
NN I I I I + I I OH.N MOO NH Ow«Nm «NH m
HOOHOV

m.m + + I + + I + mm.H Om.H z Nw«Nm HH m

NN I I I + + I I O«.N NN.H z Om«Nm «OH O

III III III III III III III III «« MH.H z «O«Nm O N

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IOOOOOOO um mOOHm IHOHQ InoO< IOHOOH IOHOOH AJMXV .OB Ovom OOOOOOOZ me HOOOH
Owns Ow< IOH mmouv IOOH
.O uOOaHHOOxm OH OO>HOOOO mOme HOOHOHHO OOO meO HO OOHOOOHHOOHQ .O OHHOH

89
the principals at 45 hours after the first exposure to E, 2911, With the
latter group of rectal swabs, clean swabs were collected from Isolators
2 and 3, and curdled liquid diet from Isolator l for microbiological

examination and all were sterile on culture.

Clinical observations. All 3 pigs in Isolator 2 were exhibiting signs

of illness at 40 to 48 hours after exposure. The body temperature of

Pig 8 was 106.4 F., Pigs 7 and 8 had diarrhea, and Pigs 6, 7 and 8 had
transient anorexia. In Isolator 3, Pigs 10 and 13 also ate slowly the sec-
ond day after exposure to E, 52;}, Three more of the pigs developed
fevers in excess of 106.0 F., Pig 11 at 4 days, Pig 13 at 5 days, and

Pig 7 at 9 days, with recurrence in Pig 7 at 16 days after exposure.
Fever and incomplete anorexia persisted in Pig 11, which died at approxi-
mately 5.5 days after being fed from a tray that had been contaminated
with E,‘ggEE, All the other pigs survived the exposure to E, 22;; and
were euthanatized to terminate the experiment when the principals were

22 days and the controls were 23 days of age.

Hematologic findigg_. Leukopenia was not observed in the 44 postexposure
samples from the principals, except that relative neutrOpenia at 4 days
after exposure followed relative neutrOphilia, which had occurred at
approximately 2 days after exposure and in which there was also an abso-
lute increase in numbers of nonsegmented neutrophils. In that first
postexposure group of samples the differential neutrOphils, including the
nonsegmented neutrophils, ranged between 49 and 76 for the principals

and 39 to 46 for the controls. The nonsegmented neutrOphil range in the
controls was 0 to 3, while the principals had 3 to 21. Two principals

had total corrected WBC counts at least 50% above the mean of the controls;

90

they were: 10,693 for Pig 10 at 42 hours after exposure, and 19,400
for Pig 11, which had been exposed to E, £2EE_only 24 hours previously.
In 5 samplings, a total of 30 blood samples was collected from the 6
surviving principals between 6 and 16 days after.exposure. The lowest
individual corrected total WBC count was 8020 and the highest was
29,612. Both extremes occurred in samples from pigs that had been fed
anti-hog cholera serum. In 20 comparative samples from the 4 controls,
the corrected total WBC range was 1974 to 6820, and both extremes were
from pigs that had not been fed anti-hog cholera serum.

Hemoglobin levels in all 11 pigs at 5 days of age and just prior to
exposure ranged between 7.7 and 10.5, with a mean of 8.8 Gm./100 ml.
The PCV range was 24 to 32% for the group of 11 at the same time. The
4 pigs that remained the controls had an average mean corpuscular hemo-
globin concentration (MCHC) of 29.75%, and the 7 principals averaged
33.53% MCHC. Hemoglobin and packed cell volumes reached their peak at
12 days of age, when the controls had means of 10.6 Gm. Hb./100 ml. and
33.75% PCV. Comparative mean values for the 6 surviving principals were:
Hb. - 9.8 Gm./100 ml. and PCV - 32.5%. Conversely, at 12 days of age,
the values for MCHC were reduced (controls - 31.4%, principals - 30.15%)
to the lowest in the 8 groups of samples taken from the pigs between 5
and 21 days of age, excepting the 29.75% MCHC in the first sampling from
the controls. At 21 days Of age the MCHC averages and the hemoglobin

and PCV ranges were:

Controls Principals
MCHC average (%) 33.55 32.64
Hemoglobin range (Gm./100 ml.) 7.2-8.6 5.5-9.0
Packed cell volume (%) 22.0-24.0 18.0-30.0

Only 2 hemoglobin estimations in the 83 samples were below 7.0 6m./100 ml.

