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thesis entitled

"A Partial and Comparative Evaluation in Nice
of an Aluminum Phosphate Adsorhed Anti-Swine
Erysipelas Bacterin Prepared by the Sonic
Pacterioclasis of Erxsipelothrix RhusiOpathiae"

 

presented by

Loyd w. Tiffany

has been accepted towards fulfillment
of the requirements for

ML.— degree in Mica

A/F/C‘ézA/wx:

/Major professor

Date October 31, 1955

 

0-169

A PARTIAL AND COMPARATIVE EVALUATION IN MIC! 01" AN ALUMINUM
PHOSPHATE ADSORBED ANTI-SX‘FINE ERYSI‘PELAS BACTERIN PREPARED
BY THE SONIC BACTERIOCLASIS OF ERYSIPELOTHRIX BITUSIOPATHIAE

Loyd layne Tiffany

A THESIS
Submitted to the School of Advanced Graduate Studies of Michigan
State University of Agriculture and Applied Science
in Partial Fulfillment of the Requirements
for the Degree of

DOCTOR OF PHILOSOPHY

Department! of Microbiology and Public Health

1955

ACKNOWLEDGEMENT

The author wishes sincerely to acknowledge the direction, counsel,
and assistance affbrded to this work and person by Dr. J.J. Stockton.
Further, the author wishes to express his gratitude to Dr. R.D.
Earner for his interest in, and support of, this problem. Assist-
ance rendered by Mr. W. Gebhard and Dr. R. Dresene of the Michigan
Department of Health also commands a large portion of the author's
recognition.

In all sincerity, acknowledgement is to be made to the many others
who have aided the author in lesser roles, and who, though anonymous
in this acknowledgement, merit and receive the author's most candid

appreciation.

Loyd Wayne Tiffany

AN ABSTRACT
of

A Partial and Comparative Evaluation in Mice of an Aluminum

Phosphate Adsorbed Anti-Swine Erysipelas Bacterin Prepared

by the Sonic Bacterioclasis of Erysipelothrix rhusiopathiae.

Attention was directed to an apparent increase in swine erysipelas
and to the economic loss which accompanied this infection. The need of
more acceptable prophylactic and therapeutic measures for controlling
this disease was stressed. Procedures for challenging eXperimental
animals and methods of prophylaxis available at the present time have
been discussed.

The object of this problem was the production and evaluation of a
bacterin which consisted of formalin killed, sonically disrupted cells
of Erysipfilothrix rhusiopathiae adsorbed onto the surface of aluminum
phosphate particles.

Three strains of E, rhusiopathiae were selected for the production

 

of bacterins. The cells were killed by the addition of 0.5 per cent
formalin to the cultures and were separated from the medium by centrifuga-
tion. These cells were then disrupted by sonic energy and adsorbed onto
aluminum phosphate. The medium used for cultivation of the cells was also
adsorbed with aluminum phosphate and the adsorbate products combined with
the cell adsorbate. White mice were used to test these products against
a challenge of undiluted virulent culture and against a challenge of
500LD5Q.

Strain 14774 did not protect any of the test animals and strains
SE-9 and SE-25 protected 100 per cent of the animals, injected with the

undiluted bacterin, against the challenge of undiluted culture. Against

Loyd Wayne Tiffany

a challenge of §OOLD5O, bacterin SE-9 protected 100 per cent of the
animals in a dilution of 1-4 and SE-25 protected 100 per cent of the
animals in a dilution of 1-2 against a similar challenge.

A commercial bacterin was evaluated for the purpose of comparison
and proved to offer greater protection than either of the experimental
products. It protected 100 per cent of the animals in a dilution of 1—2
against a challenge with undiluted culture and 100 per cent of the animals
were protected against a challenge of 500LD5O in a dilution of 1-8 of
the commercial bacterin.

Considering the variables present, for which more refined evaluation
is indicated, the most important development of this work was the promise

which this procedure offers for the deveIOpment of an acceptable bacterin.

ii

TABLE OF CONTEHTS

Page
Acknowledgement

Abstract

Introduction ................................................... 1

Review of Literature ........................................... A
Review of Swine Erysipelas Vaccines ....................... 5
Review of Bacterioclastic Action of Sound Waves ........... 1#

methods and Procedures ......................................... l6
Brief’History of Cultures ................................. 16
Preparation of Medium ..................................... 17
Preparation of Seed Cultures and Seeding of Medium ........ 17
Sonic Disruption of Cells ................................. 18
Preparation of Aluminum Phosphate ......................... 19
Description of Animals Employed ........................... 20
Determination of LD5o ..................................... 20
Administration of Bacterins ............................... 21
Challenge of vaccinated Animals ........................... 22
Evaluation of Commercial Bacterin ......................... 22

Results and Discussion ......................................... 25

Summary ........................................................ 34

Literature Cited 0.0...OO...OOOOOOOOOOOOOOOOOOOOOOO0.00.00.00.00 55

Introduction

Since the beginning of organized research relative to swine
erysipelas (about 1880), a considerable amount of knowledge has
been accummulated regarding the nature and control of the causa-

tive organism, Erysipelothrix rhusiopathiae. However, though

 

many important characteristics of this organism and its associated
disease have been clearly elucidated, a satisfactory understanding
of the facts sufficient for the establishment of an adequate con-
trol program remains, as yet, obscure.

Kaplan (l9h8) has estimated that the economic loss due to
swine erysipelas exceeds that of hog cholera in Eur0pe, and Ray
(1952) has reported it to be the second most prevalent disease of
swine in the United States. Ray (1952) reported that in 1951 this
disease was prevalent in 22 states and that 17 states reported an
increase in the range of occurrence of this infection. COOper
(1954) has reported that the prevalence of infection in turkeys
has increased to epizootic proportions in several states of the
western United States, and reports by Marsh (1951), Breed (1958),
and Ray (1950), attest the losses inflicted on the sheep industry
by this disease entity.

Also to be acknowledged are reports of vaccine erysipelas,
Fluckiger (1949, 1952, 1952); i.e. cases of this disease which
occur after vaccination with live culture and antiserum and which
are probably a result of the inoculation.

