• TABLE OF CONTENTS
HIDE
 Front Cover
 Front Matter
 Table of Contents
 Introduction
 Procedure
 Experimental results - Injection...
 Experiments with endotoxin of S....
 Experiments with S. aertrycke
 Experiments with S. aertrycke and...
 Vulnerability of tissues
 Discussion
 Conclusions
 Summary
 Literature cited














Group Title: Bulletin - University of Florida. Agricultural Experiment Station ; no. 422
Title: The etiology of fowl paralysis, leukemia and allied conditions in animals
CITATION THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00015134/00001
 Material Information
Title: The etiology of fowl paralysis, leukemia and allied conditions in animals
Series Title: Bulletin University of Florida. Agricultural Experiment Station
Alternate Title: Mechanism by which Salmonella aertrycke induces hemocytoblastosis in the chicken
Physical Description: 48 p. : ill., charts ; 23 cm.
Language: English
Creator: Emmel, M. W ( Mark Wirth ), b. 1895
Publisher: University of Florida Agricultural Experiment Station
Place of Publication: Gainesville Fla
Publication Date: 1946
 Subjects
Subject: Marek's disease   ( lcsh )
Chickens -- Diseases   ( lcsh )
Chickens -- Experiments   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Bibliography: Bibliography: p. 47-48.
Statement of Responsibility: by M.W. Emmel.
General Note: Cover title.
Funding: Bulletin (University of Florida. Agricultural Experiment Station)
 Record Information
Bibliographic ID: UF00015134
Volume ID: VID00001
Source Institution: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: aleph - 000925492
oclc - 18251261
notis - AEN6143

Table of Contents
    Front Cover
        Page 1
    Front Matter
        Page 2
        Page 3
    Table of Contents
        Page 4
    Introduction
        Page 5
        Page 6
    Procedure
        Page 7
        Page 8
    Experimental results - Injection of heat-killed and filtered broth cultures of S. aertrycke
        Page 9
        Page 10
    Experiments with endotoxin of S. aertrycke
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
    Experiments with S. aertrycke
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
    Experiments with S. aertrycke and its endotoxin
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
    Vulnerability of tissues
        Page 38
    Discussion
        Page 39
        Page 40
    Conclusions
        Page 41
        Page 42
        Page 43
        Page 44
    Summary
        Page 45
        Page 46
    Literature cited
        Page 47
        Page 48
Full Text



Bulletin 422


UNIVERSITY OF FLORIDA
AGRICULTURAL EXPERIMENT STATION
HAROLD MOWRY, Director
GAINESVILLE, FLORIDA







THE ETIOLOGY OF FOWL PARALYSIS,

LEUKEMIA AND ALLIED CONDITIONS

IN ANIMALS

XI. The Mechanism by Which Salmonella aertrycke
Induces Hemocytoblastosis in the Chicken

By M. W. EMMEL









TECHNICAL BULLETIN








Single copies free to Florida residents upon request to
AGRICULTURAL EXPERIMENT STATION
GAINESVILLE, FLORIDA


July, 1946










BOARD OF CONTROL


J. Thos. Gurney, Chairman, Orlando
N. B. Jordan, Quincy
Thos. W. Bryant, Lakeland
M. L. Mershon, Miami
J. Henson Markham, Jacksonville
J. T. Diamond, Secretary, Tallahassee




EXECUTIVE STAFF

John J. Tigert, M.A., LL.D., President of the
University3
H. Harold Hume, D.Sc., Provost for Agricul-
ture
Harold Mowry, M.S.A., Director
L. O. Gratz, Ph.D., Asst. Dir., Research
W. M. Fifield, M.S., Asst. Dir., Admin.
J. Francis Cooper, M.S.A., Editors
Clyde Beale, A.B.J., Associate Editors
Jefferson Thomas, Assistant Editors
Ida Keeling Cresap, Librarian
Ruby Newhall, Administrative Managers
K. H. Graham, LL.D., Business Manager3
Claranelle Alderman, Accountants




MAIN STATION, GAINESVILLE


AGRONOMY

W. E. Stokes, M.S., Agronomist'
Fred H. Hull, Ph.D., Agronomist
G. E. Ritchey, M.S., Agronomist'
G. B. Killinger, Ph.D., Agronomist
W. A. Carver, Ph.D., Associate
Roy E. Blaser, M.S., Associate5
H. C. Harris, Ph.D., Associate
Fred A. Clark, B.S., Assistant




ANIMAL INDUSTRY

A. L. Shealy, D.V.M., An. Industrialist1
R. B. Becker, Ph.D., Dairy Husbandman3
E. L. Fouts, Ph.D., Dairy Technologists
D. A. Sanders, D.V.M., Veterinarian
M. W. Emmel, D.V.M., Veterinarians
L. E. Swanson, D.V.M., Parasitologist4
N. R. Mehrhof, M.Agr., Poultry Husb.3
G. K. Davis, Ph.D., Animal Nutritionist
R. S. Glasscock, Ph.D., An. Husbandman
P. T. Di:: Arnold, M.S.A., Asst. Dairy Husb.3
C. L. Comar, Ph.D., Asso. Biochemist
L. E. Mull, M.S., Asst. in Dairy Tech.
Katherine Boney, B.S., Asst. Chem.
Ruth Taylor, A.B., Asst. Biochemist
J. C. Driggers, B.S.A., Asst. Poultry Husb.
Glenn Van Ness, D.V.M., Asso. Poultry
Pathologist


ECONOMICS, AGRICULTURAL

C. V. Noble, Ph.D., Agri. Economist'1
Zach Savage, M.S.A., Associates
A. H. Spurlock, M.S.A., Associate
Max E. Brunk, M.S., Associate5
I. E. Alleger, M.S., Associate
Wade P. Young, Ph.D., Associate

Orlando, Florida (Cooperative USDA)
G. Norman Rose, B.S., Asso. Agr. Economist
J. C. Townsend, Jr., B.S.A., Agr. Statistician2
J. B. Owens, B.S.A., Agr. Statistician2

ECONOMICS, HOME

Ouida D. Abbott, Ph.D., Home Econ.1
R. B. French, Ph.D.. Biochemist

ENTOMOLOGY

A. N. Tissot, Ph.D., Entomologist and Act-
ing Head of Dept.
H. E. Bratley, M.S.A., Assistant

HORTICULTURE

G. H. Blackmon, M.S.A., Horticulturist1
A. L. Stahl, Ph.D., Asso. Horticulturist
F. S. Jamison, Ph.D., Truck Hort.
Byron E. Janes, Ph.D., Asso. Hort.
R. A. Dennison, Ph.D., Asso. Hort.
R. K. Showalter, M.S., Asso. Hort.
R. J. Wilmot, M.S.A., Asst. Hort.
R. D. Dickey, M.S.A., Asst. Hort.
Victor F. Nettles, M.S.A., Asst. Hort.
J. Carlton Cain, B.S.A., Asst. Hort.5
F. S. Lagasse, Ph.D., Asso. Hort.2

PLANT PATHOLOGY

W. B. Tisdale, Ph.D., Plant Pathologist1
Phares Decker, Ph.D., Asso. Plant Path.
Erdman West, M.S., Mycologist and Botanist
Lillian E. Arnold, M.S., Asst. Botanist

SOILS

F. B. Smith, Ph.D., Chemist1 '
Gaylord M. Volk, M.S., Chemist
J. R. Henderson, M.S.A., Soil Technologist
J. R. Neller, Ph.D., Soils Chemist
Nathan Gammon, Jr., Ph.D., Soils Chemist
C. E. Bell, Ph.D., Associate Chemist
L. H. Rogers, Ph.D., Associate Biochemist
R. A. Carrigan, B.S., Asso. Biochemist
G. T. Sims, M.S.A., Associate Chemist
H. W. Winsor, B.S.A., Assistant Chemist
Geo. D. Thornton, M.S., Asst. Microbiologist8
R. E. Caldwell, M.S.A., Asst. Soil Surveyor4
Wade McCall, B.S., Asst. Chemist
J. B. Cromartie, B.S.A., Soil Surveyor

SHead of Department.
2 In cooperation with U. S.
3 Cooperative, other divisions, U. of F.
SIn Military Service.
6 On leave.










BRANCH STATIONS


NORTH FLORIDA STATION, QUINCY

J. D. Warner, M.S., Vice-Director in Charge
R. R. Kincaid, Ph.D., Plant Pathologist
W. H. Chapman, M.S., Asso. Agron.
R. C. Bond, M.S.A., Asso. Agronomist
L. G. Thompson, Ph.D., Soils Chemist
Frank D. Baker, Jr., B.S., Asst. An. Husb.

Mobile Unit, Monticello
R. W. Wallace, B.S., Associate Agronomist

Mobile Unit, Milton
Ralph L. Smith, M.S., Associate Agronomist

Mobile Unit, Marianna
R. W. Lipscomb, M.S., Associate Agronomist


Mobile Unit, Wewahitchka
J. B. White, B.S.A., Associate Agronomist


CITRUS STATION, LAKE ALFRED

A. F. Camp, Ph.D., Vice-Director in Charge
V. C. Jamison, Ph.D., Soils Chemist
J. B. Redd, Ph.D., Insecticide Chemist
W. L. Thompson, B.S., Entomologist
J. T. Griffiths, Ph.D., Entomologist
R. F. Suit, Ph.D., Plant Pathologist
E. P. Ducharme, M.S., Plant Pathologist
J. E. Benedict, B.S., Horticulturist
B. R. Fudge, Ph.D., Associate Chemist
C. R. Stearns, Jr., B.S.A., Asso. Chemist
T. W. Young, Ph.D., Asso. Horticulturist
J. W. Sites, M.S.A., Asso. Horticulturist5
H. O. Sterling, B.S., Asst. Horticulturist
J. A. Granger, B.S.A., Asst. Horticulturist
H. J. Reitz, M.S., Asso. Plant Path.


EVERGLADES STA., BELLE GLADE

R. V. Allison, Ph.D., Vice-Director in Charge
J. W. Wilson, Sc.D., Entomologist4
F. D. Stevens, B.S., Sugarcane Agron.
Thomas Bregger, Ph.D., Sugarcane
Physiologist
G. R. Townsend, Ph.D., Plant Pathologist
B. S. Clayton, B.S.C.E., Drainage Eng.2
W. D. Wylie, Ph.D., Entomologist
W. T. Forsee, Jr., Ph.D., Asso. Chemist
Robt. L. Cassell, Ph.D., Asso. Plant Path.
R. W. Kidder, M.S., Asst. An. Husb.
T. C. Erwin, Assistant Chemist
R. A. Bair, Ph.D., Asst. Agronomist
Earl L. Felix, Ph.D., Asst. Plant Path.
C. C. Seale, Asst. Agronomist
L. O. Payne. B.S.A., Asst. Agronomist
R. C. Ladeburg, B.S.A., Asst. Hort.
Russel Desrosiers, M.S., Asst. Plant Path.


SUB-TROPICAL STA., HOMESTEAD

Geo. D. Ruehle, Ph.D., Vice-Director in
Charge
H. I. Borders, M.S., Asso. Plant Path.
D. O. Wolfenbarger, Ph.D., Asso. Ento-
mologist
R. W. Harkness, Ph.D., Asst. Chemist


W. CENT. FLA. STA., BROOKSVILLE

Clement D. Gordon, Ph.D., Asso. Poultry
Geneticist in Charge2


RANGE CATTLE STA., ONA

W. G. Kirk, Ph.D., Vice-Director in Charge
E. M. Hodges, Ph.D., Associate Agronomist
D. W. Jones, B.S.A., Asst. An. Husb.
E. R. Felton, B.S.A.. Asst. An. Hush.


CENTRAL FLORIDA STATION, SANFORD
R. W. Ruprecht, Ph.D., Chemist in Charge
A. Alfred Foster, Ph.D., Asso. Hort.
J. C. Russell, M.S., Asst. Entomologist
Ben F. Whitner, Jr., B.S., Asst. Hort.


FIELD STATIONS

Leesburg
G. K. Parris, Ph.D.. Plant Path. in Charge

Plant City
A. N. Brooks, Ph.D., Plant Pathologist

Hastings
A. H. Eddins, Ph.D., Plant Pathologist
E. N. McCubbin, Ph.D., Truck Horticulturist

Monticello
S. O. Hill, B.S., Asst. Entomologist2 <
A. M. Phillips, B.S., Asst. Entomologist2


Bradenton
J. R. Beckenbach, Ph.D., Horticulturist in
Charge
E. G. Kelsheimer, Ph.D., Entomologist
A. L. Harrison, Ph.D., Plant Pathologist
David G. Kelbert, Asso. Horticulturist
E. L. Spencer, Ph.D., Soils Chemist
Robert O. Magie, Ph.D., Hort., Glad. Inv.

Lakeland
Warren O. Johnson, Meteorologist2

1 Head of Department.
2 In cooperation with U. S.
SCooperative, other divisions, U. of F.
In Military Service.
6On leave.