91
and only one was above 11.6 6m./100 ml. The principals had slightly
lower hemoglobin levels than the controls in all except the samples taken
on Day 21. Also, the principals had the lower MCHC values in the last 5
samplings. Trends were similar and ranges overlapped in measurements of
hemoglobin, packed cell volume, and MCHC in the principals and controls,

whether they had or had not been fed anti-hog cholera serum.

Serologic findings, In 9 successive samplings, the total serum protein
was found to range between 2.8 and 4.0 6m./100 ml. in the controls and
2.6 to 5.1 6m./100 ml. in the principals. The total serum protein quan—
tities were similar in range in the controls and principals irrespective
of the previous feeding of anti-hog cholera serum.

In paper strip electrophoresis the mean gamma globulin in 21 samples
from principals not fed anti-hog cholera serum was 5.0%, and in 36 samples
from the principals fed anti-hog cholera serum the mean was 4.92% of
the total serum protein. In 18 samples from 2 controls that had been
fed anti-hog cholera serum there was 4.33% gamma globulin as compared
with a mean of 3.66% in the same number of samples from the 2 controls
not fed anti-hog cholera serum. The gamma globulin range in 36 samples
from the controls was between 1 and 9%, and in 57 samples from the prin—
cipals the range was 0 to 13%.

The principals had higher beta globulin mean values than the con-
trols in all days except at 2 days after exposure to.E,'ggEi. Also, with
the exception of that one day, whenever the beta globulin value was in-
creased or decreased in the principals, it was paralleled in the controls.
Ninety-three individual samples ranged between 10 and 25% beta globulin,
while the means of the groups ranged from 11.5 to 18.5% in the controls

and 11.7 to 21.5% of the total serum protein in the principals.

92

Alpha-l-globulin decreased in all pigs as their age increased, and
declined from a range of 6 to 14% in 11 pigs at 115 hours of age to a
range of 2 to 5% with a mean of 3.2% for 10 pigs at 22 and 23 days of age.

Alpha-2—globulin and albumin were approximately equal in the pre-
exposure and first postexposure groups of blood samples. However, by the
twelfth day of age (7 days after exposure) the albumin:a1pha-2 ratio had
been increased to 2.38:1 in the controls and was only 1.12:1 in the
principals. Also, at the seventh day after exposure, the 2 principals
not fed antiserum had 28% albumin and the antiserum-fed principals had an
average of 49% albumin with albumin:a1pha-2 ratios of 0.58:1 and 1.53:1,
respectively. By 16 days after exposure, the ratio of albumin to alpha-
Z-globulin had declined to 1.66:1 in the controls and to 0.96:1 in the
principals. The 2 principals not fed anti—hog cholera serum now had the
higher albumin:a1pha-2 ratio of the 2 groups of principals, 1.02:1 for
those not fed antiserum and 0.93:1 for the antiserumrfed principals.

Albumin reached a peak at a mean of 57.25% of total serum protein
in the controls and 42.16% in the 6 principals at 12 days of age. After
12 days of age the albumin fraction fluctuated. There were means of 58%
in the controls and 37% in the principals at 23 days and 22 days of age,
respectively, when they were anesthetized and exsanguinated.

Serum potassium increased in all the pigs as their age increased.
The means at 2 days after exposure were: 4 controls - 6.4 mEq.K/liter,
7 principals - 6.14 mEq.K/liter. At 16 days after exposure the means
were: 4 controls - 9.25 mEq.K/liter, 6 principals - 9.2 mEq.K/liter.
The extreme range in individual measurements of 93 samples was 4.2 to
9.8 mEq.K/liter. Serum sodium concentrations were similar among all

groups of pigs, ranging between 136 and 154 mEq.Na/liter in 72 samples

93
from pigs between 7 and 21 days of age. In 10 additional samples at 22
and 23 days of age, 2 of the principals had lower concentrations of 133
and 128 mEq.Na/liter.

