'—

 

A CO‘MP‘ARUS’SGN OF ME MORPHOLOGW
FERMENTATI‘QN; AND SERGLGGY OF
[ELEVEN MYCQPLA$MA AND TWO

QQEENEBACTER§UM g;
REVERE-[ON mowers;

 

 

Thesis {or ”we Degree of M. 5.
MICHKGFLN STATE UNWERSETY

Richard! B“. Dardas
1959

 

TR!”

 
 

 
  

LIBRARY

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University

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BY

“ichard B. Dirfias

$ |Dfir'1‘ Af‘im
A N: [xi/D luau;

Submitted to the School of Science and Arts of Hichigen
State University of Agriculture and Apylied Science
in rartial fulfillment of the requirements

for the degree of

7" ”‘7‘“, ”‘1”? 1fl*"z"“’ '1
i"J.‘LJ.L.L;lL Ul' Qulichib

Department of Microbiology and Public Health

1959

. \
Anproved by u”

Abstract Richard B. Dardas

Two Corynebacteria isolated from culture flasks con-

 

taining previously pure cultures of the 86 strain of
MyCOplasma gallinarum, handled under highly controlled

conditions, were compared to the 36 strain of M. callin-

 

arug and ten other PPLOs to determine if morphological,
fermentative and serological relationships existed to a
great enough degree to suspect the phenomenon of rever-
sion to have occurred. On the basis of morrhological and
fermentation studies three groups were discovered to exist
among the group being tested. Serological studies showed
only one group to be present. Both Corynebacteria showed
fermentation similarities to the PLLOS and differences
from unrelated Corynebacteria which were agglutinated by

36 antisera. Cne of the two_Corynebacteria susyected to be

 

a reversion from 36 showed great serological similarities
to 36, but the other's similarities were not as striking.
On the basis of that is thought to be a group antigen of

Corynebacteria in 36 and the serological and fermentation

 

similarities, a PPLO, formerly thought to be stable, was
found to give indications of reverting to a bacteria of

the genus Corynebacterium.

 

A COHPAmISCE OF TEE IOnEHOLCGY, FEn;LJTATIOK, AND

SEmOLOGY OF E EVER XICCPLASMA AND

 

T‘O CLflY JEAoiLnIL“ SP. “EVLnoiLI anD “C 'S

 

BY

Richard B. Dardas

& TEE

UL:
Fl
(I;

Submitted to the School of Science and Arts of Michigan
State University of Agriculture and Applied Science
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIEMCE

Department of Microbiology and Public Health
1959

/

My” 1 .r ,1 f 1 1
Approved by “I " (7’ k1/(_I{,, (Hui/La-

AC I‘ZNO‘ .‘LEDGI-IEZITS

The author wishes to thank Dr. D.E. Schoenhard
for his assistance and council throughout the course
of this research.

The help and advice of Drs. C.H. Cunningham,
R.C. Belding and J... hay toward the completion of

this study is also very deeply appreciated.

TABLE OF CONTENTS

PAGE

INThODUCTION ................................... 1
IvIATEl-JALS mm masons 9
RESULTS ........................................19
DISCUSSION .....................................h8

STTVHIMY .OOOOOOOOOOOOOIOOOOOOO00.00.00.00000000061

BIBLIOGLXAPIEY VCOOOOOOO...0..OOOOOOOOOOOOOOOOOOOOO61‘.

APPENDIX .......................................68

”ITACDUCTIO}

In the year 1398 Uocard and Roux reported success
in culturing the filterable agent of bovine pleurOpneu—
monia in the absence of living cells. This report stands
today as the classic in research on organisms of the
pleurOpneumonia group. Bordet (1910) added to the know—
ledge of tie new organism by publishing an article on its
morphology. This article appeared at the same time as a
similar one by Borrel, Dujardin-Beaumetz, Jeantet and
Jouan (1910) who named the organism Asterococcus mycoides.
Bridre‘ and Donatien (1923) discovered the causative agent
of agalactia in sheep and goats to be very similar to the
organism of Nocard and Roux (1898). In 1929 Elford con-
tributed significantly by determining the size of the
agent of bovine pleurorneumonia to be between 125 and.lSO
millimicrons (mu). This work was done with Gradicol fil-
ters. Shoetensack (1934) believed that he had found the
filterable virus of canine distemper to be a member of the
pleurOpneumonia group.

{lieneberger (1935) reported the amazing discovery of
pleurOpneumonia-like organisms growing in apparent symbi-

osis with Streptobacillus moniliformis.

 

Laidlaw and Elford (1936) reported on pleurOpneumonia-
like organisms isolated from sewage which, unlike those

already mentioned needed no serum in the medium used to

culture them.

In 1936 J. B. Nelson described a symptom complex in
fowl as "an uncomplicated coryza”. This is probably the
first report on a disease known at the present time as
chronic respiratory disease (ChD)._ An avian strain of
pleuropneumonia-like organism has been repeatedly iso-
lated from cases of ChD, and infections have been pro-
duced eXperimentally with these organisms and followed
by reisolation of the organisms, (Smibert, Forbes, Faber,
Gabreten and DeVolt 1959). Koch's postulates seem to have
been satisfied. The avian strain of pleur0pneumonia-like
organism (PPLO) appears at present to be the etiological
agent of chronic respiratory disease in domestic fowls.

Since the time of this early research much work
has been done on the PPLO. A review of the literature
reveals a broad spectrum of susceptible hosts and an even
more formidable array of organism variants. Although
this paper will draw references from the entire field of
PPLO research, the author has confined his research pre-
sentation to organisms of avian origin with two organisms
of porcine origin to serve as representatives of mammalian
parasites.

While working with the Zander strain of FPLO, note
was taken of an unusual frequency of contamination occur-
ring both during routine egg passage of the organism and

in broth passage. Elaborate aseptic techniques did not

-2-

seem to affect the persistence of the contamination. At
the time there was reason to suSpect that reversion was
taking place, so eXperimentation was started to approach
a solution.

Our observations on the phenomenon of reversion are
not the first of their kind. It has been known for many
years that so called L forms of bacteria are transformed
into the parent species of bacteria upon media passage
Sabin, 19H1; Dienes and Weinberger, 1951; Edward, 195%;
Gilmore and Burnett 1959. There is always the danger that
one is working with an L form when reversion is considered.

In 1953 Minck reported the reversion of a number of
PPLOs isolated from the human genital tract to bacteria of
the genus ggggnebactegia. Since reversion occurred Minck
considered the PPLOs to be L forms. McKay and Taylor
reported in 195% that what had previously been thought to
be a PPLO had reverted to an organism resembling flem0philus
gallinarum. These authors, as did Minck, balked at the
idea of reversion of a true PPLO. PeOples, Smith and
Morton (1955) reported the presence of ggrynebacteria in
stock cultures of PPLO as reputable as the Campo strain.
E. C. Wittler (1956) reported the transformation of PPLO
in tissue culture to a member of the genus Corynebacterium
and the redifferentiation of the bacteria to an L form,
but not completely back to the original PPLO. The isola-

tion of eighteen diptheroids from one hundred cultures of

-3-

the Campo strain of PPLO was reported by Smith, PeOples
and Morton (1957). Twenty cases of reversion were
reported by Kelton and Gentry (1957). The PPLOs involved
reverted primarily to Staphylococcus.

As in most fields of scientific endeavor an abundance
of morphological studies have been carried out on the PPLO
group of organisms. The life cycles of PPLOs have been
exhaustively treated by several very competent workers in
the field. Klieneberger (1934) described the morphology
of the classic bovine, ovine and caprine strains prior to
her discovery and description of the first L form (1935).
The same author (Klieneberger, 1938) described the mor-
phology of the first true PPLO which she isolated from
the lung of a rat in her laboratory. Later, Klieneberger
(1938) found the same PPLO in a wild rat. A. B. Sabin
in 19#1 reviewed the literature up to that time and in-
cluded his personal unpublished observations. Dienes and
Weinberger (1951) published a review and included recent
morphological findings. Freundt (1952) in two fine re-
ports criticized the findings of many morphologists in
the light of his own discoveries and stressed the impor-
tance of branching mycelia as a criterion for inclusion of
organisms in the pleurOpneumonia group. His work on large
bodies and elementary granules of the group holds the sum-
mit position among work of this type although this author
does not agree with his ideas on the implications of the

.n-

discoveries.

Dienes (1953) in two articles contrasts the growth
differences of L forms on solid media and in broth and
shows the validity of bright field microscope observa—
tions on L forms and three PPLOs by demonstrating identi-
cal structures with electron microsc0py. Again in 195%
Freundt published an article on PPLOs. He divided PPLOs
into three groups on the basis of morphology. The clas-
sification of the pleurOpneumonia group on the basis of
morphology was stressed by Edward (195%) because of the
diversity of the group. Morton and Lecce (1954) published
on the morphology of human and avian PPLOs. Their work
with the electron micrOSCOpe capably discounted the clas-
sification of Freundt (1952, 195%) by showing that his
preparation had altered the configuration of the cells
he photographed and in so doing gave an inaccurate picture
of the structure. Klieneberger and Cuckow (1955) showed
structural dissimilarity between the classic pleurOpneu-
monia organism of cattle and the L3 organism, a true PPLO.
Wittler (1956) compared the colony morphology of a PPLO,

a member of the genus Corynebacterium which had been de-
rived from the PPLO, and an L form of the Corynebacterium.
The pleurOpneumonia group was discussed in length as
regards mycelial structure, means of reproduction, classi-
fication and relation to the L phase organisms by Edward

(1956). Yamamoto (1958) discusses cell shapes and colony

-5-

configuration of eight strains of avian PPLOs and uses
these results as partial justification for groupings
which he presents.

Attempts at classification of the pleuropneumonia
group have been made from time to time by the use of
fermentation techniques. Edward (1950) used thirteen of
the common sugars and alcohols to characterize the r ac-
tions of the classical pleurOpneumonia organism of cattle,
three saprOphytic strains, the L forms of Klieneberger,
two PPLOs pathogenic for mice and four PPLOs of the human
genital tract. heduction of the dye, methylene blue, was
also used as a classification method. In 195% Freundt
grouped a large number of EPLOs of human origin by fermen-
tation reactions on fourteen different carbohydrates.
Three groups were suggested by the results. Korphological
and serological studies indicated similar groupings.
Freundt's group III was in all ways identical to the Paris
variety of the bovine strain of pleurOpneumonia.

In 1956 Wittler found metabolic difference between a

PPLO and two Corynebacterium reversion products when they

 

were grown on three carbohydrates. Adler (1957) used ten
carbohydrates and found an identical fermentation pattern
in four avian PPLOs, each isolated from different organs
of domestic fowls. Although only one group is indicated
from the fermentation reactions two definite groups were

found serologically. Smith (1957) showed the metabolic

-6-

similarity of a group of Corynebacteria to the PPLOs from
which they were derived by carrying out fermentation tests
on eight carbohydrates. Yamamoto (1958) checked twelve
carbohydrates on each of eight strains of avian PPLOs and
established four separate groups by this method.