CONTENTS

Page
INTRODUCTION ----..................--- ----------............ 5
PROCEDURE ...............--------- -------------------------- 7
EXPERIMENTAL RESULTS --.......-----.........-------.------------ 9
INJECTION OF HEAT-KILLED AND FILTERED BROTH CULTURES OF
S. aertrycke ................................---........ ------------- --. -- -- 9
Intravenous and Intraperitoneal Injection of Heat-Killed Broth
Cultures ........ ...-------- --------- ---- ----- 9
Intravenous and Intraperitoneal Injection of Filtered Broth Cultures 10
Repeated Intravenous and Intraperitoneal Injection of Heat-Killed
and Filtered Broth Cultures ........................ .........- 10
EXPERIMENTS WITH ENDOTOXIN OF S. aertrycke .......-............................. 11
Immediate Reaction Following Intravenous Injection Into Day-old
Chicks .. -----..........---..---.-------.-.------. .. 11
Repeated Intravenous Injection Into Young Chicks ............................. 13
Single and Repeated Intracerebral Injection Into Young Chicks........ 13
Single and Repeated Intraperitoneal Injection Into Young Chicks...... 14
Effect of Endotoxin on Agglutination ..................--...................---- 15
Effect of Endotoxin on Blood Cells .............................- .......--- ---. .... 18
Effect of Endotoxin on the Growth of S. aertrycke on Solid and
Liquid Media -.. ...... ........---------........ ... 20
Effect of Endotoxin on Phagocytosis ..........-....-............. ......-- 22
EXPERIMENTS WITH S. aertrycke .......----------------....... --- ............------- 23
Fate of S. aertrycke Following Intravenous Injection ...................... 23
Repeated Intravenous and Intraperitoneal Injection of the Heat-
Killed Microorganism ......... ........................... 27
Ability of Various Strains to Induce This' Group of Diseases ........... 29
EXPERIMENTS WITH S. aertrycke and Its Endotoxin .............................. 32
Intravenous Injection of S. aertrycke Alone versus S. aertrycke
plus Endotoxin ......... .----------- --......... 32
Susceptibility to Fowl Paralysis as Indicated by Susceptibility to
the Neurotoxic Action of Endotoxin ....-----...... --....................- ------ 35
Virulence of Microorganism versus Potency of Its Endotoxin ........... 36
VULNERABILITY OF TISSUES .................... ---...--------------. 38
DISCUSSION -...---..--..--..... -- -........... 39
CONCLUSIONS ............---...... --....... ................- --- ... 41
SUMMARY ......--- ---- -----............-..------ --------... 45
LITERATURE CITED .... ...................... ..................... 47









THE ETIOLOGY OF FOWL PARALYSIS, LEUKEMIA
AND ALLIED CONDITIONS IN ANIMALS

XI. The Mechanism by Which Salmonella aertrycke
Induces Hemocytoblastosis in the Chicken

By M. W. EMMEL
Experiments have been reported which show that the micro-
organisms, Salmonella aertrycke, S. enteritidis and S. schott-
miilleri can induce, under certain conditions of infection, a large
number of pathologic manifestations, viz. hemocytoblastosis,
septicemia, "light", anemia, fowl paralysis (Neurolymphoma-
tosis gallinarum), erythroblastic erythroleucosis, lymphatic ery-
throleucosis, myeloid leukemia, lymphomatosis and leukemic
lymphocytoma in the chicken (7-13).1 Preliminary investiga-
tions have indicated that S. typhimurium, S. psittacosis, S.
suipestifer and S. anatum also are capable of inducing this
group of manifestations. S. aertrycke and S. enteritidis, how-
ever, have been encountered more frequently under natural
conditions on poultry farms than other members of the para-
typhoid group.2
A number of factors have been enumerated which appear to
have a direct relation to the development of specific pathologic
manifestations resulting from infection by the above species
of the parathyphoid group: (a) the resistance of the bird,
(b) the severity and duration of enteritis most frequently
associated with intestinal parasitism, (c) the number of micro-
organisms, their rate of entrance and the period over which
they gain access to the blood stream of the bird from the in-
testinal tract, (d) the properties of the particular strain of
micro-organism acting as the exciting agent and (e) the vul-
nerability of the tissues of the bird. It is logical to assume that
1 Italic figures in parentheses refer to Literature Cited.
2 As progress has been made in the continuation of these studies, it has
become apparent that some of the pathologic manifestations under con-
sideration can be induced by a number of other agents and under more
widely varying conditions than has been set forth heretofore in this series
of publications. Regardless of the original stimulus, hemocytoblastosis
is a basic condition which leads to the development of many of these mani-
festations. The development of hemocytoblastosis under other stimuli
offers an additional avenue of approach to studies involving this group
of diseases. Although a number of stimuli have been found capable of
inducing hemocytoblastosis, micro-organisms of the paratyphoid group
appear to be the principal stimulus involved in the occurrence of this
group of diseases when the birds are reared on soil.







Florida Agricultural Experiment Station


the relationship of these factors may vary in flocks as well as
in individual birds. Apparently, the inter-relationship of these
factors forms the basis for the ability of a specific strain of
micro-organism to induce such a large number of closely allied,
yet distinct, manifestations. It has been reported in previous
papers (7-13) that oral exposure to S. aertrycke, S. enteritidis
and S. schottmillleri has resulted in the development of a higher
percentage of cases of this group of diseases when the birds
were affected with chronic intestinal parasitism3 than in para-
site-free birds of the same age. It has been shown also that
the rate of dosage, amount of dosage and period of time over
which these micro-organisms were injected intravenously are
factors which tend to induce certain pathologic manifestations
more readily than others. Likewise, it has been observed that
certain strains of micro-organisms are more capable than others
of inducing this group of diseases.
The failure to isolate any of these micro-organisms from the
tissues of birds following the development of the various patho-
logic manifestations (septicemia excluded) under experimental
conditions also has been reported in previous publications (9, 11,
13). Likewise, none of these micro-organisms have been iso-
lated with any regularity from the tissues of birds in naturally
occurring outbreaks of this group of diseases. Other investi-
gators have failed to report the isolation of micro-organisms
regarded of etiologic significance from the tissues of naturally
affected birds. The author has been able, however, to isolate
micro-organisms of the paratyphoid group from the intestinal
tract of birds affected with enteritis associated with intestinal
parasitism and from the intestinal tract of a few birds affected
with fowl paralysis in which the incubation period presumably
was short (10).
There are but few diseases which can be induced by bacteria
in which one fails to isolate the offending micro-organism
from the tissues in a reasonable percentage of instances. It
would seem that the mechanism of action of the micro-organism
as far as this group of diseases is concerned must be considerably
different from that of most bacteria which induce disease.
Most of the bacteria of the paratyphoid group which are re-
garded as of greatest significance in this group of diseases have
been known for many years to induce paratyphoid fever and
3 This refers to the type of parasitism which occurs over a considerable
period of time, usually based upon reinfestation.







The Etiology of Fowl Paralysis


food poisoning in the human. The first known species, S.
enteritidis, was isolated in Germany in 1888; it was demon-
strated that a toxic substance derived from the micro-organism
induced symptoms similar to those induced by the living bac-
teria. As a result, many of the investigations involving bacteria
of this group have centered around the production of toxic sub-
stances by the micro-organisms.
It is commonly agreed that S. aertrycke, S. enteritidis, S.
schottmiilleri, S. pullorum and S. suipestifer contain intracellu-
lar toxic substances, hereinafter termed endotoxin, which are
liberated only upon destruction of the bacterial cell. There
is considerable divergence of opinion as to whether these micro-
organisms are capable of producing true soluble toxins. The
literature, on the nature and properties of the toxic substances
produced by these micro-organisms is extremely voluminous,
and often conflicting results have been reported under various
experimental conditions.
A review of the literature on this subject will not be under-
taken, since most of these investigations reported deal with
the nature and physical properties of endotoxin. Endotoxin,
however, seems to be of particular importance in the mechan-
ism which eventually leads to the production of this group
of diseases. Only those properties which seem pertinent to
this mechanism will be discussed in this paper.
Hemocytoblastosis has been described as a basic process lead-
ing to the development of many of the pathologic manifesta-
tions. This paper will be confined to a study of the mechanism
by which S. aertrycke induces hemocytoblastosis. S. aertrycke
has been found to be associated more frequently with naturally
occurring cases of fowl paralysis and intestinal parasitism than
other micro-organisms of the paratyphoid group. Consequently,
this micro-organism has been used in the experiments reported
herein. The results of these experiments are applicable to many
of the other species of the genus Salmonella which induce mani-
festations similar to those induced by S. aertrycke.

PROCEDURE
The methods of injection, rearing and management of ex-
perimental birds were described in a previous publication (9).
The experiments reported herein are of such variable nature
that the various procedures will be described in each experiment.







Florida Agricultural Experiment Station


Preparation of Endotoxin.4- Mention has been made that
S. aertrycke, as well as a number of other species of the genus
Salmonella, contain intracellular toxic substances which are
liberated on destruction of the bacterial cell. The preparation
of endotoxin from S. aertrycke was accomplished as follows:
The micro-organism was grown in Kolle flasks containing
nutrient agar (pH 6.8), the medium being inoculated with 1
ml. of a 24-hour nutrient broth culture. After 48 hours incu-
bation .at 370 C. the growth was removed by washing with a
small quantity of 0.85 percent sodium chloride solution, the
washings being transferred from flask to flask.
Using the technique described by Douglas (2), the suspension
was placed in 100 ml. round-bottom centrifuge tubes with an
equal volume of acetone. Flocculation of the micro-organisms
usually resulted immediately. After centrifuging at a rela-
tively high speed the supernatant fluid was removed, the tubes
loosely stoppered with cotton plugs and placed in the incubator
at 370 C. for desiccation. After 48 hours the desiccated micro-
organisms were removed as a thin scale from the bottom of the
tube. In the various phases of this study in which desiccated
micro-organisms were used, desiccation was accomplished in
this manner.
Endotoxin was obtained by tryptic digestion of the desiccated
micro-organisms by the technique of Hanks and Rettger (14).
One hundred milligrams of the desiccated micro-organisms were
placed in 70 milliliters of sterile 0.85 percent sodium chloride
solution. One hundred mg. of trypsin were placed in 29 ml. of
physiological salt solution, filtered through a Berkefeld N can-
dle and added to the suspension of the micro-organisms. The
suspension was adjusted to a pH of 8.2 by the addition of 10
percent sodium carbonate solution. A few drops of ether were
added, the flasks were sealed with moisture-proof cellophane
and incubated at 370 C. for 12 to 16 hours. The preparation
was partially clarified by centrifuging, filtered through a Berke-
feld N candle, 1 ml. of a 5.0 percent solution of phenol added,
and stored in the refrigerator in rubber-stoppered bottles. All
of the endotoxin preparations used in these studies, unless
otherwise stated, were prepared in this manner.
Test for Potency.-The endotoxin preparations were tested for
potency by injecting 1 ml. intraperitoneally into a 20-gram
Endotoxin as used in this paper will refer to the intracellular toxic
substances derived from the micro-organism, S. aertrycke.






The Etiology of Fowl Paralysis


mouse, or the equivalent amount into a mouse of lesser or
greater weight. One milliliter of the most potent endotoxin
prepared killed a 20-gram mouse in 5 minutes; some prepara-
tions proved entirely impotent; most of the endotoxin prepara-
tions were lethal to mice in from 3 to 12 hours. There are a
number of factors subject to variation in the above technique
probably responsible for variations in the potentcy of the final
product and which no doubt are responsible for the variable
results reported in the literature regarding some of the prop-
erties of intracelluar toxic substances of bacterial origin.

EXPERIMENTAL RESULTS
INJECTION OF HEAT-KILLED AND FILTERED BROTH
CULTURES OF S. AERTRYCKE
Intravenous and Intraperitoneal Injection of Heat-Killed Broth
Cultures.-Four -cultures of different strains of S. aertrycke
were incubated in veal infusion broth (pH 6.8) in Erlenmeyer
flasks for 11 days at 370 C. after being sealed to prevent evapora-
tion. The flasks were placed in boiling water for 20 minutes,
after which the cultures were tested for sterility. The heat-
killed cultures were of such an endotoxin potency that 1 ml. in-
jected intraperitoneally killed a 20-gram mouse in approximately
12 hours.
Many investigators have shown that broth cultures of species
of the genus Salmonella do not become toxic until many of the
micro-organisms have died and autolysis has taken place; it
would appear that the toxicity is due to liberation of toxic sub-
stances from the bacterial cells by the process of autolysis.
The sterile heat-killed cultures were injected intravenously
or intraperitoneally into 16 birds 6 weeks of age in amounts
varying from 2 to 4 ml. Veal infusion broth was injected simi-
larly into 6 control birds.
Two birds died in the group receiving intravenous injections
of the killed cultures within 24 hours; the cause of death was
undetermined. None of the birds developed fowl paralysis, leu-
kemia or an allied condition during a period of 4 months fol-
lowing injection. Many of the birds injected with the heat-
killed cultures showed. considerable depression for 4 or 6 hours
after injection. Some birds showed poor feathering and a
general unthrifty condition as long as 4 weeks after injection;
a few developed roup. The control birds appeared normal.