Tube agglutination tests employing systems of 0.1 ml. gnotobiotic
pig serum diluted 1 to 10 in isotonic sodium chloride aqueous diluent
before adding 1.0 m1. of diluted whole-cell E, ggli 083 somatic antigen,
which made the final serum dilutions 1 to 20, failed to demonstrate O
agglutinins for E, 32;; 083:K.:HNM in 43 serums collected from the gnoto-
biotic pigs at 5 to 12 days of age and tested in duplicate. Eleven
serums from the S-day-old (115 hour) pigs were also tested at final
dilutions of 1:10 and 1:20 with combined E, gglgylatex particle antigen,
and with the plain E, 22;; antigen in glycine-buffered saline solution
diluent. All the gnotobiotic pig serums were negative for agglutination.
Positive control serums from immunized conventionally reared weaned pigs

agglutinated fully at the above dilutions.

Necropsy finding_. There were no lesions in the controls. Four of 7
principals had lesions Of polyserositis which varied from serofibrinous

to fibrinOpurulent and, in one pig, organizing fibrinous serositis with
firm intestinal adhesions. In the latter case a peritoneal swab pro-
duced no bacterial growth on culture, whereas E, ggll.was recovered in
culture of peritoneal swabs from the other 3 pigs which had gross lesions,
and only from those pigs with lesions of serositis. One of the pigs with
lesions had been fed anti-hog cholera serum but had diffuse serofibrinous
peritonitis with effusion, pleuritis with approximately 10 ml. of gray

serous exudate, and severe fibrinous pericarditis.

94

Histopatholggic finding_. Lymphoid follicular development was more dis-
tinct in the 3-week-old controls than it had been in younger pigs in
previous experiments, and secondary nodules were becoming distinguishable.
However, no differences were noticed between those controls fed anti-hog
cholera serum and those not fed the antiserum. There was less distinct
follicular development in the lymph nodes and spleens of the principals
which had gross lesions than in those without gross lesions. Granulo-
cytes were prominent in the lymph nodes of the controls and principals.
EosinOphils were less numerous than neutrOphils but were obvious because
of the brightly staining granules in their cytoplasm.

Pale, vesicular, parenchymal cytOplasm in hematoxylin-eosin stained
liver sections was characteristic of all except 2 pigs. Moderately large
amounts of glycogen were visible in those livers with pale parenchyma
when they were fixed in Carnoy's solution and stained with Best's carmine.

Moderate numbers of mixed leukocytes in the dilated sinusoids were
accompanied by eosinophils and lymphoid cells in the hepatic portal
areas of Pig 7. It also had extensive fibrinopurulent exudates on the
serosal surfaces of all levels of the intestines and stomach, spleen,
liver, testicle and epididymis, lung and heart. Although Pig 10 had
no grossly visible lesions, there was an area of organizing purulent
debris on the serosa of the jejunum, and there were increased numbers
of leukocytes in the lamina propria of the jejunum. In the middle and
terminal jejunum the villi were broader and shorter than in the controls.
Some glandular crypts in the terminal jejunum and ileum in Pig 10 and in
the ileum of Pig 7 contained cellular debris and neutrophils. In Pig 11
there was prominent post-mortem decomposition in addition to lesions of

fibrinopurulent polyserositis. There was severe fibrinopurulent serositis

95

in one of 2 liver sections from Pig 13. In addition, there was a thick
layer of mucus covering the mucosa of the eSOphageal and cardiac regions

of the stomach, and fibrinopurulent serositis was present in the fundic
region. The small intestine was essentially normal, but the epithelium

of the lower jejunum was greatly vacuolated and resembled hydropic degenera-
tion as it had in the controls. There was very mild serositis of the

spiral colon, and moderate subacute serositis of the terminal colon. There
was fibrinopurulent serositis in sections of the testicle, epididymis,

and lung .

DISCUSSION

Bacterial Status

In 6 experiments, each of the first 3 experimental litters had a
Staphylococcus _p, in their isolator environment. The fourth litter was
entirely free of any demonstrable contaminant bacteria. Modifications
of equipment and technique, as described in the section on materials
and methods and by Waxler‘ggugl. (1966), controlled the problem of
staphylococcic contamination. Experiments 5 and 6 were contaminated by
a spore-forming Bacillus g2, which had seemed to survive in the cages in

single passage through the steam sterilizer.

