Table Of ContentThe Pennsylvania State College
The Graduate School
Department of Bacteriology
AN ANTIGENIC ANALYSIS OP PLANT AND SOIL CORYNEBACTERIA,
AND THEIR RELATIONSHIP TO ANIMAL STRAINS OP THIS GROUP
A dissertation
by
Stanley A. Rosenthal
Submitted in partial fulfillment
of the requirements
for the degree of
Doctor of Philosophy
August 1952
Approved:
August 4, 1952
- , , ol/
August 4, 1952
Professor and Chairman
Department of Microbiology
University of South Dakota
In Charge of Major Work
TABLE OF CONTENTS
Page
I. INTRODUCTION AND REVIEW OP THE LITERATURE . . . . 1
| II. METHODS . . . . . ........................... . 7
I
1 A. Cultures.................................. 7
j
B. Preparation of Antigen Suspensions ........ 7
1. Plant and soil attains............... 7
2. Animal strains. ................... 7
C. Preparation of Antisera............... 11
D. Adsorption of Antisera . .......... 12
' E. Procedures for Tube Agglutination............ 12
1. Plant and soil strains.................. 12
2. Animal strains......................... 12
P. Procedures for a Serological Survey of the
Corynebacteria....................... 12
III. RESULTS AND DISCUSSION........................... 14
A. Intra-species Studies........ ............ 14
1. £. michiganense .................. 14
2. £. poinsettiae........................35
3. C. insldiosum................. 35
4. £. flaccumfaoiens . . . 39
5. £. sepedonicum............... 39
B. Inter-species Studies....................... 41
C. Antigenic inter-relationships between the
Plant and -Animal Corynebacteria..............65
37504;;
Page
IV. GENERAL DISCUSSION. . . . . . ' . 75
V. SUMMARY . . . . ................. 79
VI. BIBLIOGRAPHY................ ;. . . . . . . . . . 81
VII. ACKNOWLEDGEMENTS. . . . . . . . . . . . . . . . . 85
I. INTRODUCTION AND REVIEW OP THE LITERATURE
Corynebacterium diphtheriae has received considerably
more attention by bacteriologists than other corynebacteria
because of its great medical importance. Early interest was
primarily focused on the soluble toxin of this organism, and
it was not until the therapeutic and prophylactic properties
of antitoxic serum had become firmly established that the or
ganism itself became the subject of serological investigation.
The earlier work in this field has been summarized by Morton
(1940) and McLeod (1943), who concluded that the 3 physio
logical types are antigenically distinct, with each sero
logical group composed of a number of sub-groups.
Hewitt (1947) found a total of 53 sero-types within
the physiological varieties, with the mltis type being the
most heterogeneous. Hoyle (1942), using an alcohol-soluble
antigen in the complement-fixation test, concluded that mltis,
gravis and intermedlus types contained the same group antigen
shared by C. hofmannl, in addition to an antigen specific for
C. diphtheriae. By means of the slide agglutination test,
Perris (1950b) was able to group 794 C. diphtheriae strains
into 14 sero-types, with only 3 strains remaining unclassified.
Both Oeding (1950) and Lautrop (1950) have demonstrated
the presence of thermolabile and thermostable antigens in C.
diphtheriae. The group-specific "0" antigens were heat-stable
and were present in all 3 physiological varieties. Lautrop
(1950) found the same "0" antigen present in over 300 strains
of £. diphtheriae, with most strains containing an additional
"0* antigen. The heat-labile antigens were apparently type-
specific.
Substances responsible for serological specificity
in C. diphtheriae have been isolated. Wong and T'ung (1938)
first reported that they had isolated a polysaccharide which
was shared by all 3 physiological varieties; however, they
(Wong and T*ung, 1939b) later demonstrated 2 group-specific
polysaccharides. Their report is in accord with the findings
of Oeding (1950) that the heat-stable group antigen was re
sistant to treatment with trypsin and absolute alcohol.
Morton (1940) suggested that Wong and T*ung (1938), in their
first paper, used strains which were of the same serological
group, although they differed culturally. Shortly thereafter,
they (Wong and T»ung, 1939c; 1940) isolated an alkali-soluble
protein from diphtheria organisms. Five serological types of
C. diphtheriae each yielded an alkali-soluble protein which
precipitated only with homologous antiserum. These data
strongly indicate that an alkali-soluble protein is the sub
stance responsible for type-specificity in C. diphtheriae.
Freeman and Minzel (1952) have recently suggested that
C. diphtheriae strains be grouped on the basis of glucose and
starch fermentation in heart infusion broth and then typed
serologically, using type-specific antisera.
The interesting and Important problem of the sero
logical differentiation of C. diphtheriae has been hindered
by the auto-agglutination of a large number of strains. This
property has undoubtedly contributed to the large number of
conflicting reports which have appeared in the literature.