Norman, Saslaw and Kuhn (1950) used serological
methods to establish an antigenic relationship between six
PPLOs from the human genital tract. deciprocal cross ag-
glutination was discovered upon testing of the six antigens
and six antisera. In 195% White, Kallace and Alberts re-
ported antigenic similarity between PLLOs causing CfiD and
infectious sinusitis in turkeys using hemagglutination and
hemagglutination inhibition methods. heciprocal cross
reactions were again noted with the antisera when used in
the hemagglutination inhibition test. Gianforte, Jungherr
and Jacobs (1955) showed a serological relationship between
all of the seven avian PPLO strains he used in his work.
Slide agglutination, tube,agglutination, agglutinin absorp-
tion, cross agglutinin absorption, complement fixation,
precipitin, hemagglutination and hemagglutination inhibi-
tion test methods were used.

Adler (1958) made use of the similar antigenic struc-
ture of CED agents and infectious sinusitis agents to
develOp slide agglutination tests for the detection of
these organisms by using a univalent antigen preparation

which reacts serologically with most pathogenic strains.

-7-

Smith, PeOples and Morton (1957) proved that PPLOs and

the diptheroids derived from them have antigens in com-
mon. Yamamoto and Adler (1958) discovered that the agents
of GED and infectious sinusitis, both PPLOs, could be an-
tigenically classified into four different and distinct
types. These groups correSpond to the groups determined

by carbohydrate fermentation which were mentioned earlier.

MATERIALS AND METHODS

For the routine prOpagation of all of the organisms
to be mentioned, Bacto PPLO Agar and Bacto PPLO Broth with-
out crystal violet were used. One per cent Bacto PPLO
Serum Fraction was added to both media after sterilization
by autoclaving at 121° C for fifteen minutes. Routine
stock cultures were maintained by transferring ten milli-
liters of three day old cultures to two hundred and fifty
milliliter flasks containing one hundred milliliters (mls)
of PPLO culture broth. The temperature of incubation in
all cases was 37° C.

Constant checks for viability and pure cultures
were accomplished by plating and spreading two tenths
milliliters of the %8 hour old cultures on 1 centimeter
(cm) thick, serum enriched PPLO agar petri dishes. After
incubation of the plates for 2% hours,colonies of PPLO could
be easily seen by examination of the inverted plates with
the stereoscopic microscOpe using 80X magnification. PPLO
agar blocks stained by the method of Dienes (Morton, Smith,
Williams and Eickenberg, 1951) were examined with a com-
pound microscope, using a magnification of 970x at every
third broth passage for configuration of the colonies.

All of the PPLO cultures used in the fermentation,
serological and morphological observations were derived

from lines which had survived nineteen consecutive broth

-9...

transfers without complication. These PPLOs were all ob-
tained from different sources and were not eXposed to L
form inducing compounds after their initial isolation in
penicillin broth. PC-l, PC-2, PC-3, PC-%, PC-5, PC-7,
PC-8 and PC-lO were each isolated from a chicken showing
clinical symptoms of chronic respiratory disease. P-6
and P-9 were each isolated from a pig showing clinical
symptoms of acute pericarditis (Glasser's Disease). .The
above organisms'were obtained from the diagnostic labor-
atories at The Michigan State University College of Vet-
ernary Medicine. The Zander (S6) strain of PPLO was given
to us by Dr. H. E. Adler of The University of California.
The two gppynebactegig cultures (CS and CL) were isolated
in our own laboratories from flasks containing the S6
strain of PPLO.

Because the medium used in the culture of PPLO is
so easily contaminated and the isolation of a reversion
product is so vulnerable to the charge of contamination,
Special care was taken to assure a pure culture of S6
with which to begin eXperimentation. One 100 milliliter
(m1) portion of PPLO broth was inoculated with 10 mls of
a three day old culture of S6 and subcultured every three
days for a total of three passages. Agar plates were in-
oculated each day for the nine day period with material
from the subcultures to check for PPLO growth and the pres-

enxre of contamination. At the end of this period the third

-10-

subculture was filtered through a sintered glass, bacteri-
al grade filter and the filtrate recovered. Ten milli-
liters were injected into each of three 100ml portions of
sterile PPLO broth. The filtrate was withdrawn with a
sterile syringe and injected through rubber closures fit-
ted on flasks that had been sterilized with aluminum foil
over the tOp. This technique was continued until the

isolation of the two Corynebacteria was completed. A

 

PPLO agar plate was also inoculated at this time with 0.2
m1 of filtrate. The filtrate, petri dishes and subculture
flasks were incubated at 370 C. In four days the filtrate
was slightly turbid and four colonies of PPLOs were dis-
covered on the agar. Each colony was separately cut out
of the plate on a block of agar and aseptically trans-
planted into 100ml of PPLO broth. Only one flask from the
above inoculation proved to contain a PPLO population at
the end of six days. With the material from this flask
five successive three day subcultures were made. At the
end of three weeks incubation subculture #2 and #% showed
excessive turbidity and were reOpened, plated onto PPLO
agar without serum fraction, and inoculated into tubes of
brain-heart infusion broth without serum fraction.

The organisms from both sources appeared to be in
pure culture in both cases, but were subjected to further
pure culture techniques. Brain-heart infusion agar and

broth without serum fraction were imployed in conjunction

-11-

with the streak plate technique for obtaining pure cul-
tures. Material was picked from single colonies with the
aid of the stereosc0pic microscOpe using 80X magnifica-
tion and inoculated into broth. After growth was evidenced
in the broth, material was restreaked onto Brain-Heart

Agar without serum fraction. This process was repeated
several times to assure a pure culture.

Photomicrographs were taken of each of the cul-
tures under consideration using lOOX, 2OOX, %OOX and 970K
magnification and white light. The Specimens used for the
photomicrographs were forty-eight hour old agar cultures
prepared by the agar block and staining method of Dienes.
Specimens used for the photomicrographs of Cozynebactez-
1pm were agar block preparations as described above and
impression films from forty-eight hour old agar cultures
stained with methylene blue.

The Bacto Phenol Red Broth used as the basal
medium for the determination of PPLO and Corynebactegigm
fermentation ability was rehydrated as directed and dis-
pensed in 16 X 150 millimeter tubes. Inserts and either
one or one half per cent of the desired carbohydrate were
added and the medium was autoclaved at ten pounds steam
pressure for ten minutes (Appendix Table 23). After the
media had cooled, one per cent serum fraction was added
and the tubes were incubated for 2% hours to check for

possible contamination.

Except for the S6 strain of PPLO none of the stock
PPLOs were filtered to assure freedom from bacterial con-
tamination. There is, however, no reason to believe that
contamination was present as each of the strains had under-
gone nineteen subcultures with scrupulous examination
along the way without a discovery of such a nature.

A final check on the homogeneity of the cultures
prior to inoculation into carbohydrate tubes was carried
out. Three days before the tubes were to be inoculated
one petri plate from each of the different cultures was
inoculated with O.2ml of the reSpective material that
was transferred to become subculture #19. After incuba-
tion for three days the plates were examined for contam-
ination.

The carbohydrate tubes were inoculated with 10%
of their liquid volume from the reSpective source stock
cultures. The source flasks were then reincubated to
check for contamination during procedure. A rack of unin-
taculated.carbohydrate controls containing one per cent
serum fraction was incubated along with the test group for
the duration of the eXperiment.

All tubes were checked at frequent intervals for
changes in color and the appearance of gas. At each ob-
servation each tube was checked against its control and
recorded as negative, 1, 2 or 3. A negative reaction is

indicated by an unchanged tube. The term, 1, is used to

-13-

indicate a subtle change toward the acid side. The term,
2, is used to indicate an acid reaction producing an
orange color. The notation, 3, is used to indicate acid
production as shown by a yellow color. Only 2 and 3 re-
actions will be considered definite and conclusive evi-
dence of acid production. The ability of the organism to
multiply in the basal medium was checked by inoculation of
each organism into phenol red broth enriched with one per
cent serum fraction. Positive evidence for the effect of
growth in these tubes was obtained by noting the slight
turbidity of the tubes after three days incubation and
whether subculture on PPLO agar produced growth. Presence
of contamination in the test tubes was checked by plating
all materials appearing excessively turbid.

For the production of antiserum against the stock
PPLO and Corynebacterium cultures, ten rabbits were obtain-
ed. Unfortunately funds were not available to purchase
the required number of rabbits from controlled litters.
Seven of our rabbits were of diverse providence and in
most cases of such age that high quality antibody produc-
tion could not be eXpected. Three rabbits were six month
old animals from the same litter. Jith these facts in
mind it was deemed necessary to use the most effective
immunization process possible. Two antigen preparations
were used for this purpose.

The first antigens were concentrated three

-1h-

day old cultures from twenty-fourth subculture of stock
organisms corresponding to our #1 nephelometer. The for-
mula for this tube is shown in Table 2% of the Appendix.
The antigen was heat killed at 560 C for thirty minutes
and mixed in equal preportions with a prepared adjuvant
mixture of 10% Aracel and 90% Bayal F 011.

The second antigens for injection were simply live,
unaltered three day old cultures of the reSpective PPLOs.
In the case of the gprynebacteria antigens the material
was heat killed by exposure to 56° C for thirty minutes
and adjusted to the turbidity of our #1 nephelometer tube.

Before any injections were given, all rabbits were
prebled from the heart to secure normal serum. Twenty
milliliters were taken from each rabbit. The blood was
allowed to clot and the serum decanted and centrifuged
for clarification. The sera were inactivated by heat and
frozen. Two rabbits were then checked for sensitivity to
the PPLO cultures by subcutaneous injection of 0.1 milli-
liter of the adjuvanted antigen on the shaved lateral as-
pect of the animal at the diaphragmatic margin.

All rabbits were injected on three consecutive days:
day two, 1 ml by the intraperitoneal route; day three, 0.3
ml by the intramuscular route; day four, 0.3 ml by the int-
ramuscular route. On the nineth, eleventh, thirteenth,
fourteenth, eighteenth and twentieth day all rabbits being

immunized against PPLO received 6 mls by the intraperi-

-15-

toneal route. 0n the same days as above, Corynebacter
ium rabbits received 1 ml, 2 mls, 3 mls, 6 mls and 6 mls,
respectively by the intraperitoneal route. This schedule
is in table 27 of the Ajpendix.

Seven days after the last injection, 50 cc of
blood were withdrawn from all animals by cardiac punc-
ture. The blood was allowed to clot and shrink in test
tubes before the serum was aseitically drawn off and cen-
trifuged at 2,500 revolutions per minute for ten minutes
to effect clarification. Serum was diseensed in Hahn
tubes, inactivated at 560 C for thirty minutes and fro-
zen for storage.