The actual incidence of swine erysipelas in Michigan is
thought to be very low, but evidence is accummulating which sug-

gests an increase at least in the number of recognized cases of

- 1 -

swine erysipelas infection within this state Veterinary Reporting
Service (195A). For the most part, the chronic form of this disease
has been predominant in Michigan; however, during the past year
several acute cases have been observed of which a number have re-
sulted in death losses.

Several methods of control have been developed which have been
very promising in their early evaluations. The simultaneous method
of administration of antiserum and live culture, the inception of
the use of formalin killed, aluminum hydroxide adsorbed bacterin
and recently, the lysebacterin, the use of antibiotics both separ-
ately and mixed with antiserum, and vaccines consisting of avirulent
strains of g, rhusiopathiae have been developed. Each has been ac-
companied by rather disappointing limitations which were revealed by
repeated field and laboratory applications of these products.

Continued, and perhaps increased, economic losses due to swine
erysipelas serve as a stimulus to more intensive investigational
efforts regarding control and prevention of this infection. Concern
is justly directed to the lack of satisfactory control measures and
the continued appearance of the infection in the animal and bird
populations. The continued demand on the swine, sheep, and turkey
industries for human sustenance in itself emphasizes the pressing
need for more acceptable methods of understanding and control.

The present investigation, of which this thesis is a report,
was undertaken in order to determine the value, in mice, of a bac-
terin prepared by the sonic disruption of g, rhusiOpathiae for
protection against a challenge dose of this same organism. A numr

ber of techniques and numerous bits of information have been adopted

from the available literature and utilized in the production of
the exPerimental bacterins and, though the nature of the problem
invites a more prolonged investigation, the limitations of time
and economics permit only a brief examination of this one greatly

delimited phase.

Review of the Literature

Prior to the great swine plague, the abstruse nature of swine
erysipelas is evidenced by the numerous colloquial synonyms which
were applied to this infection. Swine typhus, wild fire, the rose,
St. Anthony's fire, spotted fever and spotted fire were some of the
commoner terms used to describe a syndrome, the chief characteristic
of which was an erythematous condition of the skin. No doubt, the
nature of the symptoms was the great motivating force which resulted
- in the lumping together of several distinct diseases under one or
more of the mentioned terms. The term "swine erysipelas" was no
exception to this practice since it was also applied at times to dis-
eases which were accompanied by redness of the skin. However, a rather
inclusive examination of the old literature by Friedberger and Froh-
ner (1910) revealed that the term “swine erysipelas' usually referred
to the disease, swine erysipelas, as we recognize it today. This
would indicate that swine erysipelas as such has been a more or less
covert disease in Europe for many centuries.

The great swine plague of hog cholera which in 1877-78 swept
the United States, England, the Scandanavian countries and most of
Europe, probably provided an impetus to the early investigation of
swine diseases.

Subsequently, Robert Koch (1880) isolated an organism from a
mouse which he had previously injected with a quantity of putrefying
blood. He named this organism.Erysipelothrix muriseptica. However,
Koch was not known to have associated this organism with swine
erysipelas.

Following Koch's work, Thuillier in 1882, Pasteur and Thuillier

-4-

(1885), isolated an organism from pigs dying of rouget. This or-
ganism was poorly described by Pasteur and Dumas (1885).

About four months after Thuillier's observation, Loeffler
(1885) cultured an organism from the blood vessels of the skin of
a pig which had died of swine erysipelas. He gave a more accurate
and inclusive description of the etiologic agent and the clinical
manifestations of swine erysipelas than did his contemporaries, and
though he did not publish his findings until 1885, received the cre-
dit for describing this organism for the first time and associating
it with swine erysipelas.

Pasteur and Thuillier (1885) observed the existence of an in-
verse relationship between the virulence of the swine erysipelas
bacillus in rabbits and pigs. As the organism increased in virulence
for the rabbit, it decreased in virulence for the pig. This observa-
tion was the basis for the first vaccine against swine erysipelas.
The modified attenuated culture was administered to pigs and followed
twelve days later by an injection of a culture which had been en-
hanced in virulence by passage through pigeons. Since this was the
only prophylactic procedure available at that time, it was adopted
and used rather extensively in Europe. Forthcoming from the prac-
tical evaluations of this vaccine, were numerous objections based,
in part, on the anxiety created by the use of live culture as a
vaccine. The period of actual protection was questionable and a
suitable means of challenging experimental animals was not at their
disposal. Thus the search for a more acceptable control agent con-
tinued.

A second phase of the historical investigation of swine erysi-

pelss control agents began with the observations of Emmerich and
Mastbaum (1891), who demonstrated the protection afforded by the

blood serum of rabbits which had been immunized against the swine
erysipelas bacillus. Subsequently, Lorenz, (1895, 1894, 1896) Le-
clainche (1897) and Schutz and Voges (1898) began to eXperiment

using culture and antiserum and in 1896, Lorenz published a descrip-
tion of a “Simultaneous Method" of immunization against swine ery-
sipelas. This method consisted of the injection of live virulent
culture simultaneously with a specific antiserum. This procedure

was supplemented by a second injection of virulent culture only,

about 1% days following the primary injection if a more prolonged
immunity was desired. Leclainche (1897) also offered a method which
was very similar to that proposed by Lorenz. The methods differ in
that the Leclainche procedure requires the vaccine and antiserum to

be mixed prior to injection, and followed in 10-12 days by a second
administration of vaccine alone. The Lorenz method necessitated the
injection of antiserum and culture simultaneously into different parts
of the body. The “simultaneous method" of immunization against swine
erysipelas has undergone numerous field and laboratory evaluations and
though its use is still limited in the United States and certain other
countries, remains one of the most effective control procedures avail-
able at the present time. For the most part, the limitations imposed
on this technique are based on an anxiety created by the necessity of
using live culture to combat the infection, since this involves the
possible spread of the disease and poses a potential hazard to the
person administering the vaccine. Further, the economics involved

in the production of the antiserum calls fbr a more desirable method.

Live culture conferring only a relatively short and uncertain
duration of protection, plus the economics involved in the pro-
duction of antisera, are the main objections to the all-out accept-
ance of this technique. However, these objections of themselves serve
to emphasize the need of better control products. The existent anxiety
may, in part, be unjust, in that too much may be eXpected from an
otherwise effective control product. This product, if properly uti-
lized, is a powerful supplement to the present poorly-stocked arsenal
of control procedures. In areas of known soil contamination e.g.,

one could hardly prohibit the use of this vaccine technique since it
is one of the most successful methods at one's present disposal.
However, in areas where there has been little history of the disease,
one would be justified in exercising caution in introducing such a
procedure.