Florida Agricultural Experiment Station


Intravenous and Intraperitoneal Injection of Filtered Broth
Cultures.-Six cultures of different strains of S. aertrycke were
incubated in veal infusion broth (pH 6.8) in Erlenmeyer flasks
for 11 days at 370 C. after being sealed to prevent evaporation.
The cultures were filtered through Berkefeld N candles, and
the filtrates were tested for sterility. The filtrates were of such
an endotoxin potency that 1 ml. injected intraperitoneally killed
a 20-gram mouse in approximately 10 hours.
The sterile filtrates were injected intravenously or intra-
peritoneally into 18 birds 5 weeks of age in amounts varying
from 2 to 4 ml. Veal infusion broth was injected similarly
into 6 control birds.
None of the birds developed fowl paralysis, leukemia or an
allied condition during the 4 months the birds were held for
observation. Considerable depression often was observed for
about 4 hours following injection in the filtrate-injected birds.
As in the preceding experiment, many of the filtrate-injected
birds showed poor feathering and an unthrifty condition with
the development of a few cases of roup 6 to 8 weeks after in-
jection. The control birds remained normal.
Repeated Intravenous and Intraperitoneal Injection of Heat-
Killed and Filtered Broth Cultures.-Since single intravenous
and intraperitoneal injections of heat-killed and filtered broth
cultures of S. aertrycke failed to induce fowl paralysis, leukemia
or an allied condition, it was deemed advisable to administer
in a similar manner repeated injections of such cultures.
A veal infusion broth culture of S. aertrycke was prepared
as in the 2 preceding experiments; 1/2 of the culture was heat-
killed while the remaining portion was filtered through a Berke-
feld N candle. The endotoxin potency was such that 1 ml. in-
jected intraperitoneally killed a 20-gram mouse in approximately
14 hours. The heat-killed culture and the filtrate each were ad-
ministered as in the preceding experiments to 2 groups of 6
Barred Plymouth Rock chickens 6 weeks of age.
One milliliter was administered at each injection 3 times a
week for 3 weeks. Six control birds received similar injections
of veal broth.
None of the birds developed fowl paralysis, leukemia or an
allied condition during an observation period of 4 months. Many
birds in both groups showed poor feathering, unthriftiness and
roup. These conditions were decidedly more marked than in
the 2 previous experiments and at the end of the 4-months






The Etiology of Fowl Paralysis


period the injected birds were visibly more poorly developed
than the control birds which remained normal.
As the results in the preceding 3 experiments were negative,
none of the birds were studied from the standpoint of hemo-
cytoblastosis.
EXPERIMENTS WITH ENDOTOXIN OF S. AERTRYCKE
Immediate Reaction Following Intravenous Injection into
Day-Old Chicks.-The intravenous injection of 1 ml. of reason-
.ably potent endotoxin into day-old chicks often caused the im-
mediate development of paralytic symptoms. In many instances
these symptoms were not unlike those of typical fowl paralysis;
lameness, paralysis of both legs and of the auditory nerve, wry
neck, complete paralysis, and spastic cervical muscles often re-
sulted (Fig. 1). Some chicks showed nervousness and twitch-
ing of the muscles, while others appeared sleepy and droopy.
Other chicks appeared unaffected except perhaps for indications
of slight depression. These symptoms, however, were transient;
1 to 2 hours after intravenous injection the affected chicks
again were apparently normal. Chicks injected in this manner
with endotoxin were raised to maturity with no indications that
they had been so treated.
Older chickens required the intravenous injection of larger
amounts of endotoxin to obtain comparable effects. Six-week-
old chicks of the same breed and strain as those injected as
baby chicks required the injection of 4 to 8 times as much
endotoxin to induce paralytic symptoms. In the instance of 12-
week-old chickens, 8 to 12 times the amount required to induce
paralytic symptoms in baby chicks was required. Adult birds
required increasingly larger dosages of endotoxin to induce the
same effect. In the case of birds above 6 weeks of age paralytic
symptoms were not observed as frequently as in birds under this
age; however, symptoms of depression usually occurred.
The intraperitoneal injection of similar amounts of endotoxin
often produced temporary depression but only occasionally in-
duced paralytic symptoms. This probably was due to slow ab-
sorption from the peritoneal cavity.
This experiment indicates that the endotoxin of S. aertrycke
is neurotoxic and that young chickens are more susceptible to
this action than older ones. Of more than passing interest is
the observation that the intravenous injection of endotoxin
induced transient and varied paralytic symptoms and that the







12 Florida Agricultural Experiment Station


Fig. 1.-The reaction of baby chicks to the intravenous injection of
endotoxin extracted from S. aertrycke: 1, no reaction; 2, depression but
no paralytic symptoms; 3, wry neck; 4, paralysis of both legs and wry
neck; 5, paralysis of one leg; 6, spastic paralysis of the cervical muscles;
7, paralysis of both legs; 8, paralysis of both legs and wry neck. Affected
chicks appeared normal within 2 hours after injection.






The Etiology of Fowl Paralysis


symptoms induced closely approximated the wide range of symp-
toms observed in outbreaks of typical fowl paralysis. Endo-
toxin prepared from S. enteritidis, S. schottmillleri, S. typhi-
murium, S. psittacosis and S. pullorum yielded similar results.
It was observed that the more potent preparations of endo-
toxin often caused nervous symptoms and paralysis of the pos-
terior extremities in the mouse when these preparations were
tested for potency.
Repeated Intravenous Injection Into Young Chickens.-Ten
8-week-old Rhode Island Red pullets were given repeated in-
travenous injections of endotoxin, 2 ml. per dose 3 times a week
for 4 weeks. None of the birds developed fowl paralysis, leu-
kemia or an allied condition during the 4-month period follow-
ing the last injection of endotoxin.
The experiment was repeated on a like number of Single
Comb White Leghorn chicks 5 weeks of age; the period of in-
jection was extended to 10 weeks. None of the endotoxin-
injected chicks developed any of the pathologic manifestations
with the exception of mild hemocytoblastosis during the 4-month
period they were held for observation.
A mild degree of hemocytoblastosis developed shortly after
the first injection of endotoxin. The total erythrocyte count
of these birds ranged from 2,600,000 to 2,850,000 during the
period of injection and was somewhat lower than that of the
control birds. Many degenerative erythrocytes were observed
in blood smears taken from the peripheral circulation. The
highest leucocyte count during the period of injection was 82,000.
The percentage of degenerative and immature leucocytes ob-
served was comparatively small. The lymphocyte was the
first type of leucocyte to show degenerative changes; the change
most commonly noted was that in which the cell and nucleus lost
their spherical shape and became enlarged, the riucleus be-
coming an eosin-staining homogenous mass (12).
Single and Repeated Intracerebral Injections Into Young
Chicks.-Pappenheimer (20) has shown that the intracerebral
injection of emulsions of affected nerves of paralyzed birds re-
sulted in the development of fowl paralysis in 25 percent of
the young chicks so injected. Following the technique of this
investigator (19), 7 day-old Barred Plymouth Rock chicks were
given single intracerebral injections of 1 minim of endo-
toxin while 10 day-old chicks of like breed were given in-
tracerebral injections of 2 minims of the same endotoxin







14 Florida Agricultural Experiment Station

preparation every other day for 2 weeks. The intracerebral
injection of 2 minims often caused paralytic symptoms
from which recovery usually occurred in from 1 to 2 hours.
These symptoms were similar to those induced by the intra-
venous injection of endotoxin into day-old chicks.
Two chicks died from pullorum disease. The remaining birds
were held for observation until they were 3 months of age;
fowl paralysis failed to occur in either group.
A third group of 20 day-old Barred Plymouth Rock chicks
were given repeated intracerebral injections of endotoxin. Ten
chicks were given 1 minim and the remaining 10 chicks were
given 2 minims 3 times a week for 3 weeks. One chick died
on the ninth day showing congestion of the cerebrum. The
remaining birds were held until 3 months of age. No cases of
fowl paralysis developed within that period.
Single and Repeated Intraperitoneal Injections Into Young
Chicks.-Twenty-four day-old Barred Plymouth Rock chicks
were injected intraperitoneally with single doses of endotoxin
varying in amount from 2 to 5 ml.; the endotoxin was derived
from 6 strains of S. aertrycke, 4 birds in each of 6 groups re-
ceiving the same endotoxin preparation. Those birds receiving
the more potent endotoxin preparations as well as the larger
doses usually showed temporary paralytic symptoms from which
recovery occurred in 2 to 3 hours. Absorption following this
mode of injection apparently was not rapid and consequently
fewer birds showed paralytic symptoms following the injection
of the endotoxin than in the previous experiment in which it
was injected intravenously. The birds were held for 3 months
after injection. No cases of fowl paralysis occurred.
Since endotoxin failed to induce fowl paralysis in any of the
preceding experiments, it was considered advisable to inject
endotoxin into day-old chicks and to repeat and increase the
dosage. An effort was made to regulate the dosage so that
paralytic symptoms did not appear. The initial dose consisted
of 0.2 ml. of endotoxin. Twenty-two day-old Single Comb White
Leghorn chicks were injected 3 times a week as follows.:. one
group of 7 chicks received 15.3 ml. of endotoxin intraperitoneally
over a period of 29 days; 8 chicks received 10.2 ml. in 32 days,
while another group of 7 chicks received 20.3 ml. of endotoxin
in 33 days. Injection of the endotoxin at times caused complete
paralysis, twitching, nervousness and indisposition, with re-
covery being complete in 2 to 3 hours following injection. Three






The Etiology of Fowl Paralysis


chicks in the various groups died from pullorum disease, but
fowl paralysis failed to occur; the birds were held for 4 months
following final injection of endotoxin. One bird developed lym-
phatic erythroleucosis 87 days after injection. At death, 22 days
later, the total erythrocyte count was 1,740,000, while the leu-
cocytes numbered 78,000 per cu. mm. Sixteen control chicks, 4
in each group, received similar injections of a mixture of phenol
and 0.85 percent sodium chloride equivalent to that in the en-
dotoxin preparation. All control birds remained normal. Rou-
tine total and differential blood counts were not made.
Ten 8-week-old Barred Plymouth Rock pullets were given
.repeated injections of endotoxin intraperitoneally over a 3-
month period, 3 ml. per dose 3 times a week. None of these
birds developed symptoms of fowl paralysis during the 4-month
period following the last injection of endotoxin. The 4 con-
trols remained normal.
Total and differential blood counts of the birds in these 2
groups were made every 2 weeks. It was found that a mild
degree of hemocytoblastosis developed shortly after the first
injection. The erythrocytes became considerably decreased in
number and numerous degenerative erythrocytes were observed
in blood smears from the peripheral circulation. The total
and differential leucocyte counts were similar to those observed
in the birds receiving repeated introvenous injections of endo-
toxin in a previously described experiment.
Effect of Endotoxin on Agglutination.-Eight series of tubes
were used, each series containing sufficient tubes for the titer
of the immune serum under test. The agglutination tests were
conducted in the usual manner, each tube containing 1 ml. of a
suspension of S. aertrycke and immune serum dilutions begin-
ning at 1 to 25 and extending serially upward. The 8 series
of tubes were prepared exactly alike. The first series served
as the control; 0.01, 0.025, 0.05, 0.075, 0.1, 0.25 and 0.5 ml. of
endotoxin (pH 6.8) was added to each tube in each of the 7
remaining series, respectively. In the first experiments all of
the tubes in the first 7 series were brought to the same volume
as those in the last series by the addition of the proper amount
of 0.85 percent sodium chloride solution (pH 6.8); it Was found
that results were the same when this was not done. Volumes
were not equalized in subsequent experiments. Contents of
the tubes were mixed thoroughly. Results were recorded 12












TABLE 1.-RESULTS OF AGGLUTINATION TESTS ON S. aertrycke IMMUNE CHICKEN SERUM WHEN ANTIGEN AND VARYING
AMOUNTS OF ENDOTOXIN PREPARED FROM THE SAME STRAIN OF THIS MICRO-ORGANISM WERE USED.
Amount Agglutination Titer
Endotoxin ________
Added 1-25 1-50 1-100 1-200 1-400 1-800 1-1600 1-3200
(ml.) 12* 24 12 241 2 2 24 12 24 12 24 12 24 12 24 12 24
+ + ++ +. + P
+ ++
None + + + + +


.02 + + + + + + + +
+ + + + + p --

.05 + + + + + ++ +
.05 +_ + + + + +
+ + + + p
.1 + + + -

.25 + + + _
+ + +
.5 + + + -
.5
Incubation period in hours.
Positive.
Negative.
p Partial.










TABLE 2.--RESULTS OF AGGLUTINATION TESTS ON S. aertrycke IMMUNE CHICKEN SERUM WHEN ANTIGEN AND VARYING
AMOUNTS OF ENDOTOXIN PREPARED FROM ANOTHER STRAIN OF THE MICRO-ORGANISM WERE USED.