The production of PILO antigen for use in serolo-
gical tests was accomplished by inoculating PPLO broth
with organisms from the twenty-fourth subculture of the
reSpective PkLOs. The broth used was autoclaved and fil-
tered through paper to remove all particulate matter,
diSpensed in 500 m1 quantities and reautoclaved. One per
cent serum f'action was added, and the flasks were incu-
bated for twenty-four hours to assure sterility. Agar
plates were seeded with 0.2 ml of inoculum, prior to inoc-
ulation of the antigen culture flasks, to check the purity
of the inoculum. Ten per cent inoculum was added to the

antigen production flasks with sterile 50 ml volumetric

pipettes. The flasks were incubated at 370 C for four

days. Antigens of the Corynebacteria were grown as indi-

-16-

cated above using Brain Heart Infusion Broth without
serum fraction. At the end of the incubation time the
cultures were killed by adding 1.% mls of formalin to
each 500 ml portion. The cultures were centrifuged in
a Servall angle centrifuge at 12,000 revolutions per min-
ute for forty-five minutes and the supernatant decanted.
The PPLOs and Corynebacterium were resuSpended in saline
buffered to pH 7.3. PPLO antigen was adjusted to a tur-
bidity allowing %8% transmittance on a Bausch and Lomb
Spectronic "20" colorimeter set at a wave length of 525
millimicrons (mu). Corynebactegig antigens were adjusted
to a turbidity corresponding to our #1 nephelometer tube.
The antigen was diSpensed into Kahn tubes and stored at
10° C.

Slide agglutination tests were done on each anti-
gen using normal serum and homologous immune serum.
Cross agglutination slide tests were performed also. One
drOp of standardized antigen was placed on a clean slide
and mixed with one drop of antiserum. The mixture was
allowed to react for three minutes and was observed under
100x magnification. A positive test was indicated by
clumping of the antigen within five minutes. Controls
were done by replacing normal serum for the immune serum
and by mixing buffered saline with the immune serum in
equal pr0portions.

The immune and normal sera were also tested for

-17-

the ability to agglutinate unrelated organisms by per—
forming slide tests on thirteen unrelated bacterial anti-
gens prepared as described for the Corynebacteria. Five
Corynebacteria of known species, Sarcina lutea, £3939-
bacillus planarum, Streptococcus pyogenes, Streptococcus
faecalis, Alcaligenes faecalis, Staphylococcus aureus,
Bacillus cereus, and Proteus morganiiwere employed in
this manner.

The titres of all PPLO and Corynebacteria were de-
termined by employing standard methods with the tube
agglutination test as described by Boyd (1956). Cross
agglutination titres were determined between S6 and CS
by determining zones of Optimal proportion and following
this with a standard tube agglutination test using the
antigens and antisera in all possible combinations.

This method is described by Boyd (1956). Absorption and
reciprocal cross absorption were performed with the anti-
gens and followed by agglutination and cross agglutination
tests as described by Boyd (1956).

-18-

RESULTS

The results of this study include observations made
on eleven organisms belonging to the genus Mycoplasma and
two organisms derived from them belonging to the genus
Corynebactggigm. These organisms were placed in the genus
Corynebacterium on the basis of their positive Gram Stain
reactions, positive catalase reactions, metachromatic
granules, palisade arrangement of the cells, ability to
reduce potassium tellurite and pleomorphism as typified
by club shaped forms.

The results obtained from morphological Studies will
be found on the succeeding pages in the form of photomi-
crographs. The photomicrographs will be presented in
groups representing the organisms as they will be grouped
by fermentation patterns. Organisms which best represent
the characteristics of each group were chosen for these
photographs. Since the morphological characteristics
vary so little, redundancy is reduced to a minimum by the
use of representative organisms.

Figuresl through 15 of PC-3 and P-6 are represen-
tative of the first group (A) determined by carbohydrate
fermentation. The colonies of PC-3 rarely exceed 55 mi-
crons (u) in diameter and grow deep into the agar. The
colony is round in most cases and has the appearance of

a mound of pepper. Vacuoles of regular shape are rep-

-19-

resented by clear areas evenly diSpersed throughout the
colony. No membranous edge is present at the periphery

of the colony.

__ ., --

Figure 1. Photomicrograph of PC-3 with a colony
diameter of 52 microns (u). Magnification.100X.

 

Figure 2. Photomicrograph of PC-3. Same colony as
above with a magnification of %00X. '

   

 

VF" ’ “fluff “V. #56 ,‘ fluff-C" “"77 _ a"
C-r O 7‘. 0' t . " ,“ ' qt,“
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I ‘ ‘ . finn-
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9' .9 x;-*
. 1.. 0 '.
s J" ' A
4 Q ,
t . " .; \ _. .
‘ 0
k. v; tel ‘
[s .
. O “ o .
i 4‘] .

Figure 3. Photomicrograph of PC-3. Same colony
as above with a magnification of 970x.

 

Figure %. Photomicrograph of a P-6 colony with
a diameter of 38u. Magnification of %00X

The colonies of P-6 represented in Figures % and 5

are the smallest of those studied, rarely growing to a

.21-

diameter greater than 38u. The colony grows into the
agar making it difficult to photograph without blurred
edges. Vacuoles of constant size are distributed uni-
formly throughout the colony. Colonies of P-6 tend to
have a more uneven margin than the other members of the

group.

 

Figure 5. Photomicrograph of P-6. Same colony

as above with a magnification of 970x.

Organisms PC-5, P-9, $6 and 08 (Figure 6 through
20) are the organisms which make up another group by vir-
tue of the similarity in their fermentation patterns.
PC-5 (Figures 6, 7 and 8) grows in colonies which have
irregular borders, many dark inclusions and a great var-
iety of sizes. The colonies rarely exceed 89u even on

aparcely pepulated plates. The vacuoles, present in

-22-

large numbers, are a dominant colony feature. These
bodies lack the tendency to be grouped as in L forms and
are generally constant in size, although one colony pic-

tured contains a large clear area l6u in diameter.

& I . v‘___. __7 ____ _ ‘ ,4; 'M'li

Figure 6. Photomicrograph of a PC-5 colony
with a diameter of 89u. Magnification 100x.

 

-Figure 7. Photomicrograph of the PC-5 colony
showed above. Magnification %00X.

-23-

Figure 8. Photomicrograph of the colony of PC-5
showed in the previous figure. Magnification 970x.

Figure 9 Photomicrograph of a P-9 colony with a
diameter of 115u. Magnification 100x

P-9 (Figures 9-11) grows in colonies which have

fissured surfaces and a diameter not exceeding 115u.

-gh-

Vacuoles may be more numerous at the edges of the colon-
ies. The colonies have an extremely deep center and

peculiar cauliflower-like edges.

 

 

 

.-
I - - O .I
o ‘ ' ‘ ‘ . . . | J
“in- arm. ‘z-b "4'

Figure 10. Photomicrograph of the colony of P-9
showed in the previous figure. Magnification.%OOX.

 

Figure 11. Photomicrograph of the colony of P-9
showed above. Magnification.970X.

-25-

86 colonies may reach a diameter of lhlu when
distributed far enough from one another to allow Optimal

conditions for growth. (Figures 12 and 13)

 

Figure 12. Photomicrograph of an 86 giant colony
with a diameter of 151u. Magnification hoax.

 

Figure 13. Photomicrograph of the 86 giant colony
pictured above. Magnification 970x.

-26-

The usual colony size of 36 (Figures lh through 16)
rarely exceeds 9lu and is almost always round with dense
but not well demarcated centers. The edge of the colon-
ies contains many small vacuoles, but few large bodies.

Areas of greater density are evident throughout the col-

onies.

9
. . .0

Figure 1%. Photomicrograph of a 86 colony with
the usual colony diameter of 9lu. Magnification

lOOX.

The colonies of CS (Figures 17 through 20) may
reach a diameter of 0.6mm after several days. After
twenty-four hours the size of the colony rarely exceeds
302u in diameter (Figure 17). The edges of the colony
are entire with a center slightly more dense than the
border. There is a slight tendency for the colony to

grow into the agar. The homogeneity of the colony does

-27-

not suggest the presence of large vacuoles or large bod-
ies. The impressions of the colonies stained with methy-
lene blue (Figures 18, 19 and 20) are naturally diminished
in size and cannot be used as a true picture of colony

diameter. These colonies show the pleomorphic elements

typical of the genus W.

9
ah‘
‘3

- 1.: o
_ 1'3 -

 

L

 

'7‘

Figure 15. Photomicrograph of the 86 colonies
pictured in the previous plate. Magnification 2001.

.23..

 

Figure 16. Photomicrograph of the 86 colony
pictured above. Magnification.97OX.

L———-_——-———— __ _— _.._ - _-

Figure 17. Photomicrograph of a 2% hour old col-
figgxof 03 with a diameter of 302a. Magnification

 

{fit . ,
V _ ’I‘ . 4 a; '9
.3] ‘
t"
:: ’3‘ k _ J”
:3 e .
$3
W $1!" 11'
' w.
a ‘ : fl. ’
1‘ > 1
a e
t
5":
‘ ..
a
.. I.

*3“ I M! _V i, (_i, ‘

Figure 18. Photomicrograph of an agar impression

preparation of CS stained with methylene blue.

Magnification 2OOX.

Another group based on carbohydrate fermentation
is represented by PC-h and PC-8 (Figures 21 through 26).
This group contains organisms which produce the largest
colonies. PC-h (Figures 21 through 26) deve10ps the
largest colonies of the group with which we are concerned,
but even this large colony rarely exceeds 15lu in diameter.
On low magnification this colony appears to have a thin
membrane bordering the even margin of the colony. This
membrane on higher power has the appearance of many de-
ve10ping vacuoles. The margin of the colony contains
many uniform clear areas and is not as dense as the cen-
ter.

PC-8 (Figures 2% through 26) has a colony diameter
of l32u and a very irregular shape. This irregularity

-30-

along with vacuoles of many sizes and the large size of
the colonies is a constant feature of PC-8. The colony
grows deeply into the agar and makes photography diffi-
cult eSpecially at higher powers.

 

Figure 19. Photomicrograph of the agar impres-

sion preparation of CS resented in the previous
figure. Magnification OX.

 

Figure 20. Photomicrograph of the agar impres-

sion preparation of CS presented in the previous
figure. Magnification 97ox.

 

Figure 21. Photomicrograph of a colony of PC-h
with a diameter of lSlu. Magnification lOOX.

CL, (Figures 25 and 26) whose diameter is 0.2H mil-

limeters at twenty-four hours, becomes 1.5mm after several

-32-

days and is large in comparison with the other organisms
previously described. The photomicrographs show a dense
centered colony with no tendency to grow into the agar.

.A band of less dense material is seen at the periphery

of the colony. The center of the colony seems to contain
vacuole-like bodies up to lOu in diameter. Very small
vacuole-like bodies are scattered throughout the colony.
0n.low power the central vacuoles look.like dense homo-

geneous inclusions and are easily picked out..

 

Figure 22. Photomicrograph of the colony of PC-h

Eggiented in the preceeding plate. Magnification

 

Figure 23. Photomicrograph of the colony of P04
presented in the preceeding plate. Magnification
9701.

 

 

Figure 2%. Photomicrograph of a colony of PC-8
with a diameter of 132u. Magnification 1001.

 

Figure 25. Photomicrograph of the colony of PC-8
gggsented in the preceeding plate. Magnification
X.

‘ .- We.