Muromcev and Mhtvyenko (1954), in attempting to evaluate the
effects created by the changes in density, surface tension, and os-
motic pressure, brought about by the addition of varying amounts of
agar to liquid medium, felt that this addition of agar created an
environment i§.zit£g_which more closely approximated that present in
the animal body than did a solid or broth medium. They found that g,

rhusiOpathiae grew very well in their "agaro-bullion' and thus they

 

observed a basis for the production of a vaccine against swine
erysipelas. Probably attempting to circumvent the objections to the
use of live culture vaccines, they produced a killed bacterin through
the addition of 0.5 per cent formalin to the cultures of g, ghggigr
pathiae. The promise of such an agent was quickly recognized and

after preliminary investigations of the vaccine in 1954 and 1955,

large scale inoculations of the Muromcev vaccine were carried out

in 1956 and 1958 (Jastrzebski 1945). Again, a means of challenge

of swine, supposedly protected by this vaccine, was not available

and results were confined to field observations of the incidence of
infection. Also, control animals were few and not necessarily lo-
cated in the same herd for which they were to be the standard.

Though much promise was evidenced from these observations, the lack
of the original Russian literature and the existent world conditions
at that time necessitated a further evaluation, which was accomp-
lished with the aid of a more recent technique. This vaccine was
not abandoned, however, as Kaplan (1948) reported on extensive trials
of this vaccine in Poland. Some aspects of the procedure (preparation
of the vaccine) have doubtless influenced other more recent develop-
ments in the field of erysipelas prophylaxis.

Honda and Sugimura (1955) attempted to produce a vaccine against
swine erysipelas by culturing the organism in increasing amounts of
trypaflavin and, though the organisms lost their pathogenicity for
mice, they regained it after culturing on agar. This was a step to-
ward the selection of a culture of very low or lacking in virulence
for swine.

Staub (1940) isolated a strain of E, rhusippathiae which ap-
peared to be an avirulent mutant. This strain was used quite ex-
tensively in France and in the French colonies as well as in Poland,
and, though some authors reported success, there were not enough
reports of experiments with controls to prove the value of this
vaccine. Staub maintains that the strain is practically avirulent

for mice but Gladhill (1949) was able to demonstrate virulence for

mice after several passages in serum broth.

Fortner (1944) provided a significant advance toward the eval-
uation of the level of immunity induced by various vaccine prepara-
tions through the introduction of the “Scarification Technique” for
challenge. Prior to the inception of this technique, the knowledge
of levels of immunity attained by vaccines was based primarily on
gross field and laboratory observations of control and immunized ani-
mals. No satisfactory means of determining the degree of protection
conferred by various vaccine products or the susceptability of unex-
posed animals existed. The advent of this technique was timely and
welcome. Fortner (1946, 1947) was able to demonstrate that little or
no immunity was produced in response to the formalin killed vaccine
of Muromcev (1954). Since this method constitutes a severe challenge
and necessitates the implication of a number of animals, (see descrip-
tion of method below) it stands as the most acceptable means of eval-
uation of immunity level available today.

This method consisted of the production of parallel scarifica-
tions on the lateral surface of the thorax and abdomen of a number
of swine (Fortner used eight swine) not deep enough to induce hem-
orrhage, but of sufficient depth as to yield an effusion of tissue
fluid. A culture to be tested was then rubbed into one line on each
of the pigs and the response noted. The response may be local edema
along the line of scarification, accompanied by a rise in tempera-
ture, or a generalized septicemia or chronic arthritis may result.

Since this method of Fortner did not lend itself to the pre-
ciseness desirable in laboratory procedures, Hare and Delpy (1955)

developed a method which consisted of the titration of strains with

antiserum in the pig skin by means of intradermal inoculations.
Hars's and Delpy‘s experiments demonstrate a significant value for
this test though it does involve the use of antiserum which is of
economic importance.

Following the introduction of the scarification technique de-
veloped by Fortner (1944), a means was then available for the eval-
uation of existing and subsequent vaccine and antibiotic preparations.

Traub (1947), Dinter (1948-1949), and Manninger (1951), working
more or less independently and with different objects in mind, evolved
a vaccine which offered a good immunizing potential and was safe in
that it was a killed preparation. Preliminary investigations re-
vealed that only a small number of strains of E, rhusiopathiae were
capable of inducing immunity in host animals, but that these strains
induced a good immunity against all strains of E, rhusiqpathiae.
Dedie (1949) was able to classify the strains of this organism into
groups on the basis of their acid soluble antigens and was able to
distinguish three such groups which he termed A, B, and N. He also
noted that group B strains, which were the good immunizers, composed
about 10 per cent of all the strains, and that groups A and N which,
though they contained very virulent strains, induced little or no
immunity. Dinter and Bakes (1947, 1948, 1949) observed that about
10 per cent of all strains possessed the capacity to agglutinate
chicken erythrocytes. It thus appeared that the group B antigen,
the immunizing antigen, and the haemagglutinating antigen were all
present in the good immunizing strains, though separate from each
other.

Traub (1947) selected good immunizing strains by exposing ani-

mals to selected strains and challenging them by the method developed

- 10 -

by Fortner (1944), which he cultured in liquid medium and killed
with formalin. He then added aluminum.hydroxide to the medium with
the object of adsorbing the "specific immunizing substances" (3.1.5.)
which were elaborated into the medium. This aluminum hydroxide ad-
sorbed, formalin killed vaccine, produced from strains selected by
Partner's method of scarification, has several advantages. It is a
killed preparation, good immunizing strains are present, it is con-
centrated, it is less eXpensive than the simultaneous serum culture
method, and is, because of its nature, slowly adsorbed by the tissues,
thus providing a longer period of antigenic stimulation. Numerous
favorable reports have followed its application. Mass (1948) re-
ported that the aluminum hydroxide formalin killed vaccine was 64
times more effective than the Muromcev vaccine. Possibly this can

be explained in that the Muromcev vaccine consisted of a pool of
many strains thus including poor immunizers with the good, and at

the same time, diluting out any soluble antigens in the medium to the
point of little value. Partner (1947) was able to demonstrate good
protection afforded by the Traub vaccine, using his own scarification
procedure as a challenge method. Bergman (1949) also reported fa-
vorable results and demonstrated a greater potential than with the
simultaneous method of Lorenz. Shuman (1952) of the United States
Bureau of Animal Industries performed a number of experiments de-
signed to test and evaluate the adsorbed vaccine and concluded that
though his results could not be justly compared with those obtained
in Europe, this vaccine was of definite value in protecting animals
against swine erysipelas.