Experiments 1, 2, 3, and 4

Confirmation of the hypothesis that E. _c_o_1_i_ 083:K.:NM would be
pathogenic for gnotobiotic pigs was achieved in the first 4 experiments,
when clinical signs of illness occurred in 100% of the 25 principals
exposed to E, 22;; but in none of the 10 controls. Twenty-one of the
25 principals (84%) died between 10 and 162 hours after exposure to
.E,.ggEi, and all 25 (100%) had gross lesions at necrOpsy. Twenty-four
pigs had lesions of polyserositis, and the remaining pig had fibrinous
peritonitis accompanying polyarthritis when it was euthanatized. Dunne
(1959) doubted the significance of E, 22;; isolations from pigs which had
died or were killed while in a moribund condition. The present work,
however, agrees with the report of Jubb and Kennedy (1963) that the in-

fection can be septicemic. Escherichia coli was isolated from the serous

96

97
cavities and/or synovial cavities of pigs which were euthanatized while
they were still ambulant, as well as from all the exposed principals that
died. The outstanding lesions of serofibrinous to fibrinopurulent poly-
serositis were the result of inflammatory reaction, which could occur
only in the living host.

Overt polyarthritis was first observed in Experiment 2, when lameness
in one or more swollen leg joints was observed in 4 of the 6 principals.
The post—mortem recovery of E, ggli_in cultures of the elbow synovial
fluid confirmed the suspicion of infectious arthritis in 16 of the 19
principals in Experiments 2, 3, and 4. The problem of localization of
.§:.£Qll in the joints as a complication in colibacillosis of pigs was
recognized by Dunne (1958, 1964) and Jubb and Kennedy (1963).

Lesions of gastritis were never more severe than catarrhal. Moon
.2£N§i° (1966b) reported that the lesions of enteritis in diarrheal dis—
ease of pigs were usually limited to hyperemia of the jejunum, and several
pigs had no morphologic evidence of enteritis. Lesions of enteritis in
the pigs in these experiments were usually minor and were overshadowed
by the lesions of serositis. Subserosal edema of the spiral colon was
a frequent lesion. Ventral abdominal edema in several of the pigs resembled

I

lesions which occurred in gnotobiotic pigs experimentally infected with

E, coli 0138:K81 by Christie (1967).

 

Varying the volume of the inoculum from 1.25 ml. to 5.0 ml. of E, 221;.
and the age of the pigs at exposure from 69 to 148 hours of age did not
establish a relationship between the volume of inoculum or the age of the
pigs and susceptibility to infection. The pigs which were youngest at
exposure had the greatest mean survival time. Developmental age of the

pigs, however, may not have coincided with their chronologic age based

98
on the number of elapsed hours or days from the time they were delivered
by hysterotomy. Feeding medroxyprogesterone to the sows in Experiments
2 through 5 allowed the hysterotomies to be performed at more advanced
stages of gestation than might have been possible without the progesta-
tional hormone in the sow's diet. In order of decreasing mean postexposure
survival time, Experiments 2, 4, l, and 3 consisted of litters delivered
at 115, 114, 112, and 112 days' gestation, respectively. This small
number of experiments can hardly be conclusive, but it serves to illus-
trate that fetal maturity may be one of the variable factors in gnotobiotic
research. Altered placental permeability related to the feeding of a
progestational hormone could constitute another variable, particularly
if larger molecules such as gamma globulin were transferred across the
placental barrier. Pigs in Experiment 3, which were from a sow fed pro-
gesterone, were at least as susceptible as those pigs in Experiment 1,
in which the sow had not been fed medroxyprogesterone. Saunders SE al.
(1963b) cited reports to indicate that pigs from gilts are more susceptible
to infection than pigs from sows. In Experiments 1 and 3, the pigs,
which were from sows, did not survive as long as pigs from a gilt in
Experiment 4.

Clinical signs were not sufficiently characteristic to be pathogno-
monic for infections with'E,'gg;E_but conformed to the nonspecific effects
ascribed to the endotoxins of the Enterobacteriaceae by Smith §£_§;,