 

 

 

L__.__ . ._--_.___-- ______ __ __ __ _.____

 

.Figure 26. Photomicrograph of the colony of PC-8
presented above. Magnification 970x.

 

 

 

————- —

Figure 25. Photomicrograph of colonies of CL whose
largest member has a diameter of 0.2%mm. Magnifica-
tion lOOX.

 

.“ . '. . ~ . IL...‘ .'-'. I ’1 s-\
L \i.“. o 2: 7 -. 9.. 4‘ 30.9

 

 

Figure 26. Photomicrograph of the group of CL
colonies showed above. Magnification.h00X;

Over a seventeen day period observations of fermen-
tation were made that may be stated generally in order to
better orient the reader. These generalizations are:

1. All the test organisms except CL ferment arabinose,
galactose, levulose and xylose. Only one organism fails
to ferment glucose.

2. The action on arabinose and xylose is slow and incom-
plete, never appearing before nine days incubation and
never giving more than a 2/ reaction.

3. None of the organisms ferment dulcitol, inositol,
inulin, lactose, mannitol, raffinose, salicin, or sor-
bitol. PC-H is the only organism that showed action on
sucrose.

h. CL ferments five of the test carbohydrates. Three of
these carbohydrates - dextrose, levulose and xylose - are
included in the group fermented by all the other organ-
isms tested. The two remaining substances, starch and
maltose are regularly fermented by CL as well as by a
number of the other test organisms.

On the basis of the carbohydrate tests it is possible
to place each of the PPLOs and the CS into one of three
.groups.

The organisms in Group A were found to be PC-l,
PC-2, PC-3 and P-6. The organisms in Group B were found
to be PC-S, P-9, S6 and CS. The organisms in Group C were

found to be PC-h, PC-7, PC-8 and PC-lO. CL was not in-

-37-

cluded in these groupings due to its aberrant pattern
of fermentation. The results of these tests are sum—
marized in Table 1.

Group A was found to consist of organisms with the
ability to do the following:

1. Ferment the pentose sugars arabinose and xylose with
the production of a 2/ acid reaction and no gas.

2. Ferment the aldohexose sugars dextrose and galac-
tose with the production of acid and no gas.

3. Ferment the ketohexose sugar, levulose,with the pro-
duction of acid and no gas. None of the organisms of
this group ferment dulcitol, inositol, inulin, lactose,
maltose, mannitol, mannose, raffinose, rhamnose, suc-
rose, salicin, sorbitol, starch or trehalose.

Group B was found to consist of organisms which
fermented all of the sugars of Group A and in addition:
1. Ferment the methyl pentose sugar, rhamnose, with the
production of acid and no gas.

2. Ferment dextrin and starch with the production of
acid and no gas.

3. Ferment only one disaccharide, maltose, a reducing
sugar. None of the organisms of this group ferment dul-
citol, inositol, inulin, lactose, mannitol, raffinose,
salicin, sorbitol, or trehalose.

Group C was found to consist of organisms which

ferment all of the sugars fermented by Group B and in

-33-

addition:

1. Ferment the disaccharide, trehalose, which is a non-
reducing sugar. None of the organisms of this group
ferment dulcitol, inositol, inulin, lactose, mannitol,
raffinose, salicin, sorbitol or sucrose. 1

It was found that within each group the times re-
quired by members of the group to show a positive reac—
tion on a particular carbohydrate were not always uniform.
This observation is further complicated by the fact that
each organism within a group did not react to the same
degree on a particular carbohydrate. Table 1 represents
the time required to ferment the various carbohydrates
and the degree to which each organism used each carbohy-
drate.

The results obtained from carbohydrate tests on the
two "unrelated" Corynebacteria which reacted serologically
with CS and 86 are shown in Table l. The two reactors
are quite divergent in their patterns. CL pyogenes showed
a similarity to group B in that it fermented arabinose,
dextrose, dextrin, galactose, levulose, maltose, mannose
and xylose and did not ferment dulcitol, inositol, inulin,
mannitol or raffinose. The same organism is dissimilar
to Group B on the basis of its ability to ferment car-
bohydrates not included in the fermentative Spectrum of
Group B. These carbohydrates include lactose, salicin,

sorbitol, sucrose and trehalose. Starch is not fermented

-39-

TABLE .1.

The reactions of PPLOs and Corynebacteria on
twenty carbohydrates eXpressed in terms
of time and relative acid production*

 

Carbohydrate Organism:

PC:;¥ PC-2 PC-3 PCéh PC-53 P—6’ PC-7 PC-8
Arabinose 2-13 2-153 2-17 2—11 _2-17 2-15’2-11 2-13
Dextrose 2—15 2—15 0 2-11 2-17 2-15 3-9 2-13
Dextrin O O 0 2-11 3-11 0 3-6 0
Dulcitol O 0 0 0 0 0 0 O
Galactose 2-13 2-13 2-17 2-11 2-11 2-11 3-9 2-11
Inositol O O O O O O O O
Inulin 0 0 0 0 O 0 0 0
Lactose O O O O O O 0 0
Levulose 2-11 2-13 2-13 3-11 2-13 2-11 3-9 3-15
Maltese O O 0 2-11 3-17 0 3-9 2-13
Mannitol 0 0 O 0 O O O O
Mannose O O 0 2-15 0 0 3-9 3-9
Raffinose O O O O 0 0 O O
Rhamnose 0 0 0 2-15 0 0 0 2—15
Sucrose 0 O 0 2-15 0 O 0 0
Salicin 0 0 O O O O O 0
Sorbitol O O O O O O O 0
Starch O 0 O 0 2-17 0 2-11 2-15
Trehalose O O 0 0 O 0 2-11 2-11
gylose 2-11 2-13 2-13 219 2-11 2-11 2-11 2-2

 

 

* The degree of reaction is indicated by the first digit
in the body of the table.
ed by the second digit in the body of the table

_hQ-

The time in days is indicat-

TABLE 1 (Continued)

 

 

Qggpohydrate Organism

P-9 130-10 8? CS CL CE? CY* C*
Arabinose 2-15 2-13 2-11 2-13 0 2-1 2~2 0
Dextrose 2-15 3-9 3-4 2-13 3e2 3-1 0 o
Dextrin 2-11 3-9 3-9 3~l3 0 3-1 0 O
Dulcitol 0 0 0 O 0 O 0 0
Galactose 2-11 3-9 3-6 2-3 2-h 3-1 2-2 0
Inositol O 0 O 0 0 O O O
Inulin 0 O O 0 O. O O O
Lactose 0 0 0 ,0 0 2-2 0 O
Levulose 3-l7 3-9 3-9 2-15 3-h 3-1 2-2 0
Maltose 3-17 3-9 3—9 2-13 3-13 3-1 0 O
Mannitol 0 O O 0 0 O O O
Mannose 2-17 3-9 3-9 O 2-% 3-1 0 O
Baffinose O O 0 O 0 O O O
Rhamnose 2-17 0 0 0 0 3-1 0 O
Sucrose O O O O 0 3-2 0 0
Salicin 0 O 0 O 0 3-1 0 O
Sorbitol 0 O O O 0 3-3 0 0
Starch 2-15 0 3-9 0 2-2 0 0 0
Trehalose 0 2—13 0 O 0 3-13 0 0
Xylose 22l3 2-l3‘ 2-11 2-11 2-2 Vigglg O 0

 

 

* CY denotes Corynebacterium.X. CP denotes Corynebacterium
pyogenes. C denotes the control tubes.

-hl-

by C. pyogenes. This fact sets it apart from Group B
organisms most of which use starch.

Corynebacterium Y was found to have a fermentative
pattern less inclusive than that which might be considered
basic for this group of organisms. The organism fermented
only arabinose, galactose and levulose.

The results obtained from the slide agglutination
tests and slide cross agglutination tests were done with
organisms PC-3, PC-H, PC-5, P-6, PC-7, PC-8, P-9, S6, C8
and CL and are given in Table 2. Serological studies
were not done on organisms PC-l, PC-2 and PC-lO due to
the inability of these strains to grow in the quantities
required for the antigen preparations.

Briefly stated the results indicate that all of the
organisms that we worked with were antigenically related.
All of the PPLOs were agglutinated with a H/ reaction
when mixed with homologous antiserum. In all cases cross
agglutination between PPLOs was of the magnitude of either
3% or hf. CS was agglutinated with a h% reaction when
mixed with CS, PC-7, PC-9, S6, and CL antisera. CS was
agglutinated with a 3/ reaction by PC-3, PC-5 and P-6
antisera. The antiserum of PC-h agglutinated CS with a
2/ reaction. The antiserum of CS failed to agglutinate
PC-3, PC-h, PC-8 and S6 antigens, but showed a h/ activity
with PC-5, P-6, P-9, CS and CL antigens and a 3/ reaction
with PC-7.

-hg-

oanop one
no anon one an n3onm mm x: Op \H 809% poponw ma soaponapsflmmo mo oonwop one *

 

 

 

I I I I I I I I I I I onfiaom
I u; x: I x: a; x: x: x: I I no
I e; } in e; is is ta a on a 8
I I I e; e; e; e; in x: e; in em
I I s; e; } c; x: c; e; e; in mi.
I I I in } e; } } c; is E was
I I a } is is i; a e; e; c; you
I I e; e; is e; } c; is c; a: we
I I c; e; e; } e; c; E e; e; Tom
I I I is e; e; e; e; x: is e; .18
I I I c; c; e; e; u; e; e; c; Tue
eeuaem no mo mm qu MMWMaeemIou ImIe ,mIom aqua ImIoa was use

ego ace we .moquu enmao no
onomfipno pno mnomfipno one near moanomnom memop noapmnfipsammo opHHw

N mqmna

-h3-

The antigen, CL, was agglutinated with a H/ reaction
by all antisera except those of PC-3, FC-N and S6. The
antiserum of CL was quite inactive on all antigens except
CS and CL in which case a H/ reaction was indicated.

When thirteen stock cultures of bacteria of various
genera were prepared as antigens and tested by the slide
agglutination method with the antisera of CS and S6, only

two of the Corynebacteria in the group showed a strong

 

positive agglutination. Alcaligenes faecalis showed a
1/ reaction when tested against CS antiserum, but no re-
action with the S6 antiserum or the saline and normal
serum controls. 9. pyogenes and Corynebacterium X both
showed a h/ reaction with CS and S6 antiserum and no re-
action with the normal serum or saline controls. The

results of these tests are shown in Table 3.

TABLE 3

Slide agglutination tests showing the reactions
of thirteen unrelated bacteria with the antisera
of S6 and CS

 

 

 

 

 

 

Antigens Antisera

86 CS
Sarcina lutea - -
Lactobacillus planarum - —
Staphylococcus aureus - -
Streptococcus pypgenes - -
Streptococcus faecalis - -
Bacillus cereus - —

 

Corynebacterium equi
Corynebacterium diptheriae
Corynebacterium S-15
Corynebacterium pyogenes
Qgrynebucterium l
Alcaligenes faecalis
froteus morgani

-hh-

l-c’et’l
II—J-F‘Jl'l

The results obtained from the tube agglutination
tests that were done with the antigens and homologous anti-
sera of all the test cultures may be seen in Table h. The
titres shown in these eXperiments are very erratic, rang-

ing from 16 to 20h8.