Manninger (1951) in Hungary deve10ped a product very similar

- 11 -

to that produced by Traub (1947), but was working with an entirely
different object in mind. Manninger was interested in knowing if
the change from smooth to rough phase was accompanied by a permanent

loss of virulence. Since E, rhusiopathiae shows quite a tendency toward

 

dissociation, this organism was selected for study. He learned that
Schoenig, Gochenour and Grey ( 1958) had immunized mice with rough
strains and this motivated him to evaluate his own rough strains for
their immunizing capacity. He found a few of his rough strains were
suitable immunizers, and also that upon injecting as much as 50 m1
of these organisms, there would be no subsequent signs of disease.
Fortner's method of scarification was also found to yield no symp-
toms when these strains were used. Manninger felt that the vaccine
products would be too rapidly eliminated from the tissues and that
it would be desirable to adsorb these products onto some agent in
order to prolong their action. He selected aluminum hydroxide. He
observed that formalin did not appear to alter the antigenicity of
his selected strains and, for the purpose of preserving the product,
added this substance.

Thus he produced a vaccine very similar to that developed by
Traub (1947) but by an entirely different approach. The one out-
standing difference in the two products, and of the greatest advan-
tage in the Manninger vaccine, is the use of rough avirulent strains,
whereas Traub's preparations probably include virulent organisms.
Even though formalin is added, some potential danger exists to a
greater or lesser degree.

In testing his own vaccine, Manninger (1951) found that a good

immunity developed after about 10 days in animals over 5-4 months

of age, this protection lasting for about 10 months.
Recent deve10pments by Housemann (1949) and Gochenour (1952)
were directed to the production of more stable vaccine products.
Housemann produced a lyOphilized adsorbate vaccine which is of value
especially in the warmer climates, and Gochenour applied the 1yophili—
zation process to the standardization and preservation of cultures
used in the simultaneous method of swine erysipelas prophylaxis and
treatment. Gochenour (1952) reported that lyOphilized vaccine dessi-
cated from the frozen state retained its original viability and viru-
lence for 2 years and remained stable in the restored state for 7 days.
Since the original recognition of g, rhusiqpathiae as the etio-
logical agent of swine erysipelas in EurOpe by Loeffler (1885) and
by Creech (1921) in the United States, several effective procedures
have been developed for the control of this disease. Though a number
of shortcomings of these procedures continue to motivate present-day
research efforts, perhaps the best summary of the available methods
is to be found in the words of McNutt (1954): ”The mistake we have
made in the use of the simultaneous method is that we expect too much
of it. If we will use it for what it is, we will get along much
better because it is truely valuable and there is nothing better
at present." Shuman (1954) observed: "This may also be true of the
adsorbate bacterin.” Shuman (1954) pointed out: 'No prophylactic
measure offers 100 per cent protection under all circumstances, and
it is therefOre the obligation of the practicing veterinarian of today
to apply effectively those measures which are of present avail in the
absence of more effective procedures which the future research efforts

will doubtless reveal."

_ 15 _

The application of ultrasonic wave action to bacterioclasis
dates from the publication of Wood and Loomis (1927) of the destruc-
tion of luminous bacteria. Some of the main contributions have been
made by Yen and Liu (1954), Stumpf, Green and Smith (1946) and
Shropshire (1947). Liu and Yen (1954) and Royert and Grabar (1947)
both observed the necessity for the presence of gas in'a liquid as
a precursor to bacterial destruction. The gas present did not need
to be oxygen, as hydrogen and other inert gases served equally well,
thus ruling out the possibility of an oxidation reaction as the dis-
ruptive agent. The actual mechanism responsible for the cellular
disruption is believed to be that of cavitation. If the intensity
of the sound waves is sufficiently high, the passage of these waves
through a liquid results in the formation of minute cavities at regions
of low pressure and their subsequent collapse in succeeding high pres-
sure phases. As calculated by Rayleigh (1917), when cavitation occurs,
pressures of several hundreds of atmospheres are developed around the
collapsing cavities. This severe localized mechanical action is be-
lieved to be responsible for the disintegration of bacterial cells.
Several investigators have reported on bacterioclasis e.g., Harvey
and Loomis (1929), Chambers and Flosdorf (1956), Stumpf, Green and
Smith (1946), Hamre (1949), and Williams and Gaines (1950).

The design of most modern equipment to dissipate the heat pro-
duced by the passage of the sound waves through the medium eliminates
another factor which might be considered effective in cellular de—
struction and further delimits the possible active factors in sonic
disintegration.

The influence of the suspending medium on the destructive effect

- 14 -

of sonic waves on bacterial cells has been evaluated in part by
Beckwith and Weaver (1936), Jacobs and Thornley (1954) and Shrapshire
(1947). In general, they have observed some protection to be afforded
the cells by such materials as milk, gum tragacanth and gelatin. Re-
sults also indicated that the power output served as an influencing
factor in bacterioclastic action. Since power is a factor of the
amplitude of the waves rather than the frequency, it was desirable in
this work to use the upper limits of the sonic spectrum,as a greater
amplitude is more easily obtained at a lower frequency. The action

as far as the disruption of the cells may be concerned, is essentially
the same. However, in using the upper limits of the sonic spectrum,
one is able to take advantage of both a high frequency and a comparatively

greater amplitude, or more generally speaking, a greater power output.

_ 15 -

Methods and Procedures

Strains of E, rhusigpathiae, SE-9 and SE-25 which were used

 

in the production of two of the three experimental bacterins were
obtained from the Corn States Laboratories located in Omaha, Ne-
braska. Strain 14774 which was used, both for the production of

one experimental bacterin and for all of the experimental challenging
procedures, was obtained from Dr. J.P. Newman of the Department of
Microbiology and Public Health, Michigan State University, East
Lansing, Michigan.