(1964) and Carpenter (1965). Fever was recorded on at least one day for
each of 18 pigs. This contrasts with the report by Saunders g£__l,
(1963a) that no fevers, but frequent subnormal body temperatures, were
observed in their "pathogen-free" pigs infected with a different serotype

of E, coli. Subnormal body temperatures recorded for several of the

99
gnotobiotes in these experiments usually were observed shortly before the
pigs died. Diarrhea was only moderately severe in most cases but was
observed in the majority of the principals. Anorexia appeared to be
related more to soreness and pain induced by movement than to decreased
alertness until near death. Some of the pigs which did not stand and
approach their trays when fed would protest being moved but would eat
after being lifted to their feet. In Experiment 1 leukOpenia followed
by leukocytosis conformed to the sequence of these reactions in laboratory
animals inoculated with endotoxins of Gram-negative bacteria as reported
by Carpenter (1965). Leuk0penia was observed in 7 of the 25 pigs and
was primarily neutropenic, with differential counts as low as 3% neutro-
phils coexisting with relative lymphocytosis. In Experiment 1 leuko-
cytosis occurred only in those pigs which became febrile. The occurrence
of leukocytosis in approximately 50% of the principals indicated that
gnotobiotic pigs are capable of a neutrophilic reSponse to oral challenge
with E,'ggli. Circulating neutrophils were the cells most depleted in
the leukopenic phase. Also, in the later experiments a frequently observed
pattern was leukocytosis followed by leukOpenia or neutrOpenia, which
often preceded death when the pig was overwhelmed by infection.

The mean hemoglobin of 8.33 Gm./100 ml. for all 35 pigs at l to 2
days of age and before exposure in the first 4 experiments was approximately
10% less than the means reported by Miller ggnal. (1961b) for 2-day-old
naturally reared pigs. However, at 7 to 9 days of age the mean hemoglobin
of 12 surviving principals in Experiments 2, 3, and 4 was 8.4 Gm./100 ml.,
which exceeded the mean values for normal pigs of similar age as reported
by Schalm (1965) and Gardiner, Sippel, and McCormick (1953); and the

mean MCHC of 30.5% was greater than those in the report of Miller ggngl.

100
(1961b). The preceding data permit the assumption that these gnotobiotic
pigs were not predisposed to infection because of anemia. Also, it is
evident that hemoconcentration was not a pathologic effect of E, ggii_
infection in the pigs, since the principals had slightly lower PCV and
hemoglobin values than controls of similar ages.

In electrOphoresis the principals in Experiment 4 did not parallel
the increase in albumin per cent of serum protein which occurred in the
controls. Frankel and Reitman (1963) and Coles (1966) included decreased
hepatic function in disease, decreased protein intake, decreased protein
digestion and absorption, and nephritis and nephrosis as causes of
decreased serum albumin levels. The relative decrease in the alpha
globulin component observed in the controls did not occur in the princi-
pals, and their beta and gamma globulin remained essentially unchanged.
The lack of results of total protein determinations for the foregoing
groups of serums prevented quantitative evaluation of the relative serum
protein changes.

Serum electrolytes, sodium and potassium, in 28 samples in Experi-
ment 2 were essentially the same in principals and controls. The range
of sodium values from 131 to 152.5 mEq./1iter in the principals and from
135 to 151 mEq./liter in the controls was similar to that presented by
Benjamin (1961). The serum potassium was of slightly less concentration
than that reported by Widdowson and McCance (1956) for newborn pigs. The
serum potassium increased with age of the pigs, and the maximum of the
extremes (4.5 to 8.45 mEq./liter) was from one of the controls. Therefore,
it seems unlikely that elevated serum potassium was associated with car-
diac arrest, as suggested by Roy ggngl. (1959) in calves which died with

localized intestinal E, coli infections. HistOpathologic examination of

lOl

tissues from the controls revealed great similarity between these gnoto-
biotic pigs and those described by Waxler (1961) and Schmidt (1961). The
lymph nodes appeared immature and did not have secondary lymphoid nodules.
Liver lobulation was indistinct. Vacuolization resembling hydrOpic
degeneration of the epithelium in the mucosa of the jejunum and ileum
occurred in controls as well as principals. Sprinz ggual. (1961) stated
that the intestinal mucosa of the germfree guinea pig at 42 days of age
resembled the mucosa of the intestine in the fetal guinea pig. Kenworthy
and Allen described the influence of diet and bacteria on small intestinal
morphology with special reference to early weaning and Escherichia coli.
They related the differences in morphology to alterations of metabolic
pathways associated with the absence of bacterial enzymes from the sub-
strate in the digestive tract. Sprinz g£_a;. (1961) discussed the
morphogenetic influence of bacteria on the intestinal mucosa and concluded
that bacteria, diet, hormones, etc., probably act as a combination of
factors influencing the intestinal mucosal morphology.