TABLE 3

Agglutinating antibody titres of the stock PPLOS
and Corynebacteria antisera as determined by the
tube agglutination test

 

Antisera rc-3 Pcefl PC-S P-6 FC-7 PC-B r-9 86 C3 CL
Titre 20MB 102M 102% 32 2oh8 16 56 32 6ho 32

Optimal prOportions studies were done on the anti-
gens and antisera of CS and S6. The results compiled in
Table 5 show the antigen concentration to be Optimal at
l/h of the strength of the standardized antigen when deal-
ing with CS and l/2 the strength of the standardized anti-
gen when dealing with S6.

TABLE 5

nesults of Optimal prOportion point tests done
with S6 and CS and their homologous antisera

 

CS Antigen CS Antiserum S6 Antigen 86 Antiserum
Dilution Titre Dilution Titre

1/1 320 l/l 16

1/2 320 1/2 32

1/h 6H0 1/# 0

1/8 6RD

l/l6 320 _

 

The results obtained from cross agglutination tests

_ug-

using the tube method and employing S6 and CS antigens

and their respective antisera are given in Table 6. The
titres were found to be highest in the zone of optimal pro-
portions established previously at 1/2 concentration of stand-
ardized FPLO antigen and l/H concentration of standardized

S antigen. Cross reaction was definitely found to be

C)

Iresent when CS antigen was crossed with S6 antiserum. A

h

very low titre was found when S6 was tested with CS anti-

serum.

TABLE 6
Results obtained when cross agglutination reactions
using the tube method were performed with S6 and CS
and their respective antisera

Antigen Antiserum

 

 

36 cs
36 32 2
cs 32 6nd

TABLE‘Z

Preparation of absorbed antisera

 

Antiserum to be Absorbing antigen Absorbed antiserum
absorbed number
86 36 1
CS S6 2
CS CS 3
36 cs LI

 

The results of the antiserum absorption studies accom-
plished with the antigens of CS and S6 and the antiserum pre-

pared according to Table 7 are represented in Table 8.

—h6-

MO titre was found when S6 antiserum absorbed with S6
antigen (Absorbed serum #1) was reacted with the antigens
S6 or CS. Io titre was found when CS antiserum absorbed
with CS (Absorbed serum # 3) was reacted with the antigens
S6 or CS. No titre was found when S6 antiserum absorbed
with CS (Absorbed serum # H) was reacted with the antigens
CS or S6. A drOp in titre from 6H0 to 32 was Observed
when CS antiserum absorbed with PLLO (Absorbed serum # 2)

was reacted with CS antigen. No S6 titre was present in

the # 2 serum.

TABLE §

Titres Obtained when homologous S6 and CS antisera
absorbed with homologous and heterologous antigens
were reacted with homologous and heterologous
antigens usinggthe tube agglutination method

 

Antisera
Antigens l 2 3 h
S6 - - - -
CS — 32 - -

 

_.h 7 ..

DISCUSSION

The appearance of the microcolonies under discussion
seems to identify them as PPLOs rather than L forms on
the basis of descriptions of these organisms given pre-
viously by other workers in the field. The following
differentiating characteristics, upon which the above
statement is based, were found in the publications Of
Edward (195%), Klieneberger-Nobel (195%) and Mittler
(1956).

Typical PPLO colonies ranging from less than 0.1 mm
to approximately 0.3 mm are considerably smaller than the
L forms which range from 0.5 mm to 1.0 mm in diameter.
PPLOs isolated from living sources require some kind of
serum for growth in nutrient material as Opposed to L
forms which can be grown in a determined medium lacking
serum. PPLOs are isolated from animal sources and main-
tained with considerable ease as compared to the diffi-
culties in isolating L forms. L forms usually grow
superficially on the surface of agar whereas PPLOs grow
deeply into the agar. L forms produce colonies with
many pleomorphic elements present which vary greatly in
size; whereas, the colonies of PPLO have an even granular
appearance. L forms cannot be held as such in routine
culture but require special additives to prevent immediate
reversion to their bacterial precursor. PPLOs can be

routinely cultured and isolated lacking L form inducing

-H8-

compounds in the culture media.

It has been our eXperience that the central button
or fried egg appearance of PPLOS so frequently mentioned
in the literature does not appear until many agar passages
have been carried out. These central structures are so
infrequent that to this author they do not constitute a
characteristic of the avian or porcine organisms so far
studied. The exact Opposite characteristic, that is,
tiny, irregular, completely homogeneous colonies seem to
be the rule. Numerous broth and agar passages must be
carried out before the colonies have entire margins and
increase in size. None of the colonies produced by the
stock PPLOS we have discussed here show significant
characteristics of L forms. The most vacuolated organ-
ism in this group, PC-8, does not have either the great
diversity of vacuole diameter or the quantity of vacuoles
shown in PPLOS photographed by Dienes (1951) or the L
forms photographed by Dienes (1953).

Colony sizes could probably be used to group these
organisms since we would arrive at the same groups re-
ported for fermentative similarities. It so happens that
the group with the smallest colonies also has the most
limited fermentative Spectrum and the group with the lar-
gest colonies the broadest fermentative Spectrum. This
may lead one to SuSpect a deficiency in viability of the

small colony organisms or an inability to produce enough

-hg-

organisms to Show a change in the tubes. The low power
photomicrograph of PC-3, however, Shows many colonies
present on the agar and demonstrates the viability of

the weakest fermenter of Group A. Yamamoto and Adler
(1958) report avian PPLOS with no known capacity for fer-
mentation which produce typical PPLO colonies in abundance.
Padgett and Schoenhard (unpublished data) found eight PPLOS
of porcine origin to be similar in morphology to those de-
scribed here. One small variety tested for fermentative
ability was found to have a Spectrum identical to P-6.
Smith PeOpleS and Morton (1957) described the Corynebac-
gggig associated with the Campo strain of PPLO as a minute
colony about five times the diameter of the PPLO. The
colony was Slightly raised, dense at the center, with an
uneven edge. With the exception of the irregular edge,

CS fits this description very nicely. CL, as Opposed to
CS, Shows little morphological similarity to Smith's
gprynepacteria.

Since the organisms producing the microcolonies fit
the descriptions given by previous authors for PPLOS, we
must conclude that these organisms are probably members
of the PPLO group. Since the groups based on colonial
configuration correspond closely with the groups made up
of organisms with Similar fermentative patterns, there is
justification for considering the groupings a tenable ap-

proach to relating the organisms under consideration.

-50-

The purpose of performing fermentation studies on
this group of organisms may be considered three-fold in
nature. First and foremost was to seek for an insight
into the relationship between the two Corynebacteria and
the PPLOS, especially 36, from which the Corynebacteria
were thought to be derived; second, to determine whether
relationships existing within the group of avian PPLOS
was desired; third, a relationship between avian and por-
cine PPLOS was sought.

The relationship of S6 and CS in terms of fermenta-
tive pattern appears to be a very close one. CS is de-
ficient only in the fermentation of starch and mannose.
This slight difference between the metabolism of a PPLO
and its suSpected ancestor is not sufficient evidence
with which to dismiss a close relationship. Such a dif-
ference was reported by Vittler (1956) to occur during
her study of a Corynebacterium and its related PPLO.
Differences in carbohydrate fermentation are frequently
displayed between strains within a Single species so that
such a difference might be expected in the case under con-
sideration.

In the case Of CL much the same type of variance was
discovered. The deficiency in this instance is CL's in-
ability to ferment arabinose and dextrin. Since all of
the PPLOS and CS have been found to use arabinose to the

same degree, this deletion from the fermentative Spectrum

-51-

of CL represents a more surprising occurrence. A diverg-
ence of such a nature, although within the realm of pos-
sibility, makes close correlation between CL and the PPLO
group being studied very difficult. Only the genetic in-
stability of this strain as evidenced by the multiplicity
of colonial variants produced by the organism offers an
eXplanation for the incongruity of the fermentation pat-
tern and the serological dissimilarity which was mentioned.
The possibility that CL is a contaminant would appear to
be small due to the precautions taken against such an
occurrence and the length of time taken for the bacteria
to appear in the original flask.

The relationship of the two Corynebactegia to the
other PPLOs differs little from that which has already
been discussed for 86. Those places where divergences in
fermentative patterns were discovered are the same for
other members of group B.

It is concluded that CS differs no more than any of
the PPLOS in the fermentation pattern we have found typ-
ical of the PPLOS studied. CL differs only slightly in
terms of the number of carbohydrates fermented but signi-
ficantly on the basis of arabinose fermentation. This dif-
ference may possibly be attributed to genetic instability
or a low mutation rate.

The relationship between members of the groups stud-

ied has already been approached in terms of groupings. It

-52-

was observed that certain carbohydrate reactions could be
eXpected from the group as a whole and that other reac-
tions served to place the organisms in one of three groups.
Group A simply represents the basic fermentative pat-
tern eXpected from members of the PPLO group. Studies
by Freundt (195%) on PPLOS from human sources reveal a
fermentative pattern including gas production by some
PPLOS from some carbohydrates. In Freundt's patterns
xylose is not fermented and gas is produced from galac-
tose. Adler (1957) in his study of avian PPLOS found a
similarity between the organisms studied on the basis of
fermentation. Adler‘s single fermentation group includes
two serological groups and differs from the groups dis-
cussed here by the uniform ability of the organisms to
ferment sucrose. Arabinose and xylose were not tested.
Yamamoto and Adler (1958) described five serologically
distinct groups which differed from each other by either
complete inaction on carbohydrates or time required for
fermentation to occur. These relations did not hold in
all cases according to Yamamoto. Three of the five groups
were represented by only one organism, and the range of
carbohydrates did not include some of those found in this
study to differentiate the organisms into groups. Without
consideration of time the basic fermentation pattern of
Yamamoto's carbohydrate active organisms are identical to

those presented here with the exception of sucrose.

-53-

Group B and Group C organisms were classified as such
mainly on their ability to ferment disaccharide sugars as
a part of their broader Spectrum. Group C can use non-
reducing disaccharide sugars whereas Group B organisms are
unable to do so. This seems a more tenable approach than
the use of methyl pentose fermentation as a distinguishing
characteristic.

Morphological studies seem to bear out the group dif-
ferences observed in carbohydrate fermentation studies and
so add a degree of validity to the scheme used.

It is interesting to note that P-6 and P-9, both por-
cine strains, are classified in different groups. At
once one might be led to believe that the porcine strains
should be more closely related to each other than to the
avian strains by virtue of a common host. But in reality
the host may have very little to do with the resemblance
or dissimilarity. The reSpective organisms from which the
porcine strains were derived may easily have been found
in avians also and serve as the ancestors for related
avian PPLOS. Perhaps this is just another example of com-
mon bacterial flora. There exists a good possibility that
the divergence in fermentation represents only a superfi-
cial difference in organisms which have a common ancestry
in a bacterium indigenous to both porcines and avians.
This latter alternative is also suggested by serological

evidence.