Amount Agglutination Titer
Endotoxin10
Added 1-25 1-50 1-100 1-200 1-400 1-800 1-1600 1-3200
(ml.) 12* 24 12 24 12 24 12 24 12 24 12 24 12 24 12 24
+ + + + + + + -
+ + + + + + +
+t + + + p s
.01 + + + + + + + +
+ .+ + p -
.025 J + + + + + + + -

.05 + + + + +_ +-
+ -+ --P
.075 + + + + -
+ + + -


.25 -


* Incubation period in hours.
+ Positive.
- Negative.
p Partial.








Florida Agricultural Experiment Station


and 24 hours after incubation at 370 C. S. aertrycke immune
chicken serum was used in all series of agglutination tests.
Results of 2 experiments are tabulated in Tables 1 and 2.
It was found that the addition of 0.01 ml. of endotoxin inhibited
agglutination in the higher titers of the immune serum after
12 hours' incubation, but that 0.025 to 0.075 ml. of endotoxin
was necessary to reduce the titer of the immune serum during
24 hours' incubation. This experiment was repeated with differ-
ent strains of S. aertrycke as antigen and as a source of endo-
toxin. Comparable results were obtained in all cases in which
the same strains of S. aertrycke were used as antigen and as a
source of endotoxin as in the instances in which endotoxin of
other strains of this micro-organism were used. When the titer
of immune serum was less than 1 to 800, endotoxin did not have
such a marked effect on agglutination, although a tendency to
inhibit agglutination and to reduce the titer usually occurred.
Two series of agglutination tests 'were conducted in which
endotoxin preparations of different potency were used. In
all cases inhibition of agglutination and reduction of agglutina-
tion titer were approximately proportional to the potency of
the endotoxin used.
Effect of Endotoxin on Blood Cells.-Since the repeated intra-
venous and intraperitoneal injection of endotoxin into 8 to 12-
week-old birds induced a mild degree of hemocytoblastosis, it
was considered advisable to determine the effect of endotoxin
on- blood cells.
One milliliter of citrated chicken blood was placed in each
of 4 test tubes. To the second, third and fourth tubes, respect-
ively, 0.1, 0.5, and 1.0 ml. of endotoxin (pH 6.8) was added.
Physiological salt-solution (pH 6.8) was added to the first 3
tubes to make the total content equal in volume to that of the
fourth. The first tube served as a control. The tubes were
incubated in a water bath at 370 C. After 15 and 30-minute
periods of incubation, smears were made from each of the tubes
and stained with Wright's stain for study.
A number of preliminary experiments were necessary to
ascertain the potency of endotoxin which would give satisfactory
results. Endotoxin used in most of the other experiments, re-
ported herein, produced such rapid degeneration of leucocytes
it was difficult to follow the changes. An endotoxin prepara-
tion, 1 ml. of which would kill a 20-gram mouse in 20 hours,







The Etiology of Fowl Paralysis


diluted with an equal volume of physiological salt solution was
found satisfactory for use in these experiments.5
After 15 minutes incubation the rod-bearing eosinophile was
the only cell to show evidence of change in the control tube. A
few rods in occasional cells appeared slightly swollen. After 30
minutes incubation this condition was slightly more marked
and a few scattered dark granules appeared in some cells.
In the second tube, in which the dilution of endotoxin to whole
blood was 1 to 10, significant degenerative changes were noted
in most of the leucocytes after 15 minutes incubation. Degen-
erative changes were more pronounced and more general in the
rod-bearing eosinophile. Many small vacuoles, often variable in
size, were present in the cytoplasm of these cells. In many
cells a few normal rods and remnants of rods were observed.
Only occasional granules were noted. The cytoplasm stained
faintly. Many lymphocytes appeared unchanged; an occasional
cell, however, showed the presence of 3 or 4 vacuoles of variable
size in the cytoplasm; other cells appeared slightly irregular
in outline, due possibly to degeneration of the cytoplasm. The
monocytes appeared slightly blurred but showed no distinct
structural change. A few erythrocytes showed degeneration;
the cell became enlarged, the cytoplasm was irregular in outline
and the nucleus became an enlarged, spherical, structureless
eosin-staining mass.
In the remaining tubes, in which the dilutions of endotoxin
to whole blood were 1 to 2 and 1 to 1, correspondingly greater
degrees of degeneration were observed in the tubes incubated
for 15 minutes. The rod-bearing eosinophiles contained many
vacuoles with little or no evidence of cytoplasm remaining; the
cells often were distorted and the nuclei appeared necrotic; a
few granules were observed. Many lymphocytes were distorted,
the nucleus and cytoplasm being involved in the process. Other
lymphocytes retained their normal shape but the outlines of
the cells were ragged; a few vacuoles were present in occasional
lymphocytes whose degeneration was not well advanced. The
monocytes appeared blurred with an occasional vacuole in the
cytoplasm; the outline of many of these cells was irregular.
5 It was found that a suitable endotoxin preparation also could be pro-
duced by thoroughly grinding 0.85 gms. sodium chloride and 100 mg. of
desiccated S. aertrycke in a dry sterile mortar. The residue was then
suspended in 100 ml. of sterile distilled water. After thorough agitation
the suspension was centrifuged at high speed. The supernatant fluid
containing endotoxin was filtered through a Berkefeld W candle, heated
at 100* C. for 15 minutes and stored in the refrigerator for use.







Florida Agricultural Experiment Station


After an incubation period of 30 minutes the leucocytes in
the tube in which the dilution of endotoxin to whole blood was
1 to 10 showed more progressed degenerative changes than were
noted in the same tube incubated but 15 minutes. These changes
were not as advanced, however, as in the tubes containing
stronger dilutions of endotoxin incubated for the lesser period.
The rod-bearing eosinophiles showed the presence of but few
vacuoles as compared with the smears made after 15 minutes
incubation. The significant change was in the observation of
many dark-stained granules whose appearance was not unlike
the granules which appear in the cytoplasm of the same type
of cells following introduction of S. aertrycke into the blood
stream of living chickens (Fig. 2).
Even in the most concentrated endotoxin dilutions the struc-
ture of the granule-bearing eosinophile remained unchanged.
In some cases the nucleus of these cells stained more faintly
than normally. This observation is of interest, since the author
has advanced evidence previously to show that at least some
granule-bearing eosinophiles probably have their origin in de-
generative rod-bearing eosinophiles. The normal granule-bear-
ing eosinophile usually is observed more frequently in recessive
hemocytoblastosis than in active hemocytoblastosis.
The experiment was repeated, using endotoxin from various
strains of S. aertrycke and the blood of different chickens. Com-
parable results were obtained in each instance.
Effect of Endotoxin on the Growth of S. aertrycke on Solid
and Liquid Media.-Thirty tubes containing 5 ml. of 3 percent
nutrient agar (pH 6.8) were prepared and sterilized. Before
cooling, 5.0 ml. of sterile 0.85 percent sodium chloride solution
(pH 6.8) were added to each of 1 series of 10 tubes; 5.0 ml. of
endotoxin (pH 6.8) were added to each of a second series of
10 tubes; 2.5 ml. of endotoxin and 2.5 ml. of 0.85 percent sodium
chloride solution, both with a pH of 6.8, were added to each
of a third series of 10 tubes. The contents of each tube were
poured into a petri dish and allowed to cool. Spot and streak
serial transfers were made after every 24-hour incubation period
at 370 C. No appreciable differences in the 3 series of plate cul-
tures could be detected by gross examination of the colonies or
by microscopic examination of smears from the colonies.
The experiment was repeated with 0.5 ml. of defibrinated
sheep's blood added to each plate. The results were similar
to those of the preceding experiment.










































Fig. 2.-Effects of endotoxin on the rod-bearing eosinophile: 1, typical cell in control tube after 30
minutes incubation in water bath; 2, 3, 4, typical cells after 15 minutes incubation with 0.1 ml. endotoxin
per ml. of oxalated blood; rods are distinguishable in many instances but many granules occur and the
cell usually is distorted; 5, 6, typical cells after 30 minutes incubation with 0.1 ml. endotoxin per ml. of
oxalated blood. The cell in 5 is considerably enlarged and shows the presence of vacuoles in many rods;
many such cells rupture. The cell in 6 approaches the size and shape of a normal cell but the rods appar-
ently are condensed into granules; this cell is similar to that observed in typical cases of leukemia.


.Y,. .


0






0 4
-ii

(cc
Tj4


C4







Florida Agricultural Experiment Station


The experiment was repeated a third time. In the instance
of those plates containing endotoxin, 0.5 ml. of defibrinated
sheep's blood was incubated at 370 C. for 2 hours with the
endotoxin to be incorporated in the media of each tube. In
the check series, 0.5 ml. of blood was added as in the preceding
experiment. The results were again negative.
Three experiments similar to those described above were con-
ducted, nutrient broth being substituted for nutrient agar. The
first series consisted of the addition of endotoxin to nutrient
broth; the second, the addition of endotoxin to nutrient broth
enriched with defibrinated sheep's blood; and the third, the
addition of an incubated endotoxin-sheep's blood mixture to
nutrient broth. Stained smears prepared from the 3 series of
tubes in each experiment after incubation of the first, fifth and
tenth serial transfers failed to reveal any changes in the growth
characters of S. aertrycke. Smears made on nutrient agar plates
prepared in the same manner as the nutrient broth series from
which the smears were made did not show any apparent varia-
tions in the growth character of the micro-organism. Dilution
plates were prepared from each series of tubes after incubation
of the first, fifth and tenth transfers; marked variations in the
growth curves did not occur.
Effect of Endotoxin on Phagocytosis.-There are a few refer-
ences in the literature pertaining to the effect of intracellular
toxic substances on phagocytosis. Dudgeon, Panton and Wil-
son (4, 5) have reported endotoxin to have a more or less spe-
cific inhibitory action on phagocytosis and suggested in a sub-
sequent paper the possibility of employing the specificity of the
reaction as a basis for the differentiation of many species of
bacteria. Wittenberg, Lederer and Mollov (25) found the en-
dotoxin of Neisseria gonorrhoeae to inhibit phagocytosis of this
micro-organism by human leucocytes and that the degree of
inhibition depended on the strength of endotoxin to which the
leucocytes or micro-organisms were exposed.
The following experiments were conducted to determine the
effect of endotoxin of S. aertrycke on phagocytosis of the same
strain of micro-organism. It has been shown that the rod-bear-
ing eosinophile of chickens readily undergoes degenerative
changes when exposed to endotoxin in vitro as well as in vivo.
Since these degenerative changes apparently result in a dis-
integration of the rods into granules, observations on phago-
cytosis by these cells were difficult. This was especially true







The Etiology of Fowl Paralysis


in many cases, since the bacterial cell often lost its rod structure
and appeared as small granules within the phagocyte. Con-
sequently, the leucocytes of sheep's blood were used in the fol-
lowing experiments.
Endotoxin was prepared in a manner similar to that in the
preceding experiments on the effect of endotoxin on blood cells.
S. aertrycke was cultured on nutrient agar slants and suspended
in 0.85 percent sodium chloride solution after 24 hours incu-
bation; best results were obtained when the suspension was
rather dense. Two drops of this suspension of S. aertrycke
and 2 drops of citrated sheep's blood were added to each of 2
tubes; 2 drops of the endotoxin preparation were added to 1
tube and 2 drops of physiological salt solution to the second
tube. The contents of the tubes were gently agitated and placed
in a water bath at 37 C. for 30 minutes. Smears from the 2
tubes were stained with Wright's stain. This technique is simi-
lar to that used by Wittenber et al. The bacterial cells were
counted in 50 leucocytes.
The results indicated that the endotoxin of S. aertrycke had
an inhibitory effect upon phagocytosis. Experiments in which
different strains of the micro-organism were used yielded simi-
lar results. The phagocytes showed a marked variation in their
ability to phagocytize S. aertrycke in the check tubes as well as
in tubes containing endotoxin. To arrive at the result the
average number of bacterial cells engulfed by 50 leucocytes
was determined. Eight determinations showed that leucocytes
exposed to endotoxin in this manner were capable of phagocy-
tizing but 1/20 to 1/40 of the number of bacteria phagocytized
by leucocytes not exposed to endotoxin.