All three strains were tested for their fermentative ability,
virulence for white mice, and haemagglutinative capacity.

The fermentative ability was determined by use of the basal
medium.described by Tiffany (1955) to which the various compounds
to be tested were added. In general, all three strains displayed
a good ability to ferment glucose, fructose, lactose, glactose and
xylose, and a mediocre ability to attack arabinose and mannose.

The test for virulence was accomplished by injecting 0.1 m1 of
an 18 hr broth culture of the respective strains subcutaneously in-
to each of five white mice. Each strain killed all five of the test
animals within three days subsequent to injection.

The Hirst test was used to ascertain the ability of these
strains to haemagglutinate chicken erythrocytes. In accordance with
this test, the three strains were found to be negative with respect
to inducing haemagglutination of the chicken red blood cells.

Pure cultures of these strains for use in this work were main-
tained at room temperature in semi-solid nutrient-tryptose broth.

Tiffany and Wheaten (1954)

- 15 -

Prgparation 2; Medium

 

Production of the vaccine necessitated the acquisition of large
numbers of cells. It was also desirable to use a medium.which was as
free of other antigenic substances as possible and which would still
support a good growth of the organism. In view of the successful use
in our laboratory of nutrient-tryptose broth for the cultivation of
E, rhusiOpathigg_(Tiffany and Wheaten 1954), and since it contained
fewer potential antigens than some other acceptable media, it was
selected for the cultivation of this organism for vaccine production.

Thirty-nine grams of dehydrated tryptose broth (Difco) and 12
grams of dehydrated nutrient broth (Difco) were added to 1500 ml of
distilled water. These substances were dissolved with gentle heating.
The dissolved medium was then filtered through four layers of gauze
and a pledget of glass wool. After filtration, the medium.was dis-
pensed into three fi-liter flasks. At this point the pH was adjusted
electrometrically to 7.6 with 0.5N NaOH, and a small (5.0 cm) glass
enclosed bar magnet was placed in the flask. The flasks were gauze-
plugged and the plug covered with a brown paper cap. Sterilization
was accomplished by autoclaving at 121 C for 50 min. This medium was
then cooled and ready for seeding.

Seeding g§_0u1ture Flasks

Seed cultures of each strain were prepared and checked for purity
of culture by microscopic examination of stained samples of the re-
spective cultures. After checking the purity of the seed cultures,
0.5 ml of this 18 hr growth was used to seed the respective flasks of
medium.prepared as described above.

The three 5-liter flasks, after seeding, were placed on separate

- 17 -

Mag Mix magnetic stirrers (Precision Scientific Company) at room
temperature (approximately 24 G) and the rotations of the bar magnet
were adjusted to about 260 r.p.m.

Incubation under these conditions was continued for 24 hrs.

After 24 hrs, sufficient formalin (40 per cent) was added so as to
produce a final concentration of 0.5 per cent. Incubation was then
continued for 48 hrs.

A check for sterility was made following the 48 hr incubation
period, during which the formalin was acting.

The cells were separated from the medium by centrifugation in
an International Equipment Company Model PRI refrigerated centrifuge.
This instrument was equipped with an angle head (R.O.F. 2787 X G).

The medium fraction was placed aside prior to the addition of aluminum
phosphate and the cells were resuspended in 50 m1 of a solution of 1
per cent Beets-peptone (Difco) and 0.5 per cent formalin. Using this
formalinized peptone as a blank, a Genco photelometer Model B-2 (using
a flue filter) was adjusted to 100 per cent transmission. The density
of the cell suspension was then adjusted to correspond with the No. 7
tube of the McFarland mephelometer (approximately 2.1 X 109 organisms/
ml). The cells were then ready for sonic disruption.

Disruption 9_f_ gel};

Bacterioclasis was accomplished with the use of a Raytheon
Magnetostriction sonic oscillator Model DF-101. This equipment is
designed to deliver 200 watts of power from the driver unit to the
stand. The working frequency is 10 KC. The temperature in the cup
(which contains the sample) is maintained at about 40-45 0 by the
use of water coolant flowing through a water jacket enclosing the

cup. Approximately 1 qt/min is passed through this water jacket.

- 13 -

The unit was allowed to warm up for a period of 15 min, and a
50-ml sample (approximated) was placed into the cup. The machine was
activated and adjusted to operating frequency. The sample was ex-
posed to the sonic waves for 50 min. At the end of this time, the
sample was removed and allowed to settle for 24 hrs at 56 F. An
even, slightly opalescent suspension, colloidal in nature, resulted.

Subsequent to settling,the liquid suspension was removed and
the debris discarded.

Aluminum.phosphate was then added to both the medium, which was
previously set aside, and the sonisized suspension.

Preparation g§_the Aluminum Phosphate

The aluminum phosphate was prepared by the addition of a solution
of sodium phosphate to a solution of aluminum.chloride.

Thirty grams of aluminum chloride was dissolved in 7,000 m1 of
distilled water. This solution was stirred continuously and 2,000
m1 of 12 per cent sodium phosphate (hexa-hydrate) was added. Suffi-
cient distilled water was added to make 15,000 ml. The flask was then
covered and allowed to stand until the precipitate settled and then
the supranatant was removed. Four 1-ga1 funnels equipped with No.

50 Watman filter paper were used to retain the precipitate while it
was being washed with 8/10 liters of distilled water to remove the
chlorides. The pH was adjusted to 6.0-6.5 using 1 N NaOH and the
preparation allowed to stand for 2 hrs, at which time the pH was
again checked. Sterilization of the precipitate was accomplished

by autoclaving at 121 0 for 50 min. During sterilization, the pH
dropped to 5.4. The sterilized preparation containing 5 mg of alumi-

num (as aluminum) per ml was stored at room temperature prior to use.

- 19 -

The large quantity of precipitate was prepared in order to have
sufficient amounts as to meet the needs of other projects.

Sufficient amounts of the precipitate were added to both the
medium and the sonisized cells, so as to make a concentration of
5 mg/ml. The pH was checked to be sure that it was 5.2-5.4 and was
so adjusted with 1 N NaOH if it was not found to be in this range.
These preparations were then incubated at 55 C for 24 hrs.