-54-

The similarity of fermentative Spectra in the PPLOS
studied suggests more than just a casual relationship be-
tween these organisms. In the light of the fermentative
similarities the organisms must belong to the same genus
and probably the same Species. The organisms placed in
the genus Corynebacterium on the basis of structure and
other tests previously mentioned must be considered close-
ly related to the PPLOS in view of fermentative similar-
ity and antigenic Similarity.

The two diptheroids showing antigenic relationship
to 86 do not have as much in common with the PPLOS in
terms of fermentation as do CS and CL and probably consti-
tute orgaiisms with similar group antigens. The mere fact
that only Corynebacteria, of all the organisms used in the
non-Specific Slide agglutination tests, showed a similar
antigen structure Speaks well for this genus as the ances-
tor of this group of PPLOS.

The serological studies presented in this paper were
undertaken in order to approach an understanding of the
relationships existing between the test organisms. The
majority of the serological studies was directed toward
demonstrating a relationship between 86 and C3.

Before a critical examination of the evidence is
made the fact that the methods used preclude any but a
qualitative analysis of the data should be well borne in

mind. On the basis of available information it is impos-

-55-

sible to know whether common, chemically identical antigens
shared by this group of organisms are reSponsible for cross
reactions or whether chemically similar antigens are pre-
sent in the group which react with heterologous antisera.
Since dilution titres are not indicative of the type and
degree of reaction between antibodies and antigens or of
their valencies, but are merely rough estimates of the
antibody dilution at which no more reaction is percep-
tible, these results cannot be interpreted in a quanti-
tative way. It is altogether possible that non-visual
combinations are present. Inability to control serum
quality further limits the analysis of the results.

Kabat and Keyer (l9h8) discuss in detail the above limi-
tations of qualitative tube agglutination techniques.

The use of only one animal for the production of each anti-
serum was a serious error and makes it impossible to accu-
rately determine whether low titre serums are the result of
unresponsive animals or organisms lacking the ability to
stimulate the formation of high quality antisera.

The results of the slide agglutination tests seem to
indicate that all of the PPLOS stimulate the production of
Specific homologous antisera. Each antiserum posesses the
ability to agglutinate all of the tested PPLOS. On the
basis of this observation one is led to the belief that
chemically identical antigens are held in common by the

test organisms or chemically similar antigens are distri-

-56-

buted throughout the group. A relationship like the

latter xists between Proteus and nickettsia. Since

cross reaction between all of the PILOS is so uniform,
chemically identical antigens probably represent the

most plausible explanation. Unlike the different anti—
genic groups found by Adler (1957) and Yamamoto and Adler
(1958) a single group is suggested by these results. Gian-
forte (1955) in a study conducted with seven avian PPLOS
reported a single serological group.

The reactions of CS antiserum cannot be eXplained with
surety in those cases where no agglutination occurred.
When one considers that Optimal proportions were necessary
for the very low titre of CS antiserum obtained from S6,
in the more sensitive tube agglutination tests, the re—
sults of the slide tests are not incongruous with the
over-all results. An eXplanation for the lack of aggluti-
nation power of CS antiserum for 86 must, however, still
be sought.

Antigens possessing a low valency, but retaining
the ability to stimulate antibody production may play a
role in this phenomenon according to Boyd (1956). A de-
crease in cell wall material containing such antigens or
combining sites is in keeping with the concept of the
PPLO. A further simplification of previously antigenic
cell wall material to the level of a simple haptene could

eXplain why antibodies of C3 are ineffective on 36.

-57-

Since the S6 haptenes stimulate no antibodies, those pro-
duced by active antigens of S6 have access to the surface
of the cell and, therefore, Show a visible titre. The
antigens in CS, if represented by haptenes in the PILO,
may stimulate antibodies which interfere with the homol-
ogous visible reaction by homologous reaction with hap-
tenes producing steric hinderance.

The organisms PC-3, PC-h and PC-8 are probably un-
able to react with CS antiserum for the reason just de-
scribed, but there is the possibility that they are not as
closely related to CS as is S6 in View of their divergence
from the group B fermentative pattern.

The inability of CL antiserum to agglutinate any of the
PPLOS is seemingly incongruous with the assumption that
CL is a precursor to S6 and related to the other PPLOS.

It will be noted, however, that CL antisera has a much
lower titre than CS antisera, which showed a minimal reac-
tion with S6 antigen. This fact coupled with the possi-
bility of a low antigen valency in the PPLOS and the
possible existence of haptenes could account for the di-
vergence. Since the animal used for the production of
this antiserum was six months old and supposedly capable
of good antibody production, either the preparation of the
antigen, the immunization procedure, or a poor natural an-
tigenicity must be suspected to be the cause of the low

titre.

The ability of all of the antisera to agglutinate CS
probably indicates that CS contains enough common antigens
of the required valency to react perceptibly.

The inability of PC-3 and PC-h antisera to react with
CL may be eXplained on the basis of a lack of common anti-
gens.

The ability of CS and S6 to agglutinate supposedly

unrelated Corynebacteria may be interpreted as evidence

 

that both organisms contain the group antigen described
for Corynebacterium by Hoyle (l9h2) and Niell, Richardson,
Flemming, Sugg, and Gaspari (1931). Since this antigen

is located in the deeper portions of the cell, it is not
surprising to find it in the PPLOS if these organisms rep-
resent bacteria with diminished peripheral structures.
This data strengthens the argument for derivation of

these PPLOS from members of the genus Corynebacterium.

 

The fact that the antiserum titres of the test
organisms vary to such a degree is unfortunate, especial-
ly when low titres such as those encountered in the anti—
serum of S6, PC-8, P-6 and CL occur. The low titres may
be eXplained by the work of Baumgartner (193%), who showed
that old animals produce antibodies of inferior quality
and in diminished quantity.

The low titres obtained when cross agglutination re-
actions were performed using S6 and CS antigens and their

homologous antisera may be interpreted in the same way as

-59..

the slide arglutination cross reactions.

u’»

1

The-implications cf the absorption tests only Serve

to verify what has previously been discussed. The re—

sults may be correctly predicted in the light of preceed-
ing interpretations. 36 antiserum would not be exp"ctcd

to react with either 36 antigen or C3 antigen ii the homo-
logous :ntibodies icn'iknrwwi those held in.Czr Jhl‘ l

. - ‘V - n 2,. . r‘ «x -1 r. A 1
were rtsorbed with an excess of 3c antigen. [he sane 1e-

1ation holds for the antiserum of C3 absorbed 'ith Co
antigen. The reiult of absorbing the antiserum of S6
\ith CS citigen would naturally be the absence of a titre

for CS. The fact that all of the visible titre for S6 is

antigens responsible for stimulating the product.
agglutinating antibodies of S6 are found in CS. ihe ob-

‘1 §O

servation of a titre for Co after absorbing CS antisera

(‘f‘
3'

cu

Cf
'3
0

Ft

C C

'3
J wt.

U]

with S6 seems to indicate that CS has i en

s4

.
tr

r
J...

g)

_L O

inciting the production of antibodies which are not pre-
sent or represented by haptenes in S6. Conclusive evi-
dence for an exylanation of the inability of CS antibodies,
stimulated by antigens held in common with S6 to aggluti-
nate S6 where homologous antiserum does cause egg
is not forthcoming, but a plausible explanation has been

offered.

_-60-

SUMMARY

The organisms isolated from chickens with CBD and
pigs with Glasser's disease proved to be members of the
pleurOpneumonia-like group of organisms on the basis of
morphological characteristics considered by many authors
to be typical and unique for the group.

The entire group of PPLOS tested showed similar fer-
mentation capacities and complete serological cross reac-
tions. Groupings arrived at by morphological studies
corresponded exactly with groupings made on the basis of
carbohydrate fermentation.

The carbohydrate reactions of two Corynebactegia
which were presumed unrelated to the 86 strain but which
were agglutinated by 86 antiserum were dissimilar from

each other and all of the test organisms.

The two Corynebacteria associated with the Zander or
86 strain of MyCOplasma gallinarum were both similar to the
PPLOS investigated on the basis of their capacity to fer-
ment carbohydrates. Agglutination tests indicated that the
two Corynebacteria have common antigens with most of the
tested strains of MyCOQlasma gallinarum. One of the 923x-
nebacteria, CS, was more closely related to 86 and the
other PPLOS in all respects than was CL.

In the light of the close fermentation patterns of
CS and 86, the serological relationship between CS and 86

and the existence of what appears to be a Corynebacterium

~61-

group antigen in 36, this author feels there is reason to
consider 86 a part of the natural life cycle of Coryne-

bacterium 3. Evidence has been presented which indicates

 

the possibility that all of the PELOs considered may be
part of a group derived from members of the genus Coryne-

bacterium and capable, under certain conditions as yet

 

undiscovered, of reverting back to these parent strains.

It is this author's Opinion that if the results and
conclusions p1es e ted he a1e valid ones, then there is
far more than scholastic import to be connected with the
phenomenon of reversion. The maintenance of health and
the prevention of disease depends primarily upon the po-
tential host's ability to use its defense mechanisms
egains t the va riety of harmful agents that endeavor to
gain entrance to the body. Since disease is a common
occurrence it is obvious that the parasite finds ways of
by-r‘esslnr host defem1 es.

nntioo 1.3 a rear to be a host oeiense mechanism,
and it may we the 1e 1cse~iitio of a successful in vivo
by-pass of this host defense that Le witness when a bac-
teria is transformed to a TPLO or a r110 reverts to a
bacteria.

1'10 living orgaiism survives that is unable to alter
itself to resist f1eviously atal environmental conditions.

we might postulate then, that the bi h;:sic nature of the

bacteria capable of transformation may be the result of

environmental conditions which are far more favorable to
one form than to the other. The host's defenses are pene-
trated when antibodies stimulated by complete bacterial
antigens become ineffective on the reduced antigens or
haptenes of the PPLO form. The host remains in danger

of relapse if the PPLO remains in the body until such a
time that the body relaxes its defenses to a point where
it again becomes suitable for parasitism by the reverted
bacterial form.

It is hOped that this research has added to the
steadily strengthening argument for reversion of members
of the genus MyCOplasma to bacteria and has been heuris-
tic to the extent of stimulating more work in this area
especially as regards the develOpment of the implica-

tions of this phenomenon and its exact mechanism.

-53-

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.: ‘.‘.
“A
_L‘- o o o O o
o , '
o
. '.
C
_‘ - 9.
5'0 1 . a
o o o * A g F.”
a. if (i r U‘ “ a J
tr
.‘.' ' '3-r'3 ’ o 'L O
2“ ‘(‘ . 2‘36 . o ' ' o I
.itfl.‘) . 0" A t 9-.
’IJQE‘Lfifi[ 9w g 'g'1v 7

“b omfl .iqflOQ o ‘11—‘11;

r 4“" 531i . :1; Lcrzu 7
v ._ ' ~ 94‘: - q‘.'--4- 7,4. "_-

v J 1‘ \ . . ' v‘,
'3‘0. wan ,11e 's1 ,.