EXPERIMENTS WITH S. AERTRYCKE
Fate of S. aertrycke Following Intravenous Injection.-It has
been shown that repeated intravenous injections of S. aertrycke,
as well as oral exposure to this micro-organism during enteritis
caused by intestinal parasitism, often leads to the development
of fowl paralysis, leukemia or an allied condition and that the
micro-organism cannot be isolated from the tissues of affected
birds at death (11). Experiments have shown also that the
micro-organism rarely can be isolated from the peripheral cir-
culation of the bird during the period of infection per orum.
Many investigators have been hesitant to consider a bacterial
theory of the origin of fowl paralysis chiefly because of inability







24 Florida Agricultural Experiment Station

to isolate micro-organisms of etiologic significance from tissues
of affected birds.
It should be understood that S. aertrycke, as well as many
of the other species of the genus Salmonella, is capable of in-
ducing septicemia in chickens. Doyle (3), Edwards (6), Beau-
dette (1), Johnson (15) and Rettger et al (22) and others have
reported S. aertrycke as the cause of an acute disease in young
birds in which little or no difficulty was encountered in isolating
it from the tissues at death. Similar results have been reported
in a previous paper in this series. Johnson (16) also was able
to induce a septicemic disease in 6-month-old birds by the oral
administration of 1 ml. of a broth culture of S. aertrycke as
well as by the subcutaneous injection of 0.5 ml. of a suspension
of the same micro-organism.
In order to induce the various pathological manifestations
(with the exception of septicemia) under consideration in this
study it is necessary that the micro-organisms enter the blood
stream of the bird over a period of time in numbers insufficient
to cause septicemia.
The question arises as to the fate of the micro-organism in
the circulatory system of the bird. To determine this point a
number of birds were given intravenous injections of varying
amounts of S. aertrycke, observations being made during the
following 1 to 3 days.
A suspension of S. aertrycke in physiological salt solution was
injected intravenously into a number of birds of the same age
and breed. The birds were killed at intervals and cultures
were made from the heart, peripheral blood, kidneys, lungs,
spleen, liver, ovary or testicle and the bone marrow on nutrient
agar plates. After incubation at 370 C. for 48 hours the results
were recorded according to the relative number of colonies de-
veloping on the plates.
Results shown in Table 3 are typical examples of observations
made on 7 groups comprising 29 birds. One and a half to 4
hours after injection S. aertrycke was isolated in most instances
from all of the tissues cultured, the bone marrow and the ovary
or testicle being most variable. These latter tissues were gen-
erally the first to yield negative results. As the fifth and sixth
hours after injection were approached greater difficulty was
experienced in isolating the micro-organism from the heart,
peripheral blood and kidneys. The failure to isolate the micro-
organism from the lungs was next experienced, while the micro-




'I'-- THE ISOLATION OF S. aertryce FROM REPRESENTATIVE IRS KILLED AT INTERVALS
VENOUS INJECTION OF VARIOUS STRAINS OF THIS MICRO-ORGANISM.


Breed APe


Culture
Num-


Hours
Killed
after


FOLLOWING THE INTRA-


Isolation of S. aertrycke


Peripheral Kid-


Bone Ovaryor


IVCWeeks| Sex ber Injection Heart Blood neys Lung Spleen Liver Marrow Testicle

W. Leghorn 18 Female A5 1.5 + + + ++ +++ ++ + +
2.5 + + + +++ ++++ ++++ -
3 + ++ +++++++*- -
4 ++ ++++ ++++
6 + ++ ++ -
12 -

W. Rock 14 Female 36 2 + +++ ++++ +-+ -
14 + +++
10

W. Rock 14 Male 36 2 ++++ + -
4 ++++ ++++ -
10 --.+. -- + |-


B. Rock




R. I. Red




W. Leghorn


Female


Male 26


Female


A5


3 +
5
8 -
12 -

2 -
5 -
8 -
10 -

4 +
6 +
8 -
12 -
18 -


.+


t+


3333
+f3-+
333


S+St-
333
33


3333
3333
3333
++3-+


$+-



++++ +


W. Leghorn 16 Male 526 6 + ++++ ++++ -
12 ++ ++ -
18 -- -
24 -
i
+ Positive. Negative.


IYe


0~
0

0

03







26 Florida Agricultural Experiment Station

organism could always be isolated from the liver and spleen if
isolated from any other tissues of the body. The relative num-
ber of bacteria isolated from the liver and spleen was in excess
of that from any other tissues cultured; the number isolated from
the lungs ranked second, while there was little difference in the
number isolated from remaining tissues. Ten to 12 hours after
injection cultures from the liver and spleen indicated the dis-
appearance of the micro-organism from these organs.
Blood smears taken just before autopsy from those birds in
which the micro-organisms were localized in the liver and spleen
indicated that but few rod-bearing eosinophiles could be found
in the peripheral circulation. Smears taken from the spleen
and liver at autopsy showed large numbers of phagocytes.
These experiments indicate that the micro-organisms injected
intravenously tend to localize in the liver and spleen and that
much of the phagocytosis during infection probably takes place
in these organs.
In a series of experiments in which blood smears were taken
from birds at intervals the rod-bearing eosinophiles, after tend-
ing to localize in the liver and spleen, again invaded the peri-
pheral circulation. In some instances the number of eosinophiles
in the peripheral circulation at this time was considerably lower
than before experimental infection occurred.
In the instance of repeated intravenous injections of S. aer-
trycke on successive days it was possible to establish a tempor-
ary bacteremia in some birds. This was accomplished occasion-
ally in adult birds by the injection of a large number of S. aer-
trycke at a single injection. The establishment of a temporary
bacteremia in adult birds required the injection of a much
greater number of micro-organisms than in young birds. In
young birds considerable care was necessary that a fatal bac-
teremia did not result. Temporary bacteremia in old birds oc-
casionally lasted as long as 3 days, immediately after which the
bird did not show any particular evidence of disease; some birds
showed a slight indisposition for several days. In the case of
laying hens, egg production always was decreased.
In experiments in which repeated intravenous injections of
small numbers of S. aertrycke were administered the percentage
occurrence of rod-bearing eosinophiles in the peripheral circu-
lation materially decreased after 2 to 4 daily injections. Oc-
casional birds showed marked leucocytosis. Chickens, however,
did not develop leucocytosis following this type of infection







The Etiology of Fowl Paralysis


as markedly as has been generally observed in large animals
infected similarly with the same micro-organism. In every
instance, however, initial hemocytoblastosis was observed shortly
after infection.
The reduction of circulating rod-bearing eosinophiles after
repeated infection with S. aertrycke in these experiments was
considered to be due to the destruction of these cells during
phagocytosis. It has been shown previously in this paper that
endotoxin has a destructive action on blood cells. In vivo after
phagocytosis of the micro-organism the endotoxin is released
within the phagocyte, which should give endotoxin a greater
opportunity, for destructive action than when brought in con-
tact with the exterior of the cell. The destructive action of
endotoxin on the rod-bearing eosinophile also is evidenced by the
fact that this cell is the first type of blood cell to show degen-
erative changes following intravenous injection of S. aertrycke.
In birds which were autopsied 2 to 4 hours after the injection
of S. aertrycke smears from the spleen and liver indicated that
the eosinophiles which had accumulated in these organs showed
considerable degenerative changes.
Schalm (24) recently reported a micro-organism resembling
S. typhimurium as causing extensive losses in several outbreaks
of disease in young chicks. In experimentally infected birds
the micro-organism readily left the blood stream and localized
in the liver and spleen from which it was recovered for as long
as 125 days after infection.
In the experiments reported herein the author was unable to
demonstrate the persistence of foci of infection. Although S.
aertrycke localized to a marked extent in the liver and spleen
of intravenously injected birds, the numbers of micro-organ-
isms isolated from these organs diminished over a comparatively
short period. Eighteen to 24 hours after a single intravenous
injection of a slightly cloudy suspension of S. aertrycke into
12 to 16-week-old birds, the micro-organism could not be iso-
lated from the liver or spleen. Even in birds in which a tem-
porary bacteremia was induced the micro-organism was not iso-
lated from the liver or spleen 72 hours after the subsidence of
bacteremia.
Repeated Intravenous and Intraperitoneal Injection of the
Heat-killed Micro-organism.-As investigations conducted by
various workers have shown that some of the manifestations of
the leucosis complex can be transmitted by a filtrable agent ob-







Florida Agricultural Experiment Station


trained from the tissues of affected birds, it was deemed advisable
to conduct experiments in which birds were injected intraven-
ously and intraperitoneally with heat-killed S. aertrycke. It
has been suggested that there may be a mutual relationship
between the micro-organism found capable of inducing this
group of diseases in this study and the filtrablee agent" or
"virus" of fowl leucosis. In other words, the micro-organism
may be a vehicle for some other agent capable of inducing this
group of diseases.
Rivers (23) has reported that viruses are inactivated by a
temperature varying from 45 to 800 C. Exposing suspensions
of S. aertrycke to a temperature of 1000 C. for 10 minutes should
inactivate any virus which had formed a close association with
this micro-organism. Many investigators, including the author,
have shown that endotoxin is not destroyed at a temperature
of 1000 C. for 10 minutes. The chief difference between living
and heat-killed S. aertrycke would be in pathogenicity.
S. aertrycke was desiccated as previously described. Weighed
amounts of the desiccated micro-organism were suspended in
physiological saline solution and repeatedly injected intraven-
ously and intraperitoneally after subjection to heat of 1000 C.
for 10 minutes. The suspensions were cultured to assure the
absence of living micro-organisms. Injections were made twice
a week.
A summary of experimental data involving the 12 experiments
conducted is given in Table 4. Since there was marked con-
sistency in results there should be no necessity of describing
the individual experiments. In the first 4 experiments the
suspensions of dead bacteria were injected intraperitoneally and
yielded results similar to those obtained in the next 4 experi-
ments in which the suspensions were injected intravenously.
In these experiments, with a total of 80 birds, 4 birds developed
fowl paralysis after incubation periods varying from 82 to 101
days, lymphomatosis occurred in 21 with incubation periods
of from 60 to 131 days, leukemic lymphocytoma occurred in 5
with incubation periods of from 84 to 129 days, 1 bird developed
myeloid leukemia in 66 days and 2 birds died from undetermined
causes in the first experiment the day after the fourth injection;
the remaining 47 birds developed hemocytoblastosis and sub-
sequently recovered.
In the remaining 4 experiments the bacteria were injected in
smaller amounts but over a longer period; the birds were 16 to







The Etiology of Fowl Paralysis


20 weeks of age as compared with 6 to 10 weeks of age in the
preceding experiments. The injections were made intraven-
ously. From a total of 40 birds included in these experiments,
1 developed fowl paralysis in 117 days, 2 developed lymphoma-
tosis with incubation periods of 89 to 108 days, leukemic lympho-
cytoma occurred in 2 birds after incubation periods of 121 and
137 days, while 12 developed leukemia with the incubation per-
iods varying from 91 to 180 days; all of the remaining 23 birds
developed hemocytoblastosis and subsequently recovered.
Five non-injected birds of age and breed similar to those
included in each experiment served as controls; they remained
normal throughout the period of observation.
The total and differential blood counts of a few representa-
tive birds given repeated intravenous or intraperitoneal in-
jections of desiccated S. aertrycke and other data pertaining to
this group of experiments are presented in Table 5.
Ability of Various Strains of S. aertrycke.to Induce This Group
of Diseases.-It has been recognized that there are often wide
variations in the virulence of different strains of pathogenic
micro-organisms. In this study, experiments were conducted
with S. aertrycke isolated from various sources as follows: (a)
the intestinal contents of naturally occurring cases of fowl
paralysis and birds affected with enteritis associated with in-
testinal parasitism; (b) septicemic disease in baby chicks; (c)
septicemic disease in turkeys; (d) pigeons affected with arth-
ritis; and (e) from man.6
All strains from the above sources were capable of inducing
fowl paralysis and leukemia in chickens. Strain A5 isolated
by Rettger from turkeys affected with septicemia was the most
virulent strain for chickens as well as other animals used in
these studies; strains M2 and M5, however, also isolated from
turkeys by Rettger, were found to be much less virulent. Strains
19500 and 19525 isolated by Edwards from cases of arthritis
in pigeons were not particularly virulent for chickens. The cul-
tures of S. aertrycke isolated from the human, those isolated
from the intestinal tract of chickens and from a septicemic dis-
ease in baby chicks occupied an intermediate position and
showed but little variation in their ability to induce fowl paraly-
sis and leukemia.
"The author is indebted to Dr. L. F. Rettger, Yale University, Dr.
Philip R. Edwards, University of Kentucky, and the Michigan Depart-
ment of Health for strains of S. aertrycke isolated from turkeys, pigeons
and man, respectively.









TABLE 4.-SUMMARY OF EXPERIMENTAL DATA INVOLVING 12 EXPERIMENTS IN WHICH DESICCATED S. aertrycke WERE
REPEATEDLY INJECTED INTRAVENOUSLY OR INTRAPERITONEALLY AFTER SUSPENSION IN 0.85 PERCENT SODIUM CHLORIDE
AND KILLING BY HEAT AT 1000 C. FOR 10 MINUTES.