Lymphadenopathy in the principals was characterized by varying
severity of edema, hyperemia, and depletion of lymphocytes. Depletion
of lymphocytes also occurred in the spleen. In germfree guinea pigs
(Sprinz ggual,, 1961), after oral challenge with E, ggll, the lymph nodes
were less dense than in the controls and appeared as though the lympho-
cytes had been washed out of the lymphoid nodules leaving a reticular
network with prominence of reticulum cells. .

Serositis was noted in a majority of the sections from parenchyma-
tous organs but rarely penetrated to the subcapsular areas, as did the
serositis in experimental myc0p1asmosis reported by Roberts ggngl. (1963a).

Foci of hematOpoiesis were infrequent in the liver but more frequent in

102

the spleen. Cloudy swelling was evident in most of the kidney sections,
but they did not have signs of proteinuric casts in tubules, focal inter-
stitial leukocytic infiltration, or hemorrhages. The petechial hemorrhags
noted in gross examination were possibly blood-filled superficial glo-
meruli. Cloudy swelling was noted in many sections of the kidneys and
probably represented a mild response to toxemia or to terminal hypoxia.

The gastric sections were devoid of severe mucosal lesions, although
most of the pigs had some lesions of serositis. Lesions of enteritis in
the small intestine, cecum, spiral colon, and terminal colon were usually
mild or absent and were not prominent in comparison with the lesions of
serositis which infrequently extended into the muscularis.

In male pigs the peritonitis extended to involve the tunica vaginalis.
Other lesions of septicemia included fibrinous to fibrinOpurulent peri-
carditis and pleuritis with occasional focal suppurative myocarditis and

interstitial pneumonia as well as synovitis.

Experiments 5 and 6 .

Septicemic E, ggil_infections in colostrum-deprived artificially
reared pigs was recognized as a problem by Lecce and Reep (1962). Switzer
EEHEi- (1962) recommended the use of substitutes for colostrum to provide
biological protection to SPF pigs, which were much more susceptible to
ordinary infections than their conventional counterparts. Experiments
5 and 6 were designed to investigate (1) how much protection against E.
£2EE_infection could be obtained by feeding commercially available anti—
hog cholera serum to the pigs as a substitute for colostrum, and (2) how
the course of disease would be altered in those pigs fed anti-hog cholera

serum. Speer ggugi. (1959) demonstrated a declining rate of colostral

103

antibody absorption from the digestive tract of the newborn pig after
6 hours of age and virtual cessation of absorption of the large molecules
by 24 hours of age. Therefore, these pigs were fed anti-hog cholera
serum at 6 hours or less of age to insure maximum antibody absorption.

Four of the principals in Experiment 5 were fed anti-hog cholera
serum and 4 were not fed anti-hog cholera serum. All the principals
develOped leukocytosis, but a neutropenic phase was observed only in 2
pigs not fed anti-hog cholera serum. Among the 4 pigs fed-anti-hog
cholera serum no deaths occurred; 1 pig had clinical signs of illness
and when euthanatized had a single gross lesion (liver abscess).
Escherichig_coli was isolated in culture of peritoneal swabs from this
and one other pig fed anti-hog cholera serum. In the 4 principals not
fed anti-hog cholera serum, one pig developed clinical signs of illness
and died 4.5 days after exposure. The pig had severe lesions of poly-
serositis, and E, ggli_was isolated in culture of swabs from 3 serous
cavities; but E, ggll_was not septicemic in the other 3 principals not
fed anti-hog cholera serum.

In Experiment 6 severity of the disease following oral exposure to
E, 22;; 083 at 5 days of age was much greater in the 3 pigs not fed anti-
hog cholera serum than in the 4 pigs fed anti-hog cholera serum. The one
pig that died had not been fed anti-hog cholera serum nor a measured
inoculum. It had been given the empty feeding tray as a contaminated
fomite from the cage of another pig. Signs of illness develOped sooner
after exposure in the pig given the contaminated fomite than in other
pigs fed measured and, presumably, larger inoculums, suggesting that quan-
tity of inoculum was not a major factor in susceptibility in this

experiment.