’9 earn? 1 as“ t
.59>-8J£ .‘I

-

ammo? J not) stem ad-s'
«nsnnmnsngo'mefq '10 211:1:
0° S-OBS' (:22. 'O-[C

., ,
-0 ammo} 1 an! no uncrifix
08VS~ATS :33
Esotqofctd ex: 10 orii

( ‘9 .‘. J
1w: 1.1-} 2.1nnmuonim'msig 9d3
To aetiaotfringsr 9nd

-~'S:’~1‘1‘F .t‘ ,.

 

__-—‘ l —'

1_p—-

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-67-

A PPEND IX

TABLE 10

Fermentation reactions of PC-2
on twenty carbohydrates*

 

 

mm. d ate 2 1W
2_fi h 6 9 l1gfil3 15 12_
Arabinose - - - - l 1 2 2
Dextrose — - — — 1 1 2 2
Dextrin - - - - - - - -
Dulcitol - - - - - - - -
Galactose - - - - 1 2 2 2
Inositol - - - - - - - -
Inulin - - _ - - - - -
Lactose - - - - - - - -
Levulose - - — - 1 2 2 2
Maltose - - - - - - - -
Mannitol - - - - - - - -
Mannose - — - _ 1 1 - -
Raffinose - - - _ 1 - - -
Rhamnose - — — - 1 - - -
Sucrose - - - - - - - _
Salicin - — _ _ - - - -
Sorbitol - - - - - - - -
Starch - — - - - - - -
Trehalose - - _ - - - - -
Xylose - - - - 1 2 2 2

* The numbers in the body of the table indicate
the degree of reaction on the carbohydrate.

-69...

TABLE 11

Fermentation reactions of PC-3
on twenty carbohydrates*

 

 

 

 

Carbohydrate Incubation time in days
“”7 3':— 2 MW3 15 17
Arabinose - - - - l l 1 2
Dextrose — - - - - - - -
Dextrin - - — - - - - -
Dulcitol - - — _ - - - -
Galactose - - - - l l l 2
Inositol — - - - - - - -
Inulin - - - - - - - -
Lactose - - - - - - - -
Levulose - - - 1 2 2 2 2
Maltose - - - - - - - -
Mannitol - - -l - - - - -
Mannose - - - - - - - -
Raffinose — _ - - - _ - -
Rhamnose. - - - - - - - -
Sucrose - - - - - - - -
Salicin - - - - - - - -
Sorbitol - - - - - - - -
Starch - - - - - - - _
Trehalose - - - - - - - -
XYlose - - - 1 2 2 2 2

* The numbers in the body of the table indicate
the degree of reaction on the carbohydrate

-70-

TABLE 12

Fermentation reactions of PC-H
on twenty carbohydrates*

 

 

Carbohydrate Incubationgigme in Days
2 1+ 6 9 11 11 15 1L

Arabinose - - - - 2 2 2 2
Dextrose 1 1 1 1 2 2 2 2
Dextrin ‘ - - - - l 2 2 2
Dulcitol - - - - - - - -
Galactose - 1 1 1 2 2 2 2
Inositol - - - - - - - -
Inulin - - - - - - - -
Lactose — - - 1 1 - - -
Levulose - 2 2 2 3 3 3 3
Maltose - - - - 1 2 2 2
Mannitol - - - - - - - -
Mannose - - - - 1 1 2 2
Raffinose - - - - l 1 l -
Rhamnose — - - - 1 1 2 2
Sucrose - - - - 1 1 2 -
Salicin - - - - - 1 1 -
Sorbitol - - - - - 1 1 -
Starch — - - - 1 1 1 1
Trehalose - - — _ - 1 1 -
Xylose - l 1 2 2 2 2 2

* The numbers in the body of the table indicate
the degree of reaction on the carbohydrate.

-71-

TABLE 13

Fermentation reactions of PC-S
on twenty carbohydrates*

 

 

Carbohydrate Incubation Time in Days

2 h 6 9 ll 1 l l
Arabinose - - - - l l l 2
Dextrose ‘ - - - - 1 1 l 2
Dextrin - - - - 1 3 3 3
Dulcitol - - - - - - - -
Galactose - - - 1 2 2 2 2
Inositol - — - - - - - -
Inulin - — - - - - - -
Lactose - - - - - - - -
Levulose - - — 2 1 2 2 2
Maltose - - - - - - 2 3
Mannitol - - - - - - - -
Mannose - - - - - - - -
Baffinose - - - - - - - -
Rhamnose - - - - - - - -
Sucrose - - - - - - - -
Salicin - - - - - - - -
Sorbitol _ - - - - - - -
Starch - - - - 1 - - 2
Trehalose - - - - - - - -
Xylose - - - 1 2 2 2 2

* The numbers in the body of the table indicate
the degree of reaction on the carbohydrate

-72-

TABLE 1%

Fermentation reactions of P-6
on twenty carbohydrates*

 

 

Carbohydrate Incubatiog111me in Days
2 1+ 6 9 _1111 13 1§_12_

Arabinose - - - 1 1 l 2 2
Dextrose - - - 1 1 l 2 2
Dextrin - - - - - - - -
Dulcitol - - - - — - - -
Galactose - — - v- 2 2 2 2
Inositol — - - - - - - -
Inulin - - - - - - - -
Lactose - - - - - - - -
Levulose - - - 1 2 2 2 2
Maltose - - — - - - 1 -
Mannitol - - - - _ - - -
Mannose - - - - - - 1 -
Raffinose - — — - - - - ' _
Rhamnose - — - - - - 1 -
Sucrose _ - - - - - 1 -
Salicin — _ - - _ - 1 -
Sorbitol - - - - - - - _
Starch - - — - - - - -
Trehalose - — - - - - - -
Xylose - - - 1 2 2 2 2

* The numbers in the body of the table indicate
the degree of reaction on the carbohydrates.

J73-

TABLE 15

Fermentation reactions of PC-7
on twenty carbohydrates*

 

 

 

Cagbohydrate Incubation Time in Days

2 W1 1
Arabinose - - - - 2 2 2 2
Dextrose — - 1 3 3 3 3 3
Dextrin - - 1 3 3 3 3 3
Dulcitol - — - - - - - -
Galactose - - 1 3 3 3 3 3
Inositol — - - - - - - -
Inulin - - - - - - - -
Lactose — - - - - - - -
Levulose - - 1 3 3 3 3 3
Maltose - - 1 3 3 3 3 3
Mannitol - - - - - - - -
Mannose - - 1 3 3 3 3 3
haffinose - - - - - - - -
Rhamnose - - - - 1 1 - -
Sucrose - - - - 1 1 - -
Salicin - - - - 1 _ - -
Sorbitol -’ - - - - - - -
Starch - - - - 1 2 2 2
Trehalose — — - 1 2 2 2 2
Xylose - - - 1 2 2 2 2

* The numbers in the body of the table indicate
the degree of reaction on the carbohydrate

-7b-

TABLE 16

Fermentation reactions of PC-8
on twenty carbohydrates*

Cgrbogydrate Incubation Time n Da 5
2 h 9 11 1 1 l

 

 

Arabinose - - - l 1 2 2 2
Dextrose - - - 1 l 2 2 2
Dextrin - - - - - - - -
Dulcitol - - - - - - - -
Galactose - - - 1 l 2 2 2
Inositol - - - - - - - _
Inulin - - - - - - - -
Lactose - - - - 1 - - -
Levulose - - - 2 _ 2 2 3 3
Maltose - - - - l 2 2 2
Mannitol - - - - - - - -
Mannose - - - 3 3 3 3 3
Raffinose - - - - - - - -
Rhamnose - - - - 1 1 2 1
Sucrose - - - - - - - -
Salicin - — - - - - - -
Sorbitol - - - - - - - -
Starch — - - - 1 1 2 2
Trehalose - - - 1 2 2 2 2
Xylose - - - 2 2 2 2 2

* The numbers in the body of the table indicate
the degree of reaction on the carbohydrate.

-75-

TABLE 17,

Fermentation reactions of P-9
on twenty carbohydrates:

 

Carbohydrate Incubat on Time in Da 3
2 9 ll 1 1

 

Arabinose - - - 1 1 l 2 2
Dextrose - - - 1 1 A 1 2 2
Dextrin - - - 1 2 1 2 2
Dulcitol - - - - - - - -
Galactose - - - 1 2 2 2 2
Inositol A - - - - - _ - -
Inulin - - - - - - ‘- -
Lactose - - - - - - - -
Levulose - - - l 2 2 2 3
Maltose - - - 1 2 2 2 3
Mannitol - - - - - - - -
Mannose - - - - - 1 - 2
Raffinose - - - - - 1 - -
Rhamnose - - 1 - - - 1 ,2
Sucrose - - - - - - - -
Salicin - - - - - - - -
Sorbitol - - . - ,_ - - - -
Starch - - - _ 1 2 2 2 2
Trehalose - - - - -. - - -
Xylose - - - 1 2 2 2 2

* The numbers in the body of the table indicate
the degree of reaction on the carbohydrate.

-75-

TABLE 18

Fermentation reactions of PC-lO
on twenty carbohydrates*

 

 

 

Carbohydgate Incubation g_me 1n Days
""f 2 [r 6" 9 11 13 15 1.2..
Arabinose - - - - 1 2 2 2
Dextrose - - 1 3 3 3 3 3
Dextrin - - 2 3 3 3 3 3
Dulcitol - - - - - - - -
Galactose - - l 3 3 3 3 3
Inositol - - - - - - - -
Inulin - - - _ - - - -
Lactose — - - — - - - -
Levulose - - 1 3 3 3 3 3
Maltose - - 2 3 3 3 3 3
Mannitol - - - - — - - -
Mannose - - 2 3 3 3 3 3
fiaffinose. - — — - - - - -
Rhamnose - - - - - - - -
Sucrose - - - _ - - - -
Salicin - - - - - - - -
Sorbitol - — — - - - - -
Starch - — - - - - - -
Trehalose - — - ' 1 2 2 2 2
Xylose - - 1 1 2 2 2 2

* The numbers in the body of the table indicate
the degree of reaction on the carbohydrates.

-77-

TABLE 19

Fermentation reactions of 86
on twenty carbohydrates*

 

d ate cubat me Da

2__7 9 ll 1 1 l
Arabinose 1 1 1 1 2 2 2 2
Dextrose 1 1 3 3 3 3 3 3
Dextrin 1 1 3 3 3 3 3 3
Dulcitol - - - - - - - _
Galactose 1 3 3 3 3 3 3 3
Inositol - - - - - - - -
Inulin - _ - - - - - _
Lactose - - - - - 1 1 -
Levulose 1 3 3 3 3 3 3 3
Maltose 1 3 3 3 3 3 3 3
Mannitol - - - - - - - -
Mannose - 2 2 3 3 3 3 3
Raffinose - - - - - - - -
Rhamnose - - - - 1 1 1 1
Sucrose - - - - - - - -
Salicin - - - - - - - -
Sorbitol - - - - - - - -
Starch - 2 2 3 3 3 3 3
Trehalose - - - - - - - -
Xylose - 1 1 1 2 2 2 2

* The numbers in the body of the table indicate
the degree of reaction on the carbohydrates.