The precipitate was removed from the medium by centrifugation
and was recombined with the adsorbed sonisate, which had also been
concentrated by centrifugation. These products were then suspended
in a total volume of 10 m1 of formalinized peptone solution (0.5 per
cent formalin and 2.0 per cent Beets-peptone). A check was made for
sterility of the vaccine and if sterile, it was considered ready for
evaluation.
ngt_Animals

White, male and female mice, between 16-20 grams, were selected
for evaluation of the vaccines. These animals were provided with
similar diets of animal pellets and water throughout the experimental
work. They were observed daily, prior to, and during this work, and
those animals which did not appear to be healthy were replaced or re-
corded as ill, if the signs of illness occurred within a day or so
immediately following the inoculation of the vaccine.

Determination 2f.thg_§2§Q'

Concurrently with the vaccine evaluations, the LD5O of the strain
selected for challenge was determined. It was noted that strains of
E, rhusiopathiae when suspended in 2 per cent Bacto-peptone (Difco),

exhibited a greater virulence for mice than a similar suspension in

- 20 -

0.85 per cent saline solution. For this reason, greater attention
was devoted to the evaluation of the LD5O for strains suspended in
the peptone solution, as the challenge of an immune response was
observed to be more severe than when 0.85 per cent saline solution
was used as the suspending medium. A number of determinations of
the LD50 for suspensions of g, rhusiopathiae in 2 per cent peptone
were made. The result of each trial agreed well with the trials
made concurrently with the vaccine evaluation tests, and the LD5O
thus established was accepted for the challenge procedure.

Ten-fold dilutions of an l8~hr broth culture of g, rhusiopathiae

 

were made in a 2 per cent peptone solution, ranging from 1-10 to 1-100
million. Five mice were selected to receive 0.2 ml of each dilution,
respectively, by the subcutaneous route, and five mice receiving 0.2
ml of the peptone solution were held as controls. Deaths were recorded
per each dilution for a period of 7 days, at which time observations
were completed. The LD50 was then determined according to the method
of Reed and Muench (1958). The LD5o as determined from the above pro-
cedure was found to be at a dilution of 10-5-5. It was necessary to
use a clean pipette for making each dilution in order to avoid carrying
organisms over to the next higher dilution which materially affected
the resulting LD5o.
Inoculation gglvaccine into the Test Animals

In order to evaluate the potential of the proposed vaccine, a
single 0.2 ml dose of the undiluted preparation and 0.2 m1 of each
of serial two-fold dilutions ranging from 1-2 to 1-256 were administered
to the mice subcutaneously. Five mice were used per dilution. For-

malinized peptone solution was again used as the diluting menstruum.

- 21 -

Two-tenths of a ml of the formalinized peptone solution was injected
subcutaneously into five mice which were observed as controls along
with five unvaccinated mice. The test animals were observed daily
for deaths or ill effects of the vaccine. Two parallel groups of
mice were thus injected.
Challenge g£.the Vaccinated Animals

Fifteen days after the administration of the vaccine, the two
groups of test animals, and the controls, were challenged. One group
received 0.1 ml of undiluted l8-hr broth culture and the other group
500LD50 contained in a volume of 0.1 ml. All injections were made
by the subcutaneous route. Observations and records were completed
at the end of seven days.

Evaluation 2;.g_00mmercial Adsorbate Bacterin

 

In order to facilitate the comparison, the commercial adsorbate
bacterin was tested by means of a procedure which was identical with
the one described above for the mouse protection tests. The test ani-
mals were challenged in the same manner as were the animals used in
the evaluation of the sonisate experimental bacterin, and the results

were completed at the end of seven days.

- 22 -

Results and Discussion

The evaluation of any research concerned with the operation of
biOIOgical systems is dependent upon a number of variables such as
age, sex, weight, growth phase, etc. These variables can be more or
less controlled by the investigator. However, biological systems are
also invested with a number of unknown variables which influence the
final results, and which, because of their unknown nature, do not lend
themselves to regulation. Since this problem is concerned with a
general evaluation, the role which the unknown variables play is much
greater than if a more limited phase of the problem was evaluated. As
an example of an unknown variable, one might consider the variation in
response to antigenic stimulation of five mice. These animals may in
all respects appear to be similar in age, sex, weight, etc., and yet
give a different response to a standardized dose of an antigen. One
must therefore keep in mind that though an attempt has been made to
regulate the known variables, there are still other unknown variables
which are active in affecting the final results, and which are not
apparent in the tabulated experimental results.

It is interesting that the results of the mouse protection tests
recorded in Tables I and II for bacterin 14774 showed no protection
against the challenge doses employed. Yet the strain from which bac-
terin 14774 was made displayed a virulence for white mice which made
it most acceptable as a challenging agent. This strain also had the
strong capacity to attack certain of the carbohydrates which indicates
the presence and operation of a number of enzyme systems. Strains SE-9
and SE-25, also virulent for white mice, were strong in their fermentative

ability and provided a marked degree of protection against a challenge

- 25 -

Table I Results of challenging mice with 0.1 ml of a virulent
undiluted culture of Erysipelothrix rhusiqpathiae 15 days sub-
sequent to inoculation with 0.2 ml of the respective bacterin dilutions.

 

Bacterin SE—9

 

 

No. of Dilution of Postvaccinal Deaths from Per Cent
Mice Bacterin Deaths Challenge Survival

5 Undiluted 0 0/5 100

5 1/2 0 1/5 so

5 1/h 1 5/h 25

5 1/8 0 5/5 0

5 1/16 0 5/5 0

5 Unvaccinated O 5/5 0

Controls
5 Formalinized 0 5/5 0

Peptone Controls

 

 

Bacterin ss-25

5 Undiluted 0 0/5 100
5 1/2 0 2/5 60
5 1/4 1 4/4 0
5 1/3 0 5/5 0
5 1/16 1 h/h o
5 Unvaccinated 0 5/5 0
Controls
5 Pbrmalinized O 5/5 0

Peptone Controls

 

Results of mouse protection tests using bacterins 53-9 and 33-25.