No. of Injections
Experi- Culture Breed Age
meant Number Weeks Amt. Num- Method
S(mg.) ber
I 36 W.L. 6 20 4
0
II 526 B.R. 8 10 6

III 26 W.L: 10 10 6 1

IV 36 W.L. I 10 20 6


No. of Fowl
Birds Para-
Slysis I


1
(97)*


Lympho-
matosis


Results


Leukemic
Lympho-
cytoma


1
(93)


Unde-
term- Leukemia
ined


2
(1) [


Hemocy-
toblast-
osis

5


1 2 () 1
10 (101) (109-127) (66) 6


(60-97)
3
(64-102)


2
(84-102)
1
(117)


5

6


V 36 B.R. 8 20 4 10 -- (67-82) 6
1 2 1
VI 36 R.I.R. 8 20 4 10 (92) (80-120) (129) 6
3 |
VII 526 R.I.R. 10 10 8 o 10 (68-131) 7
1 3
VIII 526 R.I.R. 10 10 6 5 10 (82) (76-109) 6
S1 1 '3
IX 526 B.R. 16 4 14 10 (89) (121) (130-171) 5

X 36 B.R. 16 4 14 10 (101-153) 6
1 3
XI 26 B.R. 20 4 16 10 (108) (91-157) 6
1 1 2
XII 26-526 B.R. 20 4 i 16 | 10 (117) (137) (112-180) 6
Controls: Three non-injected birds of similar age and breed in each experiment all remained normal.
* Figures in parentheses represent incubation period in days.


0



3-.






0,






TABLE 5.-TOTAL AND DIFFERENTIAL BLOOD COUNTS OF REPRESENTATIVE BIRDS WHICH RECEIVED DESICCATED S. aertrycke SUSPENDED IN
0.85 PERCENT SODIUM CHLORIDE AND KILLED BY HEAT AT 100' C. F3R 10 MINUTES, REPEATED INJECTIONS BEING GIVEN INTRAVENOUSLY
OR INTRAPERITONEALLY.
Diffrental Bood oun


0
*0
o V
n


100
Female

73
Female


11
Male


21
Female


CO
'P
m
"a
*a"


E9


R.I.R.
10

W.L.
6


B.R.
8


W.L.
6


66 W.L.
Male 6


64 R.I.R.
Female 10


74 R.I.R.
Female 8


91 W.L.
Female 10


90 W.L.
Male 10


r'3
.a



12-17


12-15



12-15


12-15


12-17


12-17 1 131


12-15



1-27



1-27


I I I I


p
Po


a-s


1 I I 1


0E
5~ ~B


Polymorpho- Mono- Myelo-
nuclear Lymphocyte eyte cyte
a 0


I, P |


r ent


Premye-
locyte


I I I I


Fowl paralysis
,(right leg lame)


Fowl paralysis (wry
neck, walks in circle)


Fowl paralysis
(right wing paralyzed)

Leukemic lymphocytoma
(liver mesentery, bone
marrow)

Lymphomatosis (right
eye blind, lympho-
matous ovary)


Lymphomatosis
(both eyes blind)


Hemocytoblastosis



Hemocytoblastosis



Hemocytoblastosis


+ Positive.


I


Differential Blood Count


I I I


I


I


1-22
2-10

12-29
1-22
3-29
4-10

12-29
1-22
3-29


1-22
3-20

1-22
3-19
3-27

1-22
3-10
4-20

12-29
1-22
3-19
5-1

2-6
3-27
4-17
6-1

2-6
3-27
4-17
6-1


2,810
2,960

2,410
2,770
2,140
3,150

2,670
2,910
2,980

3,190
2,920
3,000

1,350
2,790
2,760

2,640
2,610
2,700

2,280
2,450
2,570
2,650

3,260
1,760
2,010
2,860

2,990
1,970
2,890
2,860


195,000
91,500

131,000
209,000
92,000
68,500

82,000
85,000
52,500

143,000
88,500
51,500

320,000
96,000
56,000

118,000
64,000
57,000

102,000
98,000
160,000
61,000

32,000
72,000
70,500
38,500

295,000
242,000
112,500
54,000


0 0 3
12 0 3

5 0 9
3 0 0
6 0 10
5 0 0

10 0 11
7 0 15
15 2 3

3 0 6
0 0 2
9 0 4

0 0 4
0 0 2
0 0 4

0 0 2
21 0 3
23 2 6

5 0 10
0 O 11
0 0 10
14 0 0

11 0 0
0 2 10
0 2 9
19 1 3

19 0 0
6 0 8
9 0 3
22 3 0


29
25

24
45
23
47

31
30
30

50
59
38

58
29
45

51
39
29

47
34
61
48

41
40
30
27

45
44
38
32


0 0 0 0
11 0 0 0

42 0 0 0
7 0 2 2
0 0 0 2
0 0 0 3

21 0 0 0
14 0 0 2
3 0 0 6

4 0 0 2
5 0 1 2
0 0 0 5

18 0 0 0
10 0 0 2
16 0 0 7

13 0 2 0
0 0 0 2
0 0 0 3

12 0 0 0
17 0 0 0
0 0 0 2
3 0 0 5

13 2 0 0
8 0 0 0
3 0 0 9
0 1 0 4

12 0 0 0
7 0 0 2
5 0 0 5
0 0 0 4


+ Positive.


I I I







32 Florida Agricultural Experiment Station

Some of the strains of S. aertrycke isolated in this study from
the chicken had been in stock for 8 years. Cultures kept in
stock on ordinary culture media usually experience a reduction
in potency of endotoxin as well as in virulence. After stock
cultures are held over long periods they show reduced ability to
induce fowl paralysis, most likely through loss of virulence and
potency of endotoxin, but such strains have not shown marked
reduction in abiilty to induce lymphomatosis and leukemia.
EXPERIMENTS WITH S. AERTRYCKE AND ITS ENDOTOXIN
Intravenous Injection of S. aertrycke Alone Versus S. aer-
trycke Plus Endotoxin.-It has been shown that endotoxin pre-
pared by tryptic digestion of S. aertrycke is neurotoxic and
when injected intravenously into young chickens induces trans-
ient paralytic symptoms. Therefore, it was deemed advisable to
conduct experiments to determine the effect of intravenous in-
jections of S. aertrycke plus endotoxin as compared with S. aer-
trycke alone.
In each instance 2 groups of birds of the same age and
breed equally divided as to males and females were used. The
suspensions for injection were prepared by adding a given vol-
ume of a cloudy suspension of S. aertrycke to equal volumes of
physiological salt solution and endotoxin, respectively. The 2
resultant suspensions contained approximately the same num-
ber of S. aertrycke. All of the birds in both groups were given
equal volumes of the respective suspensions, usually 1.0 ml. per
bird per injection twice a week.
The experiment was first conducted using endotoxin prepared
from the same strain of S. aertrycke as the suspension injected.
Experiments were then conducted in which endotoxin prepared
from other strains was used. Results are tabulated in Table 6.
In the 12 experiments conducted, 53 of the 105 birds which re-
ceived the micro-organism plus endotoxin developed fowl paraly-
sis, while only 22 of 108 birds which received intravenous in-
jections of S. aertrycke alone developed the disease (Fig. 3).
The remaining birds were not held to observe the development
of other pathologic manifestations. The incubation period of
the disease in birds receiving endotoxin was shorter as a rule
than in birds which received the micro-organism alone.
Experiments 2 and 7 are noteworthy in that practically the
same number of birds developed fowl paralysis in both groups.
The micro-organism was A5 in both instances and was the most






The Etiology of Fowl Paralysis


virulent strain of S. aertrycke used. This would indicate that
virulence of the micro-organism is of considerable importance
in the production of fowl paralysis. Very little difference was
noted among other strains. The endotoxin prepared from the
same strain of micro-organism injected and that prepared from
other strains produced similar results.
Two series of experiments were conducted in which birds in
various groups received intravenous injections containing ap-
proximately the same number of S. aertrycke but with varying
amounts of endotoxin. Increased amounts of endotoxin over
1.0 ml. often caused sudden deaths with no paralytic symptoms.
When postmortem examinations were made these birds showed
no gross evidence as to the cause of death. It was not infrequent
for the additional amounts of endotoxin to cause death 8 to 12


108









86


105


Fig. 3. The black area in the left column represents chicks 8 to 10
weeks of age which developed fowl paralysis as the result of the intra-
venous injection of S. aertrycke. The black area in the right column
represents chicks of the same age and breeding which developed fowl
paralysis as the result of the intravenous injection of S. aertrycke plus 1 ml.
of endotoxin.










TABLE 6.-SUMMARY


OF EXPERIMENTS IN WHICH BIRDS WERE EXPOSED TO INTRAVENOUS
ALONE VERSUS S. aertrycke PLUS 1 ML. ENDOTOXIN.


INJECTIONS OF S. aertrycke


Micro-organ-
ism from which
Endotoxin
was prepared


36

A5

19520

M5

526

526

36

526

36

36

36

36


Micro-
organism
Injected


36

A5

19520

M5

526

36

A5

26

526

M2

19520

M5


Micro-organism plus
Endotoxin
Number of
Number of Birds develop-
Birds ing Fowl
Injected Paralysis


Micro-organism Alone
Number of
Number of Birds Develop-
Birds ing Fowl
Injected Paralysis


8

10

10

8

10

10

8

10

13

5

8

8


TOTAL 105 53 108 22


Experi-
ment
Number


Age
Weeks


Breed


R.I.R.

R.I.R.

R.I.R.

W.L.

B.R.

B.R.

B.R.

B.R.

R.I.R.

R.I.R.

W.L.

W.L.


I I I






The Etiology of Fowl Paralysis


hours after injection; this occurred more frequently after re-
peated injections.
Experiments conducted as above with varying amounts of
endotoxin under 1.0 ml. per injection yielded results which were
not particularly significant in most instances. Results of great-
est significance were obtained with a dosage of 1.0 ml. of a rea-
sonably potent endotoxin.
Susceptibility to Fowl Paralysis as Indicated by Susceptibility
to the Neurotoxic Action of S. aertrycke Endotoxin.-Since it
appears that the neurotoxic action of the endotoxin of the micro-
organism plays an important part in the production of fowl
paralysis, it occurred to the author that the reaction of baby
chicks to the intravenous injection of endotoxin might be an
index as to their susceptibility to fowl paralysis when these
chicks at a later date were exposed to S. aertrycke. After a
number of preliminary investigations 7 experiments were con-
ducted, the summary of which appears in Table 7.
The potency and amount of endotoxin injected may be so
great as to cause symptoms of paralysis in a high percentage of
the chicks.' Consequently, it was necessary to conduct prelim-
inary experiments in the instance of each group of day-old
chicks to establish an approximate dosage of endotoxin. This
dosage, of course, is entirely arbitrary. However, the chicks
used in these experiments were from parent stock of known
susceptibility. All of the chicks in 1 group were given intra-
venous injections of the same amount of endotoxin.
When exposed to S. aertrycke the chicks did not receive the
same dosage of the micro-organism, as it was desired to induce
symptoms of paralysis in as many birds as possible; after the
initial dosage the reaction of the individual birds determined
subsequent exposure.
When exposed to the intravenous injection of endotoxin, 158
of 330 baby chicks showed paralytic symptoms; at 8 to 12
weeks of age, when these same chicks were exposed to intra-
venous injections of S. aertrycke, 137, or 86.7 percent of the
158 endotoxin -susceptible chicks, developed symptoms of paraly-
sis; 11 of the remaining 172 chicks which did not show suscep-
tibility to the neurotoxic action of endotoxin developed fowl
paralysis when similarly exposed (Fig. 4).
These experiments indicate that 36 to 62 percent of the baby
chicks injected according to this technique were susceptible to
the neurotoxic action of the endotoxin of S. aertrycke and that







.36 Florida Agricultural Experiment Station


17Z. 18Z















Fig. 4.-The black area in the left column represents day-old chicks
which showed paralytic symptoms following the intravenous injection of
endotoxin. The black area in the right column represents birds from
the same group which developed fowl paralysis induced by S. aertrycke
when 8-10 weeks of age and which were endotoxin-susceptible as day-
old chicks. Chicks represented by the dotted area developed paralysis
but were endotoxin-resistant as baby chicks.

there is a close relationship between the development of para-
lytic symptoms induced by endotoxin in baby chicks and fowl
paralysis when the same chicks at 8 to 12 weeks of age were
exposed to intravenous injections of S. aertrycke.
Under the conditions of these experiments, 3 of the birds
which showed paralytic symptoms as baby chicks following
the intravenous injection of endotoxin developed lymphomatosis
as a result of subsequent exposure to S. aertrycke, while 20 baby
chicks which did not show paralytic symptoms to the action
of endotoxin as baby chicks developed lymphomatosis as a result
of exposure to this micro-organism.
Virulence of Micro-organism Versus Potency of Its Endotoxin.
-Potency of endotoxin and virulence of the micro-organism
appear to be important factors in-relation to the development
of hemocytoblastosis and certain other manifestations in this
group of diseases.










TABLE 7.-SUMMARY OF EXPERIMENTS IN WHICH INDICATIONS OF SUSCEPTIBILITY TO FOWL PARALYSIS WERE NOTED BY
THE INTRAVENOUS INJECTION OF ENDOTOXIN INTO BABY CHICKS AND THE SUBSEQUENT INTRAVENOUS EXPOSURE OF THE
SAME CHICKS WHEN 8 TO 12 WEEKS OF AGE TO INTRAVENOUS INJECTIONS OF S. aertrycke.