-78-

TABLE 20

Fermentation reactions of CS
on twenty carbohydrates*

 

 

Carbohydrate Incubation Time in Days
’“C 21_ H 6 9 ll 13 15 12
Arabinose - - - - 1 2 2 2
Dextrose - - — - 1 2 2 2
Dextrin - - - 2 2 3 3 3
Dulcitol - - - - - - - -
Galactose - - - 1 1 2 2 2
Inositol - - - - - - - -
Inulin - - - - - - _ -
Lactose - - - - - 1 1 -
Levulose - - - 1 1 2 2 2
Maltose - - - 1 1 2 2 2
Mannitol - - - - - - - -
Mannose - - - - - 1 1 1
Raffinose - - - - - - - -
Rhamnose - - - - - 1 1 -
Sucrose - - - - - 1 1 -
Salicin - - - - - - - -
Sorbitol - - - - - 1 1 -
Starch - - - - 1 1 1 -
Trehalose - — - - - - 1 -
Xylose - - - 1 2 2 2 2

* The numbers in the body of the table indicate
'the degree of reaction on the carbohydrates.

-79-

TABLE 22

Fermentation reactions of Qggynebacterium Y (CY)
and Corynebacterium pyogenes (GP) on
twenty carbohydrates:

 

 

 

Carbohydgate Incubation Time in Dag;w

L gay 3 5 111 2GP 3 5_
Arabinose 2 2 2 2 2 2 2 2
Dextrose 2 2 2 2 - - - -
Dextrin 3 3 3 3 - - - -
Dulcitol 3 3 3 3 - - - -
Galactose - - - - 2 2 2 -
Inositol - — - — - - - -
Inulin - - - - - - - -
Lactose - 2 2 2 - - - -
Levulose 3 3 3 3 2 2 2 -
Maltose 3 3 3 3 - - - -
Mannitol - - — - - - - -
Mannose 3 3 3 3 - - - -
Raffinose — - - - - - - -
Rhamnose 3 3 3 3 - - - -
Sucrose - 3 3 3 - - - -
Salicin 3 3 3 3 - - _ -
Sorbitol - - 3 3 - - - -
Starch - - - - - - - -
Trehalose 3 3 3 3 - - - -
Xylose 2 2 2 2 - - - -

* The numbers in the body of the table indicate
the degree of reaction on the carbohydrates.

-81-

TABLE 23

Per cent carbohydrate and Bacto PPLO Serum
Fraction in the phenol red
culture broth

 

Per cent Per cent
gazhghydnate segum izggtiog
Arabinose 0.5 1.0
Dextrose 1.0 1.0
Dextrin 1.0 1.0
Dulcitol 0.5 1.0
Galactose 1.0 1.0
Inositol 0.5 1.0
Inulin 0.5 1.0
Lactose 1.0 1.0
Levulose 1.0 1.0
Maltose 1.0 1.0
Mannitol 1.0 1.0
Mannose 0.5 1.0
Raffinose 0.5 1.0
Rhamnose 0.5 1.0
Sucrose 1.0 1.0
Salicin 0.5 1.0
Sorbitol 0.5 1.0
Starch 1.6 1.0
Trehalose 0.5 1.0
Xylose 0.5 1.0
TABLE 2%

PrOportions of 1% barium chloride and sulfuric
acid used to prepare turbidity standards

Nephelometer Barium cloride Sulfuric acid
number

 

l 0.05Tml 9.95 ml
2 0.10 ml 9.90 ml
3 0.15 ml 9.85 ml
h 0.20 ml 9.80 ml
5 0.25 ml 9.75 ml
6 0.30 ml 9.70 ml
7 0.35 ml 9.65 ml
8 0.h0 ml 9.60 ml
9 0.50 ml 9. 0 ml
10 0.60 ml 9. 0 ml

 

-82-

TABLE 25
Results of the homologous and cross agglutination tests

performed with the antisera and antigens of
the stock PPLOS, CS and CL

Serum 3 Antigen 4 1 *
W35? 52’“ 53"” £76 $13835? {3'1" 1?? $93132
PC-3N* - - - - - - - - — - - .-
PCJ+A W W W W 4/ W W W W 2:4 - -
PC-MN - - - - - - - - - - - -
PCS-SA 1+} W V 11/ W W W W W 3% W -
PC-SN - - - - - - - - - - - -
P-6A 1+; w 11/ 1+; 3/ 1+; A; 1+; 1.; 3,; I”! -
P-6N - - - - - - - - - - - -
PC-7A 1+; 1+; 11/ A; h/ 1+; h/ A; 1+; 1+; A; -
PC-7N - - - - - - - - - - - -
PC-8A h/ h/ h; h} A; 4/ A; 4/ A; h; h; -
Pc-BN - - - - - - - - - - - -
P-9A h/ A; h; h; h/ h; h; h; A; h; h; -
P-9N - - - - - - - - - - - -

86A A; h/ u; A; h; A; h; u; u; a; - -

S6N - - - - - - - - - - - -

CSA - - h/ A} 3/ - h} h; - h; h; -
CSN - - - - - — — - - - - -
CLA - - - - - - - - - M; 11,4 -
CLN - - - - - - - - - - - -

* Sal column designates the reactions recorded for the

saline control. Suffix A indicates antiserum. Suffix
N indicates normal serum.

-83-

TABLE 26

Slide agglutination tests showing the reactions
of thirteen unrelated bacteria with the antisera
of S6 and CS

Antigen

Sarcina
lutea

Lactobacillus
planarum

Streptococcus
pyogenes

Alcaligenes
faecalis

Staphylococcus
aureus

Bacillus
cereus

Proteus
morgani

Corynebacterium
Y

Corynebacterium
equi

Corynebacterium
pyogenes

Corynebacterium
diptheriae

Streptococcus
faecalis

Corynebacterium
S-15

S6A*

w

1+;

Antisera

S6N*

CSA

1;

1+;

w

Saline

 

*Suffix, A, indicates an antiserum.

normal serum.

-84-

Suffix, N,

indicates

TABLE 27

Injection schedule for preparation of antisera

 

Day Operation Antigen Amount Hggge Preparation

1 Prebleed - 20.0ml C* -

1 Sensitiv. S6,CS 0.1ml SC* Adjuvant

test

2 Immunize All l.0ml IP* Adjuvant

3 Immunize All 0.3ml IM* Adjuvant

A Immunize All 0.3m1 IM Adjuvant

9 Immunize PPLOS 6.0m1 IP Live

9 Immunize CS,CL 1.0ml IP Heat killed
11 Immunize PPLOS 6.0ml IP Live

11 Immunize CS,CL 2.0ml IP Heat killed
13 Immunize PPLOs 6.0m1 IP Live

13 Immunize CS,CL 3.0ml IP Heat killed
16 Immunize PPLOS 6.0ml IP Live

16 Immunize CS,CL 6.0m1 IP Heat killed
18 Immunize PPLOS 6.0m1 IP Live

18 Immunize CS,CL 6.0ml IP Heat killed
20 Immunize PPLOS 6.0m1 IP Live

20 Immunize CS,CL 6.0ml IP Heat killed
27 Serum - 50.0ml C -

collection

 

* 0, cardiac withdrawal; SC, subcutaneous;

IP, intmaperitoneal; IM, intramuscular

TABLE 28

Homologous antisera titres obtained for
all test sera by the tube
agglutination test

 

 

 

 

(3 hours)
Antigen 3; l l 1 l... lintiiera 1 1 l l l
2 I? S 1132 5123255512162112611‘8158'6
PC-3 2 h A h h h A h l - - -
PC-h 2 h A H A H h h M h - -
PC-S 2 h h h h h h h 3 - - -
P-6 h A 4 3 - - - - - - - -
PC-7 2 3 H A h h A h 3 - - -
PC-8 3 3 - - - - - - - - - -
P-9 h 4 h h h H A 3 - - - -
S6 A A h 3 1 - - - - - - -
CS A h A A h A h 3 - - - -
CL A 3 2 - - - - - - - - -
(2% hours)
PC-3 3 A h A h h h h h 3 1 -
PC-h 2 h h h H h h h 2 — -
PC-5 3 A h A H H h A H l _ -
P-6 H h H h 2 - - - - - - -
PC-7 3 h H A A h h H H 3 1 -
PC-8 h h H 2 - - - - - - - -
P-9 h h A A H A 4 3 - - - -
36 1+ 1+ 1+ H 3 - - - - - - -
CS 3 h h H A A h h 3 - — —
CL A A h h 2 - - - - - - -

 

-86-

TABLE 29

Optimal preportion oint tests done with
S6, CS and their omologous antisera

 

PPLO
Ant en PPLO Antiserum "
%:Mfi%W
1/1 H H h h - - -
1/2 H h A 3 3 - -
1/H - - - - - - -

 

* Turbidity equals H8% transmittance

 

 

Aggigen* CS Antiserum I
i2tafififific
1/1 h A h h h 2 - - -
1/2 3 h h A A h - - -
l/h 1 2 h h h h 2 - -
1/8 1 l 3 H h 3 l - -
1/16 1 2 2 3 3 3 - - -

 

* Turbidity equals our #1 nephelometer tube

-87-

TABLE 30

Tube agglutination reactions using 86
antigen and CS antiserum

 

 

 

 

Agiigen CS Antigeggg
22331323125c0m
l/l - - - - - - - -
1/2 3 - - - - - - -
l/H - - - - - - - -
TABLE 31

Tube agglutination reactions using CS
antigen and $6 antiserum

 

 

Anc’éigen 36 Antiseggm
é: & 3_ B 1'61 L32 '6: fig Control,
1/1 2 h h h - - - -
1/2 h h h h - - - -
1A 1+ 1+ I+ 1. 1 - - -
1/8 h h h 3 - - - -

 

~88-

Tube
and

Tube
and

Tube
and

TABLE 32

cross agglutination reactions using antigens S6
CS with the antiserum of S6 absorbed with S6

 

 

Antigen and
d11ution Absogbed serum
1 1 l .1_ 1
E B, TB 32 63' Control
86 1/2 - - - - - -
CS l/h - - - - - -

 

TABLE 33

cross agglutination reactions using antigens S6
CS with the antiserum of CS absorbed with CS

 

 

Antigen and
di1ut10n Absorbed serum
1 l l .1_ 1
I; B 16 32 '67; Control
86 1/2 - - - - - -
CS 1/h - - - - - -
TABLE 3%

cross agglutination reactions using antigens S6
CS with the antiserum of S6 absorbed with CS

Antigen and

d1lgt1on Absorged serum
1 1 l 1 1

L: B j '3—2' 6’; Control
36 1/2 - - - - - -
CS l/h - - .. .. .. ..

 

TABLE 35

Tube cross agglutination reactions using antigens
S6 and CS with the antiserum of CS absorbed with

 

 

o6
Antigen and
dilution Absorbed serum
1 l l 1_ l
E 8 1'6 32 61? Control
86 1/2 - — - - - -
cs 1/u 3 3 3 3 - -

 

ROOM USE ONLY

\ 2
a “*1.
“CF 45,, A
w L.- . . ‘

 

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