104

Only one of the anti-hog cholera serum-fed principals had gross
lesions at necropsy, but the severity of those lesions was as great as
that in the antiserum-devoid principals, of which 100% had gross lesions.
HistOpathologic findings were correlated with the gross observations in
the pigs with gross lesions. Also, one of the antiserum—fed pigs which
had had clinical signs of illness but no gross lesions at necropsy did
have resolving subacute histOpathologic lesions of peritonitis. In
post-mortem microbiologic examination the antiserum-devoid pig which had
resolving gross lesions of firm intestinal adhesions had apparently
eliminated E, ggll_from the peritoneal cavity, since its peritoneal swab
produced no growth in bacterial culture. In fact, E, ggEE_was recovered
in culture of peritoneal swabs only from those pigs with acute lesions of
peritonitis.

Hematologic trends and ranges were similar in measurements of hemo—
globin and PCV, which reached their peak in the pigs at 12 days of age
and of MCHC, which was lowest when hemoglobin and PCV were highest in the
principals and controls, irrespective of the feeding of anti-hog cholera
serum. The increasing levels of hemoglobin‘and PCV to a peak at 12 days
of age contrasted with the reports of Gardiner g£_§E, (1953) and Miller
§£_§E, (1961b) for hemoglobin trends in conventionally reared pigs from
1 to 10 days of age and may have been related to the smaller increase of
body weight and body fluids in the gnotobiotic pigs as compared with the
conventionally reared pigs. One interpretation of the decreasing MCHC
while the hemoglobin and PCV were increasing would be that there was a
degree of macrocytic normochromic anemia. However, as mentioned previously,
the mean hemoglobin values in these gnotobiotic pigs exceeded the mean

values for normal pigs of similar age reported by Schalm (1965) and

105
Gardiner g£_gl, (1953). So there was no anemia in comparison with
available standards. Leukocytosis and transient neutrOpenia in the anti-
serumrdevoid principals were paralleled in those principals fed the
antiserum.

Gamma globulin and total serum protein values were essentially the
same for the principals and the controls and for those pigs fed or not
fed anti—hog cholera serum. The anti-hog cholera serum was a commercially
available nonconcentrated product and would have contained a relatively
small amount of gamma globulin. Miller 25 al. (1961a) found 6.5% gamma
globulin in newborn pig serum with a mean total serum protein of 2.22 Gm./
100 ml. in 47 pigs. The results of experiments by Nordbring and Olsson
(1957, cited by Miller g£_§l,, 1961a) were similar to those of Miller
g£,§;, (1961a) and to those reported here. Miller §£_§E, (1961a) called
attention to the subjective judgment required to differentiate the beta
and gamma globulin components which are characteristically incompletely
separated in electrophoresis of serum from conventionally reared pigs 6
to 48 hours of age. Indistinct separation of the beta and gamma globulin
fractions was also a problem in these experiments. Lecce and Reep (1962)
demonstrated agglutinins for E, ggli'08 and other E, ggli serotypes in
bovine and porcine colostrum by tube agglutination tests. They also
found that agglutinins were practically absent from porcine and bovine
gamma globulin. This may account for the failure to demonstrate aggluti-
nins for'E,'ggli 083 in ordinary tube agglutination tests and latex
agglutination tests of the serums from all of the pigs in Experiment 6
between 5 and 12 days of age. Carpenter (1965) discussed factors other
than agglutinins which may function in the phagocytic elemination of

bacteria in infections.

106
Serum potassium levels increased as the pigs' age increased in all
groups of principals and controls in Experiments 5 and 6, as they had in
Experiment 2. The erythrocytes were not subjected to fragility tests,
but it is possible that there was increased permeability of the erythro-
cytic membranes with resultant osmotic transfer of potassium to extra-

cellular location in the serum in the absence of actual hemolysis.

SUMMARY

In a series of 6 experiments, 58 gnotobiotic pigs from 6 litters
were utilized in investigation of the pathologic effects of oral exposure
to Escherichia coli serotype 083:K.:NM in gnotobiotic pigs. The pigs
were germfree or monocontaminated with Staphylococcus g3, or Bacillus sp,
at 69 to 148 hours of age, when they were fed E, Eflli inoculums in volumes
varying from 1.25 ml. to 5.0 m1. of brain-heart infusion or thioglycol-
late liquid mediums. The inoculums were incubated at 37 C. for 24 or 36
hours just prior to their being fed to the pigs.