- 24 -

Table I (Continued)

 

Bacterin 14774

 

No. of Dilution of Postvaccinal Deaths from Per Cent

 

Mice Bacterin Deaths Challenge Survival
5 Undiluted 0 5/5 0
5 1/2 0 5/5 0
5 1/4 0 5/5 0
5 1/3 0 5/5 0
5 1/16 0 5/5 0
5 Unvaccinated 0 5/5 0
Controls
5 Pormalinized 0 5/5 0

Peptone Controls

 

 

Commercial Adsorbate Bacterin

 

5 Undiluted 0 0/5 100
5 1/2 0 0/5 100
5’ 1/4 0 1/5 80
5 1/8 0 4/5 20
5 1/16 0 5/5 0
5 1/52 0 5/5 0
5 Unvaccinated 0 5/5 0
5 ssgfiifiiiiss 0 5/5 0

Peptone Controls

 

Results of mouse protection tests using bacterins 14774 and the
commercial adsorbate bacterin.

_ 25 -

Table II Results of challenging mice with 0.1 ml of a l-1552
(500LD 0) dilution of a virulent culture of Erysipelothrix rhusio-
pathiae 15 days subsequent to inoculation with 0.2 m1 of the
respective bacterin dilutions.

 

 

Bacterin SE-9

 

No. of Dilution of Postvaccinal Deaths from Per Cent

 

Mice Bacterin Deaths Challenge Survival
5 Undiluted 0 0/5 100
5 1/2 0 0/5 100
5 1/h 0 0/5 100
5 1/8 0 5/5 40
5 1/16 0 5/5 0
5 Unvaccinated 0 5/5 0
Controls
5 Formalinized 0 5/5 0

Peptone Controls

 

 

Bacterin SE—25

5 Undiluted c 0/5 100
5 1/2 0 0/5 100
5 1/4 0 5/5 40
5 1/8 0 4/5 20
5 1/16 0 5/5 0
5 Unvaccinated 0 5/5 0
Controls
5 Formalinized 0 5/5 0

Peptone Controls

 

Results of mouse protection tests using bacterins SE-9 and SE—25.

- 25 -

Table II (Continued)

 

Bacterin 14774

 

No. of Dilution of Postvaccinal Deaths from Per Cent

 

Mice Bacterin Deaths Challenge Survival
5 Undiluted C 5/5 0
5 1/2 0 5/5 0
5 1/4 0 5/5 0
5 1/8 0 5/5 0
5 1/16 0 5/5 0
5 Unvaccinated 0 5/5 0
Controls
5 Pormalinized 0 5/5 * o

Peptone Controls

 

 

Commercial Adsorbate Bacterin

 

5 Undiluted ' 0 0/5 100
5 1/2 0 0/5 100
5 1/4 0 0/5 100
5 1/8 0 0/5 100
5 1/16 0 4/5 20
5 1/52 0 5/5 0
5 Unvaccinated 0 5/5 0
Controls
5 Formalinized 0 5/5 C

Peptone Controls

 

Results of mouse protection tests using bacterins 14774 and the
commercial adsorbate bacterin.

- 27 -

dose of the organism.
Roots (1955) established that in the case of E, rhusigpathiae,
virulence is no criterion for antigenicity. Dedie (1949) was able
to classify strains of g, rhusiopathiae into three groups on the
basis of their acid soluble antigens. He used the precipitin test,
and designated these groups as A, B and N. He further observed that
approximately 10 per cent of all strains which were tested fell into
group B and this group included those strains which were antigenic.
He further determined that the antibodies produced in response to
these strains were also specific for strains which fell in groups A
and N. Though groups A and N contained many very virulent strains,
they were, for the most part, poorly antigenic. Roots (1955) observed
that, under proper conditions, strains which could be Classified into
group A would elicit some degree of protection. He reasoned that
since these organisms are markedly virulent and probably contain some
traces of the B antigen, the increase in numbers of these strain or-
ganisms within a host would permit an increased amount of B antigen
to stimulate the host and thus some protection was induced. Roots
(1955) further reported that the immune response to group B strains
could only proceed up to a certain level and that a higher level of
protection could not be obtained under the conditions of his experiments.
In view of these previous investigations, the results listed in
Tables I and II would seem to conform with these explanations. Strain
14774, showing no protection, was injected in the killed state, thus
eliminating its virulence and permitting only the slight traces of B
antigen to stimulate the host. This did not induce sufficient pro-

tection against the challenge doses. This strain, therefore, might

- 28 -

be considered to fall in either group A or N. Strains SE-9 and SE—25

induced a fair degree of protection, and having had their virulence

eliminated also, show a predilection for the group B classification.
Dinter and Bakes (1948, 1949, 1951) in attempting to design some

means of selecting antigenic strains of Q, rhusiopathiae, observed that

 

certain strains of this organism would agglutinate fowl erythrocytes.
He observed that approximately 10 per cent of all strains of this or-
ganism possessed this preperty. He was later informed by Dedie that
the organisms of the group B classification also had this property of
haemagglutination.

It is interesting that the strains employed in this work all
proved to be haemagglutination negative with chicken red blood cells.
On the basis of antigenicity, the work reported herein tends to sup-
port the observations of Roots and Dedie, but in regard to the haemag-
glutination phenomenon, a directly Opposite result was obtained.

The details of the haemagglutination test used by the EurOpean
investigators were not available to the author and thus, in testing
strains 88-9, 88-25 and 14774, it was necessary to use the Hirst test
employed by virelogists for diagnostic purposes. It is improbable that
the original test involved a modification which would have affected the
results to so great an extent and it is felt by the author that a more
acceptable reason for the negative results might be obtained from a
consideration of the medium in which the organisms were grown. It is
an accepted fact that the addition of serum to a medhwm designed for

the cultivation of E, ghgsigpathiae tends not only to enhance the

 

growth of the organisms, but also accents their fermentative ability.