Micro-
organism
Breed from which
Endotoxin
was
Prepared


B.R. 36

B.R: 526

R.I.R. 26

R.I.R. 36

W.L. 26

W.L. 526

W.L. 36
I


Micro-
organism
Injected




36

526

26

A5

A5

36

526


Number of Chicks showing
Paralysis after Intravenous
Exposure to the Micro-
organism


Number show-
ing Paralysis
to Endotoxin
as Baby
Chicks


Number
Chicks
Paralyzed
by Injection
of
Endotoxin


16

22

27

31

20

24

18


Number not
showing Para-
lysis to Endo-
toxin as
Baby Chicks


137


Number of Birds Develop-
ing Lymphomatosis after
Intravenous Exposure to
Micro-organism


Number
showing
Paralysis
to- Endotoxin


Experi-
ment
Num-
ber



I

II

III

IV

V

VI

VII


Number not
showing
Paralysis
to Endotoxin


0
0


01
o


o
Ccl




S.


Num-
ber
of
Chicks



30

50

50

50

50

50

50


1

0

1

0

0

0

1


3


3

3

2

3

2

3

4


20


Total 1 330


-


14

19

20

20

20

20
S" 15









Florida Agricultural Experiment Station


There appears to be no definite relationship between the
potency of the endotoxin prepared from the strains of micro-
organisms used and their pathogenicity. Endotoxin prepara-
tions from a strain of S. schottmiilleri consistently have been
more toxic than those prepared from any other strain of micro-
organism used; 1.0 ml. killed a 20-gram mouse in 5 minutes.
This strain of S. schottmiilleri has been the least virulent used
in this study. Conversely, some of the more virulent strains
used have yielded an endotoxin preparation of low toxicity.
Other strains occupied an intermediate position.

VULNERABILITY OF TISSUES
It has been shown that considerable variation exists in the
susceptibility of chicks to the neurotoxic action of endotoxin
of S. aertrycke. The vulnerability of the tissues of the nervous
system is considered to be of great importance in the develop-
ment of fowl paralysis. It has been shown that day-old chicks
showed a wide variation in their reactions to the neurotoxic
effects of endotoxin. The paralytic symptoms shown by day-old
chicks following the intravenous injection of endotoxin of S.
aertrycke are in many instances similar to the paralytic symp-
toms induced by S. aertrycke when the same chicks were infected
with this micro-organism at 8 to 10 weeks of age. These lat-
ter experiments indicate that not only do birds show individual
variation in the vulnerability of nervous tissue to injury by
endotoxin of S. aertrycke but that in individual birds this vul-
nerability is expressed in widely separated locations in the
nervous system. It is for this latter reason that there is such
a wide range of paralytic or nervous symptoms shown in flocks
in which an outbreak of fowl paralysis occurs.
The observation has been made on the large number of birds
involved in this study that individual birds have shown con-
siderable variation to the development of the early stages of
hemocytoblastosis. It appears that this point no doubt involves
a number of factors, .particularly the susceptibility of phago-
cytes to endotoxin injury and the cellular response of the bird
to the type of infection administered.
After hemocytoblastosis once has become established, indi-
vidual birds show an even greater variation in the subsequent
course of this process.
Thus, in any breeding program designed to increase the re-
sistance of birds to fowl paralysis, leukemia and allied con-






The Etiology of Fowl Paralysis


editions, it is necessary to consider not only the vulnerability of
nervous tissue to injury but also susceptibility to the develop-
ment and subsequent course of hemocytoblastosis. It would
appear that in the case of the development of fowl paralysis
there should be at least some correlation of these 2 factors.

DISCUSSION
The repeated intravenous and intraperitoneal injection of
heat-killed and filtered broth cultures of S. aertrycke indicated
that although the material contained an endotoxin of consider-
able toxicity as demonstrated by mouse-injection and by the
paralytic symptoms produced in chicks in some instances, fowl
paralysis, leukemia or allied condition failed to develop. For
4 to 6 weeks subsequent to the period of injections many of the
birds in these experiments showed a general unthrifty condi-
tion. These symptoms no doubt were the result of the injection
of the by-products of bacterial growth contained in the injected
material, since similar injections of endotoxin alone as well
as nutrient broth alone failed to produce such effects.
It has been shown that endotoxin of S. aertrycke often induced
paralytic symptoms when injected intravenously into day-old
chicks. Many of these symptoms appeared similar to those ob-
served in naturally occurring cases of fowl paralysis; there was
a wide variation in the nervous tissue affected; occasional symp-
toms were spastic. Endotoxin-induced symptoms, however, were
temporary, extending over a period of from 1 to 2 hours. The
intraperitoneal injection of similar amounts of endotoxin often
produced depression but paralytic symptoms were seldom ob-
served. The failure of intraperitoneally-injected chicks to show
paralytic symptoms was regarded as due to slow absorption
of endotoxin from the peritoneal cavity. The repeated intra-
venous injection of endotoxin did not result in the development
of fowl paralysis, leukemia or an allied condition. The develop-
ment of paralytic symptoms in baby chicks as the result of
the intravenous injection of endotoxin demonstrates the neuro-
toxic action of endotoxin of S. aertrycke. The repeated intra-
venous injection of endotoxin induced mild hemocytoblastosis.
Experiments showed that endotoxin has an inhibiting effect
on agglutination and phagocytosis in vitro. This tendency no
doubt calls for a greater cellular response on the part of the
host during the period of infection. Endotoxin also was shown






Florida Agricultural Experiment Station


to be destructive to leucocytes. The addition of endotoxin to
solid and liquid media did not have any apparent effect upon
the growth of S. aertrycke.
In a series of experiments in which birds were given an in-
travenous injection of a suspension of S. aertrycke and killed
at intervals thereafter an effort was made to isolate the micro-
organism from the tissues of the birds. Several hours after
the intravenous injection the micro-organism was isolated us-
ually from the heart, peripheral blood, kidney, lung, liver, spleen,
bone marrow, testes and ovary. In cases in which the dosage
was not too large there was a marked tendency for the micro-
organism to localize in the liver and spleen. In every instance
the micro-organism was isolated from the liver and spleen after
all other tissues were negative. Eventually all tissues became
negative. The findings of Schalm (24) that S. typhimurium
could be isolated from the liver and spleen of injected birds for
a period of 125 days were not substantiated in this study deal-
ing with S. aertrycke. These experiments indicated that the
blood of the chicken has a remarkable ability for clearing itself
of S. aertrycke. This point was observed by Kolmer (17) in
dealing with other micro-organisms.
Reinholt (21) observed that S. enteritidis could be isolated
from the tissues of the chicken when a fatal disease was induced
by intravenous, intraperitoneal or intramuscular injection of
the micro-organism; the micro-organism was never recovered
when the bird did not die as a result of infection. Mitchell
and Bloomer (18) administered 2 ml. of a suspension of Eber-
thella typhi intravenously in 2 doses 12 days apart, the birds be-
ing killed 48 hours after the last injection; the liver, gallbladder,
cystic duct, hepatic duct, gizzard, spleen and kidney did not yield
a culture of the injected micro-organism.
The repeated injection of large numbers of desiccated heat-
killed S. aertrycke resulted in but few cases of fowl paralysis
but in a greater number of other manifestations than in the
case of the injection of the living micro-organism. This would
indicate that virulence of the micro-organism is an important
factor in the development of fowl paralysis. In 1 group of ex-
periments it was found that the repeated intravenous injection
of S. aertrycke plus endotoxin, as compared with the injection
of the same amounts of this micro-organism alone, resulted in
the development of a greater number of cases of fowl paralysis.
These results indicate that endotoxin also is an important factor







The Etiology of Fowl Paralysis


in the development of fowl paralysis. Therefore, the conclusion
is reached that the virulence of the micro-organism and the
potency of endotoxin are 2 factors which are important in the
development of fowl paralysis. For the maximum number of
cases of fowl paralysis to develop it is essential that a micro-
organism of high virulence possessing a potent endotoxin be con-
cerned, providing the factors surrounding infection (a), re-
sistance of the bird, (b), the number of micro-organisms, their
rate of entrance and the period over which they gain access
to the blood stream, and (c) the vulnerability of the birds tissues
to injury are favorable to the development of this manifestation.
Another group of experiments indicated that the temporary
but positive reaction noted in day-old chicks following the in-
travenous injection of endotoxin can be used as an index of sus-
ceptibility to fowl paralysis. Subsequent exposure to S aer-
trycke when the chicks were 8 to 12 weeks of age resulted in
fowl paralysis in 86.7 percent of the endotoxin-susceptible
chicks. Many of these birds developing fowl paralysis exhibited
identical symptoms with those occurring as a result of the
injection of endotoxin. From an experimental point of view
this is considered an important observation, since the vulner-
ability of the nervous system to the neurotoxic effects of endo-
toxin may be determined to a relatively accurate degree in baby
chicks. This observation could be found of practical use in an
experimental breeding program dealing with susceptibility and
resistance to fowl paralysis.

CONCLUSIONS

The Initiation of Hemocytoblastosis.-The experiments re-
ported herein give important indications as to how S. aertrycke
initiates hemocytoblastosis in the chicken.
According to experiments previously reported, the first pre-
requisite is that S. aertrycke must establish itself in the intes-
tinal tract of the bird. Under natural conditions of infection
it appears that intestinal parasites-common roundworms, tape-
worms, capillaria and coccidia-play an important role in creat-
ing an environment favorable for such a situation. Experiments
have been reported which show that the oral exposure of para-
sitized birds-both artificially and experimentally infested-to
S. aertrycke resulted in the development of manifestations of
the leucosis complex while similar exposure of non-parasitized







42 Florida Agricultural Experiment Station

birds yielded negative results. Experiments also have been re-
ported which show that S. aertrycke, as well as other members
of the paratyphoid group credited with being capable of initiat-
ing processes resulting in leucosis, often constitutes a major
percentage of the intestinal bacterial flora in birds infested
with intestinal parasites. It is plainly evident from these
experiments that the micro-organism must become established
in the intestinal tract and multiply there. The severity and
duration of the inflammation of the intestinal mucosa resulting
primarily from parasitism and probably to a much lesser extent
from the micro-organism determine in a large measure the
number of micro-organisms, their rate of entrance and the
period over which they gain access to the blood stream of the
bird from the intestinal tract. Experiments have been reported
in which the intravenous injection of S. aertrycke in varying
numbers, at different rates and over varying periods of time
clearly demonstrate the importance of these factors.
The experiments reported herein indicate that when the
micro-organisms enter the blood stream they are phagocytized
as rapidly as the defense of the bird permits. It has been
stressed a number of times that the entrance of these micro-
organisms must take place at a rate insufficient to induce sep-
ticemia. The localization of the micro-organism in the spleen
and liver indicates that these organs together with the blood
cells receive the brunt of the injury accomplished at this time.
During the period of infection, which previous experiments have
shown must exist over an extended time, the concentration of
the micro-organisms in the liver and spleen necessitates the
phagocytic defense to be localized in these organs, since the
number of phagocytes in the peripheral circulation was greatly
decreased and smears from these organs showed large numbers
of phagocytes. The observation of degenerative changes in the
phagocytes in the liver and spleen within 6 hours after the in-
travenous injection of S. aertrycke is an indication of the rapid
action of endotoxin which has been. shown to produce similar
changes in vitro. It seems likely that in some instances some
multiplication of the micro-organisms may occur in these organs
during the period of infection. However, it appears that this
tendency cannot be marked, as reinfection under natural con-
ditions takes place from the intestinal tract and septicemia does
not develop in many cases. If marked multiplication occurred







The Etiology of Fowl Paralysis


in the liver and spleen a greater number of cases of septicemia
should occur under conditions of natural infection.
When the micro-organisms are phagocytized endotoxin is lib-
erated from the bacterial cell. When the accumulation of endo-
toxin within the phagocyte becomes sufficient the phagocyte is
destroyed. Degenerative and disintegrating phagocytes have
been observed in the blood during the period of infection. The
experiments reported herein demonstrate these degenerative
changes in smears from the liver and spleen after intravenous
injection of the micro-organism as well as in vitro when blood
cells are brought in contact with endotoxin during a 30-minute
period in a water bath. Previous experiments have shown that
it is necessary to administer repeated intravenous injections of
S. aertrycke to result in the development of fowl paralysis, leu-
kemia or an allied condition. Infection must extend over a per-
iod of time. During this period phagocytes as well as many
other blood cells constantly are being destroyed.
The destruction of cells in the circulating blood necessitates
the production of new ones. The hematopoietic system responds
with the production of young blood cells. The demand for addi-
tional cells often is so great that young cells do not have time
to mature fully and as a result immature blood cells gain ac-
cess to the peripheral circulation. Thus, varying percentages of
degenerative, mature and immature blood cells are found in the
blood stream, constituting a blood picture of hemocytoblastosis
according to the conception of this author.
The original impetus given hemocytoblastosis during the per-
iod of infection is important insofar as the development of
subsequent pathologic manifestations is concerned, as the pro-
cess of hemocytoblastosis is a basic condition which leads to the
development of most of these manifestations. These manifes-
tations cannot develop without the basic process of hemocyto-
blastosis. A number of factors have been enumerated previously
in this manuscript which have a direct relation to the develop-
ment of hemocytoblastosis: (a) the resistance of the bird; (b)
the severity and duration of enteritis associated with intestinal
parasitism; (c) the number of micro-organisms, their rate of
entrance and the period over which they gain access to the
blood stream; (d) virulence of the infecting micro-organism and
potency of the endotoxin; and (e) vulnerability of the bird's tis-
sues to injury. All are concerned directly in determining the
degree of hemocytoblastosis which results from infection.