In the first 4 experiments there was 100% morbidity and 84% mortality
among 25 pigs between 10 and 162 hours after exposure to §:.Eflll.at
ages ranging from 69 to 148 hours. The most prominent lesions consisted
of serofibrinous to fibrinopurulent polyserositis and/or polyarthritis
in 100% of the 25 principals.

Invasiveness of the organism was indicated by the septicemic nature
of the infection. Four principals were severely ill but not moribund
when they were euthanatized between 99 and 236 hours after exposure to

. coli. Septicemia was confirmed by post-mortem microbiological recovery

lm

of E. coli from the exudates in serous and synovial cavities of the

 

euthanatized principals as well as from the principals that had died.
Macrosc0pic and microscopic morphologic evidence of gastroenteritis

was usually mild or absent, and results of clinic0pathologic examinations

did not reveal evidence of anemia, hemoconcentration, or serum electro-

lyte (sodium and potassium) derangement in the pigs.

107

108

Clinical signs were nonspecific, rather than pathognomonic, and
included fever, hypothermia, diarrhea, partial anorexia, leukOpenia, and
leukocytosis. The occurrence of fever and leukocytosis indicated that
colostrum-deprived gnotobiotic pigs are capable of responding defensively
to bacterial stimuli in much the same manner as other animals.

In Experiments 5 and 6, anti-hog cholera serum was fed to one-half
of each group of principals at 6 hours of age or earlier as a substitute
for colostrum. Some biological protection was manifested by the absence
of mortality and reduced incidence of gross lesions in the pigs fed
anti-hog cholera serum as compared with the principals not fed anti-
hog cholera serum. Infection following exposure to a contaminated
fomite resulted in the only death among the principals in Experiment 6.
Evaluation of the degree of protection against E, ggl$_infection pro—
vided by feeding anti-hog cholera serum was complicated by an unexplained
reduction of mortality in the group of principals not fed anti-hog
cholera serum. Also, some pigs in both groups were able to eliminate
E, ggli_from the serous cavities after the acute septicemic phase of
infection.

Serums collected from the pigs at 5 to 12 days of age, before and
after exposure to E, ggll_in Experiment 6, were negative for 0 agglutinins
to whole cell E, 22$}.083 somatic antigen, alone or combined with latex
particles, in tube agglutination tests in final dilutions of 1:10 and
1:20.

Results of electrOphoresis indicated a range of 0 to 13% gamma
globulin with similar ranges in pigs irrespective of the feeding of anti-
hog cholera serum. Also, there was no difference between groups in

(1) hemoglobin values and packed cell volumes, which increased to a peak

109

at 12 days of age, (2) declining alpha1 globluin levels with increasing
age, and (3) serum potassium values, which increased with increasing age

of the pigs.

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VITA

The author was born August 24, 1924, in Pulaski County, Illinois.
He attended public schools and graduated from Mound City Community
High School in May, 1941. After attending Draughon's Business College
in Paducah, Kentucky, he was employed as a shop clerk in Peoria, Illinois.
He served in the U. S. Army Air Corps from May, 1943, until September,
1945.

During assignment to the College Training Detachment for aviation
students at Michigan State College he met Jean Marie Black, whom he
married at Okemos, Michigan, in December, 1944. Upon release from
active military service as a pilot of multiengine aircraft he enrolled
at Michigan State College in the preveterinary curriculum and received
the D.V.M. degree from there in June, 1950. After a year of large
animal practice at Mentfort, Wisconsin, he joined the faculty of the
College of Veterinary Medicine, University of Georgia, at Athens, Georgia,
where he remained for one year and returned to Illinois, where he worked
in bovine brucellosis and tuberculosis control for Union County and the
State of Illinois. In April, 1953, he moved to Albion, Indiana, and
conducted a private rural veterinary practice until August, 1960, when
he matriculated at the University of Michigan, Ann Arbor, Michigan, and
earned a Master of Public Health degree from the School of Public Health
in June, 1961.

In September, 1962, he enrolled as a part-time graduate student in
the Department of Pathology at Michigan State University, where he was

119

120
employed as an assistant instructor and later as an instructor. He is
currently employed as pathologist for the Florida Department of Agriculture,
Animal Disease Diagnostic Laboratory, at Kissimmee, Florida.

The author is a member of the A.V.M.A., Florida V.M.A., Phi Zeta,

and Sigma Xi.