Since serum contains some enzymes which are able to attack carbohydrates

- 29 -

of polysaccharide nature and reduce them to the less complex mole-
cules of di and monosaccharides, it is not a desirable component of
fermentative media (Seaman and Woodbine 1952). However, a true
evaluation of the effect of serum on the presence or absence of the
haemagglutinating antigen has not been made. In all probability,

the addition of serum.to the medium used for the cultivation of the
strains tested in this work might have made a difference on the hae-
magglutinative result. This is merely a supposition which occurs in
attempting to explain the difference in results of this work and that
of previous investigators relative to this haemagglutination phenomenon.
This reasoning is felt valid since the addition of serum.to a.medium

for cultivation of E, rhusiopathiae is a commoner practice in Europe

 

than in the United States, and is the only legical basis apparent at
present.
A closer inspection of Tables I and II reveals a slight variation

in the protection conferred by bacterins 88-9 and SE~25. Against a
challenge dose of undiluted culture, SE-9 afforded 100 per cent pro-
tection in the undiluted state only and 80 per cent protection in a
1-2 dilution of this bacterin. SE-25 was observed to have protected
100 per cent of the test animals against the undiluted culture chal-
lenge and only 60 per cent of the animals were protected in a chal-
lenge of the 1-2 dilution of this bacterin. This variation is more
clearly exemplified in the response of the vaccinated test animals to
a challenge dose of 5OOLD50, the results of which are presented in
Table II. Here, bacterin SE-9 is observed to protect 100 per cent
of the test animals in a dilution of 1-4, whereas SE-25 afforded 100

per cent protection in a dilution of 1-2.

- 5O -

Since both bacterins were prepared simultaneously and in a
similar manner, one might be led to disregard the procedure as a
source of variability and ascribe the variation to a difference in
antigenicity of the strains. However, one must proceed with caution
to such a conclusion, because of complexities of antigenicity and tech-
nique. Procedure, as a cause of variability, cannot be ruled out un-
til more detailed information is available regarding the effect of
such physical factors as adsorption, sonic disintegration and preser-
vation. Nor can one completely absolve the factor of antigenicity
without a more refined investigation of the two strains involved.

The magnitude of variation, therefore, might be attributed more
safely to a combination of these factors, rather than to one alone.

One other aspect of this thesis problem which.must be discussed
is the reason for diluting the bacterin preparations and holding the
challenge doses fixed. It would appear that one might hold the bac-
terin dose constant and vary the challenge doses, thus giving a more
complete picture of the protection afforded by the experimental pro-
ducts. However, in Table I, it may be observed that the undiluted
bacterins protected 100 per cent of the test animals against an un-
diluted culture challenge. For this reason, the procedure described
in this paragraph was not feasible.

To this point, the discussion has been mainly concerned with an
evaluation of the sonically disrupted, formalin killed experimental
bacterins. Including new the results of the mouse protection tests
obtained from the evaluation of the commercial bacterin, Tables I and
II show that a greater protection was conferred by this product than

by either of the two eXperimental bacterins.

- 51 -

The criterion for comparison being the degree of protection
elicited by the bacterins, it is obvious that the commercial bacterin
is the most acceptable of those tested. This is especially apparent
in the response of the test animals to a challenge of 500 LD50. The
commercial product protected 100 per cent of the animals in a dilution
of 1-8. The protection which the eXperimental products offered, though
not as great as that produced in response to the commercial bacterin,
was of considerable magnitude. In attempting to determine a reason for
the differences, one is confronted with a number of variables. These
variables which were evaluated only to an extent which would permit a
standardization of procedure, and which would require much more time
for a more complete evaluation, most probably effected a considerable
portion of the differences in results obtained. For example, the pro-
cess of sonic disruption of the cells introduces numerous variables
which have not been elucidated sufficiently for the establishment of
a set procedure. The length of eXposure time, the nature of the
suspending medium, the effect of this process on the antigenic com-
ponents of the cells, and the nature of the resulting products as per
their adsorption status, are all to be considered as a function of the
sonic process. The object in using sonic disintegration in this work
was based on an attempt to eXpose and make the antigenic components of
the bacterial cell more readily available for stimulation of the host
tissue. What actually happened, whether they were denatured or de-
stroyed, is not known since the medium used for culturing these or-
ganisms was also adsorbed and recombined with the sonisized cells. In
addition to the effect of the sonic disruption, one must consider the

time element. Perhaps a longer time period for antigenic stimulation

- 52 -

might have caused better protection. For instance, one might have
selected twenty days during which the bacterins had been permitted

to act, thus inducing greater immunity. This would suggest a sore-
logical investigation of the titer variation. Perhaps two injections
of the bacterins spaced at some predetermined interval might have
produced a more desirable protection. Also, the selection of another

more antigenic strain of E, rhusiopathiae might have been an influ—

 

encing factor. These variables which, if properly evaluated, would
perhaps result in the production of a more acceptable bacterin and
would possibly narrow the gap between the results of the experimental
and commercial products. This comparison is made with respect for
the fact that a more acceptable commercial product may be produced.
The greatest over-all value of this work is the promise offered
by the designed procedure as a means of producing an acceptable bac-
terin, and though much more work is indicated, such a promise in the

field of erysipelas prophylaxis is indeed rewarding.‘

Summary

Three strains of Erygioelothrix rhusiOpathiae were selected
for the production of bacterins. The cells were killed by the
addition of 0.5 per cent formalin to the cultures and were separated
from the medium by centrifugation. These cells were then disrupted
by sonic energy and adsorbed onto aluminum phosphate. The medium
used for cultivation of the cells was also adsorbed with aluminum
phosphate and the adsorbate products combined with the cell adsorbate.
White mice were used to test these products against a challenge of
undiluted virulent culture and against a challenge of 500LD50.

Strain 14774 did not protect any of the test animals and strains
SE-9 and SE-25 protected 100 per cent of the animals, injected with
the undiluted bacterin, against the challenge of undiluted culture.
Against a challenge of 500LD50, bacterin SE-9 protected 100 per cent
of the animals in a dilution of 1-4 and SE-25 protected 100 per cent
of the animals in a dilution of 1-2 against a similar challenge.

A commercial bacterin was evaluated for the purpose of comparison
and.proved to offer greater protection than either of the experimental
products. It protected 100 per cent of the animals in a dilution of
1—2 against a challenge with undiluted culture and 100 per cent of
the animals were protected against a challenge of 500LD50 in a dilution
of 1-8 of the commercial bacterin.

Considering the variables present, for which more refined
evaluation is indicated, the most important development of this work
was the promise which this procedure offers for the development of an

acceptable bacterin.

-524-

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PI‘OC. SOC. Exptle 81010, IIoYo, 21;, 12500

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