Florida Agricultural Experiment Station


The interrelationship of these factors in flocks and even in
individuals within flocks varies within wide limits. Conse-
quently, in given flocks birds may develop all degrees of hemo-
cytoblastosis. Since the original impetus given hemocytoblasto-
sis as concerned with these factors is important in determin-
ing, to some extent, the future course of hemocytoblastosis after
infection has ceased, these factors which are important during
infection have a definite bearing on the development of other
specific pathologic manifestations.
During the period of infection the destruction of cells gives
rise to other factors which tend to further the process of hemo-
cytoblastosis. These factors are important particularly in the
development of the various leukemias in which conditions de-
velop which allow hemocytoblastosis to become a perpetual pro-
cess with no extraneous stimulation. This mechanism will be
discussed in a subsequent publication.
Development of Fowl Paralysis (Neurolymphomatosis gallin-
arum).-The experiments which have been conducted to date
clearly indicate the mechanism by which fowl paralysis is pro-
duced. The basic process of hemocytoblastosis is initiated as
explained in the preceding discussion. It has been shown that
endotoxin of S. aertrycke is neurotoxic when injected into young
chicks and that the symptoms produced, while transient, closely
approximate those observed in naturally occurring outbreaks in
the field. During the period of infection endotoxin which is
being released not only is destructive to blood cells but also at-
tacks the nervous system, presumably at its weakest point. If
this injury becomes sufficiently severe paralysis of the affected
nerve results. Hemocytoblastosis and the neurotoxic action of
endotoxin on the nervous system proceed simultaneously. When
the incubation period is long enough pathologic cells in the blood
stream, by reason of hemocytoblastosis, are deposited in injured
nervous tissue, resulting in typical nerve enlargements frequently
observed in birds affected with fowl paralysis. Fowl paralysis
occurs most frequently in young flocks, as such flocks are more
susceptible than old ones to nerve injury by endotoxin as well
as to the initiation of hemocytoblastosis.
It should be mentioned that previous reports have shown that
S. aertrycke has been found to be associated with naturally
occurring outbreaks of fowl paralysis in a greater percentage
of instances than other members of the paratyphoid group.
However, certain other micro-organisms of this group have been


44 ,







The Etiology of Fowl Paralysis


incriminated in this study and all contain an endotoxin which is
neurotoxic to the chicken. It is certain that still other members
of this group of bacteria also are capable of inducing fowl
paralysis under the' conditions set forth in this study.
The experiments reported herein indicate that virulence of
the offending micro-organism and the potency of its endotoxin
are important in the development of fowl paralysis and that
there need not necessarily be any relationship between these 2
factors in the same strain of micro-organism.

SUMMARY
Single and repeated intraperitoneal and intravenous injec-
tions of heat-killed or filtered broth cultures of Salmonella aer-
trycke into birds 6 weeks of age did not result in the develop-
ment of any of the pathologic manifestations.
Endotoxin of S. aertrycke when injected intravenously into
day-old chicks induced paralytic symptoms similar to those
observed in naturally occurring cases of fowl paralysis and to
those induced by S. aertrycke in this study. Endotoxin is neuro-
toxic for the chicken. Recovery from endotoxin-induced para-
lytic symptoms occurred within several hours.
Single and repeated intravenous, intraperitoneal or intra-
cerebral injections of endotoxin into day-old chicks or older
birds did not result in the development of fowl paralysis nor
any of the other pathologic manifestations.
The endotoxin of S. aertrycke tends to inhibit agglutination
and phagocytosis in vitro but did not have any apparent effect
upon the growth and morphology of S. aertrycke when incor-
porated in liquid and solid media up to 50 percent in volume.
Endotoxin was destructive to blood cells in vivo as in vitro.
The intravenous injection of S. aertrycke alone into 6 to 12-
week-old chickens of 3 breeds resulted in 22 of 108 birds de-
veloping fowl paralysis. In experiments conducted on a com-
parative basis the intravenous injection of a similar number of
S. aertrycke plus 1.0 ml. of endotoxin resulted in 53 of 105 birds
developing fowl paralysis.
In a series of 6 experiments 158 of 330 day-old chicks of 3
breeds developed paralytic symptoms when injected with definite
amounts of endotoxin of S. aertrycke. When these same chicks
were 8 to 12 weeks of age the intravenous exposure to S. aer-
trycke resulted in the development of 137 cases of fowl paralysis
in the endotoxin-susceptible chicks and 11 cases in the endo-







Florida Agricultural Experiment Station


toxin-resistant chicks. In the same group of experiments 3
endotoxin-susceptible chicks and 2 endotoxin-resistant chicks
developed lymphomatosis. In many instances the symptoms of
paralysis resulting from the injection of the micro-organism
were identical with the paralytic symptoms resulting from the
injection of endotoxin into the day-old chicks.
There was no definite correlation between virulence of S.
aertrycke and potency of its endotoxin in specific strains and
both factors decreased when cultures were kept "in stock" on
the usual laboratory media.
Strains of S. aertrycke isolated from the intestinal tract of
naturally occurring cases of fowl paralysis, septicemia in baby
chicks, septicemia in turkeys, arthritis in pigeons and from man
were all capable of inducing the various pathologic manifesta-
tions under consideration in this study.
In a series of experiments in which birds were injected intra-
venously with S. aertrycke and killed at short intervals there-
after it was found that the micro-organism could be isolated
in large numbers from the liver and spleen after attempts to
isolate it from other organs failed. The micro-organisms were
finally phagocytized, disappearing from the liver and spleen.
The repeated intraperitoneal or intravenous injection of heat-
killed (1000 C. for 10 minutes) suspensions of S. aertrycke into
80 birds 6 to 10 weeks of age and of 3 breeds resulted in 4
cases of fowl paralysis, 21 cases of lymphomatosis, 6 cases
of leukemic lymphocytoma and 1 case of leukemia. In 40 older
birds in which exposure was similar but extended over a longer
period of time leukemia developed in 12 birds, lymphomatosis
and leukemic lymphocytoma each in'2 birds, while fowl paraly-
sis developed in but 1 bird.
The conclusion is reached from these and previous experi-
ments that when birds are reared on soil Salmonella aertrycke
can become established in the intestinal tract during or follow-
ing intestinal parasitism with the subsequent result that these
micro-organisms gain entrance to the blood stream, where de-
fensive blood cells attempt to destroy them. Phagocytes, and
in many instances other cells, are destroyed by accumulation
of endotoxin. The body responds to the destruction of blood
cells by the production of new ones, many of which enter the
peripheral circulation before full maturity. Thus, hemocyto-
blastosis is initiated with a blood picture of varying percent-








The Etiology of Fowl Paralysis


ages of degenerative, mature and immature blood cells in the
peripheral circulation.
The development of fowl paralysis is based upon nerve injury
by the neurotoxic action of endotoxin during infection and the
subsequent' deposition in many instances of pathologic cells,
present in the blood stream by reason of hemocytoblastosis, in
the injured nervous tissue, resulting in typical nerve enlarge-
ment frequently observed in birds affected with fowl paralysis.

LITERATURE CITED
1. BEAUDETTE, F. R. New Brunswick, N.J. Personal communication.
April 4, 1935.
2. DOUGLAS, S. R. On the characters of the cleavage products of certain
bacteria with special reference to their toxicity and antigenic
properties. Brit. Jour. Exp. Path. 2: 175. 1921.
3. DOYLE, T. M. S. aertrycke infection of chicks. Jour. Comp. Path.
and Ther. 40: 71. 1927.
4. DUDGEON, LEONARD S., P. N. PANTON and H. A. F. WILSON. The
influence of bacterial endotoxins on phagocytosis, including a new
method for the differentiation of bacteria. Proc. Roy. Soc. Lond.
Ser. B. 83: 33. 1910.
5. The influence of bacterial endotoxins on phagocytosis.
Proc. Roy. Soc. Lond. Ser. B. 82: 406. 1910.
6. EDWARDS, PHILIP R. A fatal infection of chicks due to bacilli of the
paratyphoid B group. Jour. Inf. Dis. 45: 191. 1929.

7. EMMEL, M. W. Hemocytoblastosis and its relation to the development
of fowl paralysis. Jour. Amer. Vet. Med. Asso. 88: 45. 1936.
8. The importance of endotoxin of Salmonella aertrycke
in the development of fowl paralysis. Jour. Amer. Vet. Med
Asso. 90: 627. 1937.
9. The etiology of fowl paralysis, leukemia and allied
conditions in animals. I. Introduction. History and a bacterial
theory of the etiology of these diseases. II. The intravenous injec-
tion of suspensions of Salmonella aertrycke in the chicken. Fla.
Agr. Exp. Sta. Bul. 284. 1935.
10. The etiology of fowl paralysis, leukemia and allied
conditions in animals. III. The intestinal flora of chickens affected
with enteritis associated with intestinal parasitism. IV. The patho-
logic manifestations of the causal micro-organism in the fowl. Fla.
Agr. Exp. Sta. Bul. 293. 1936.
11. The etiology of fowl paralysis, leukemia and allied
conditions in animals. V. The oral exposure of chickens infected








Florida Agricultural Experiment Station


with various species of Eimeria (coccidia) to Salmonella aertrycke.
VI. The oral exposure of chickens infested with Ascaridia, Taenia
and Capillaria to Salmonella aertrycke. Fla. Agr. Exp. Sta.
Bul. 305. 1936.

12. The etiology of fowl paralysis, leukemia and allied
conditions in animals. VII. Hemocytoblastosis in chickens as
induced by Salmonella aertrycke. VIII. Hemocytoblastosis in
naturally occurring cases of fowl paralysis and lymphomatosis, as
well as in other birds from the same flocks. Fla. Agr. Exp. Sta.
Bul. 306. 1936.

13. The etiology of fowl paralysis, leukemia and allied
conditions in animals. IX. Salmonella enteritidis as a causal micro-
organism for this group of diseases in the chicken. X. Salmonella
schotmiilleri as a causal micro-organism for this group of diseases
in the chicken. Fla. Agr. Exp. Sta. Bul. 313. 1937.

14. HANKS, JOHN H., and LEO F. RETTGER, Bacterial endotoxin; search
for a specific intracellular toxin in S. pullorum. Jour. Immunol.
22: 283. 1932.

15. JOHNSON, E. P. Blacksburg, Va. Personal communication. July
30, 1935.
16. The etiology and histogenesis of leucosis and lym-
phomatosis of fowls. Vir. Agr. Exp. Sta. Tech. Bul. 56. 1934.

17. KOLMER, JOHN A. Etiology, prophylaxis and treatment of surgical
septicemia. 'Arch. Otolaryng. 25: 59. 1937.

18. MITCHELL, O. W. H., and G. T. BLOOMER. Experimental study of the
chicken as a possible typhoid carrier. Jour.-Med. Res. 26: 247. 1914.
19. PAPPENHEIMER, A. M. New York, N.Y. Personal communication.
Oct. 23, 1930.

20. PAPPENHEIMER, A. M., L. C. DUNN and S. M. SEIDLIN. Studies on
fowl paralysis (Neurolymphomatosis gallinarum). II. Transmis-
sion Experiments. Jour. Exp. Med. 49: 87. 1929.

21. REINHOLT. Infektionsversuche mit den "Fleishvergiftern" beim Ge-
flugel. Centbl. f. Bakt. 1. Abt. orig. 62: 312. 1912.

22. RETTGER, L. F., WAYNE N. PLASTRIDGE and RUTH CAMERON. Endemic
paratyphoid infection in turkeys. Jour. Inf. Dis. 52: 272. 1933.

23. RIVERS, THOMAS M. Filtrable viruses. The Williams and Wilkins
Co. 1928.

24. SCHALM, O. W. Study of a paratyphoid infection in chicks. Jour.
Inf. Dis. 61: 208. 1937.

25. WITTENBURG, JOSEPH, MAX LEDERER and MOLLIE MOLLOV. Studies in
phagocytosis. I. Effect of endotoxin on phagocytosis of gonococci.
Jour. Immunol. 24: 135. 1933.




University of Florida Home Page
© 2004 - 2010 University of Florida George A. Smathers Libraries.
All rights reserved.

Acceptable Use, Copyright, and Disclaimer Statement
Last updated October 10, 2010 - - mvs