Various methods have been used to obtain stable suspensions
from auto-flocculating strains. Robinson and Peeney (1936)
and Hewitt (1947) used NaOH solutions to obtain stable anti
gen suspensions, while Murray (1935) was successful with
1.3$ NaHCOa ai*d mechanical separation of clumped cells. In
the slide agglutination test, Ferris (1950a,b) used 5$ saline,
although titers were slightly lower than those obtained when
physiological saline was employed. Ewing (1933) stabilized
auto-agglutinating strains by heating and shaking, and Eagle-
ton and Baxter (1923) used glycerin and heat. Minzel and
Freeman (1950) incorporated Tween 80 into broth media when
growing diphtherial antigens. After testing 200 strains of
C. diphtheriae and various diphtheroids, they concluded that
the use of Tween 80 broth gave suspensions which were more
stable than those prepared by other methods.
Relatively little work has been reported on the sero
logical relationships between £. diphtheriae and other members
of the genus. Bailey (1925), using tube agglutination pro
cedures, found no significant cross reactions among diphtheria
organisms, C. hofmanni and C. xerose. On the other hand,
Wong and T'ung (1939a) found that a polysaccharide isolated
from C. xerose reacted equally well with all antisera prepared
against the various cultural types of C. diphtheriae and that
C. xerose antiserum precipitated polysaccharides prepared from
C. diphtheriae. Antiserum prepared against C. hofmanni did
not react with the above polysaccharides, nor did heterologous
antisera precipitate C. hofmanni polysaccharide. These
data support the report of Bull and McKee (1924), who found
that C. hofmanni did not bind complement in the presence of
extracts of virulent and avirulent diphtheria organisms. Of
the 3 strains of C. hofmanni used in this latter study, 2
shared antigenic components, while 1 strain appeared to be
entirely unrelated to the others. On the other hand, Hoyle
(1942) found that a lipoid antigen was shared by C. diphtheriae
and G. hofmanni, but that C. hofmanni also contained a specific
antigen. Lautrop (1950) reported that the "O" antigen of £.
diphtheriae is also present in £. hofmanni and C. ovis.
The serological relationships among strains of C. equi
have been published and confirmed (Bruner, et al, 1939; Karl-
son, £t al, 1940; Bruner and Edwards, 1941; Woodroofe, 1950).
These organisms are highly type-specific, and species-specific
antigens can not be demonstrated by agglutination tests.
Woodroofe (1950) divided 16 strains of C. equi into 3 main
groups, with 3 strains agglutinating only in homologous anti-
serum. Bruner and Edwards (1941) found that 29 of 34 strains
fell into 4 distinct groups which were further subdivided into
types. The remaining 5 strains were not related antigenically
to the main groups or to each other.
The species-specific antigen of C. equi was demon
strated by removing the type-specif1c antigen with HC1 at
100 C. Heating the cells with acid at 60 C did not remove
the type-specific antigen (Bruner, et al, 1939). All strains
examined contained the species-specific antigen, which was
A
demonstrated by eomplement-fixation tests on the extracted
cell residues; but not all antisera contained the species-
specific antibody (Bruner, et al, 1939).
Merchant (1935) has investigated the serological
relationships among corynebacteria causing animal diseases.
He found that C. pyogenes contained a species-specific anti
gen; antisera prepared against 2 strains agglutinated all 5
of his C. pyogenes cultures. Only 1 C. pyogenes strain ag
glutinated in 2 of 4 C. renale antisera and in 1 C. pseudo-
tuberculosis antiserum. C. pseudotuberculosis apparently
contained a species-specific antigen, but species-specific
antibody could not be demonstrated in all C. pseudotuberculosis
antisera. Three C. renale antisera agglutinated 4 to 6 of 12
C. pseudotuberculosis strains. Most of Merchants C. renale
cultures contained a species-specific antigen. Marked cross
reactions were observed when G. renale antigens were reacted
with C. pseudotuberculosis antisera. Four strains classified
as £• resale on the basis of fermentation reactions, morphology
cultural characteristics and source of isolation were found
to have a closer serological relationship to C. pseudotuber
culosis than to C. renale. Merchant also found serological
cross reactions among 1 strain of C. diphtheriae, 2 of C.
renale, and 1 of C. pseudotuberculosis. In addition, Lautrop
(1950) reported on the antigenic relationship between C.
diphtheriae and C. ovis (C. pseudotuberculosis).
Feenstra, e_t al (1945) have shown the presence of a
species-specific antigen in C. renale, confirming the earlier
work of Merchant (1935), but only 1 of 5 C. renale antisera
agglutinated all strains.
Although some work has been done on the animal and
human corynebacteria, a siirvey of available literature has
failed to reveal any serological studies on the plant and
soil diphtheroids. Conn and Dimmick (1947) have objected
to the inclusion of the plant and soil diphtheroids in the
genus Corynebacterium (Jensen, 1934; Starr and Pirone, 1942).
On the basis of morphological and physiological studies, they
felt that the plant diphtheroids now classified in the genus
Corynebacterium show sufficient differences from the type
species to suggest that they do not belong in this genus.
Their opinions, nevertheless, were not based on antigenic
studies. They also felt that not all of the plant coryne
bacteria belong in the same genus. Therefore, the purpose
of this study is to investigate the antigenic structure of
the plant and soil corynebacteria and the possible occurrence
and distribution of their antigens within the animal coryne
bacteria, in an attempt to clarify the relationships among
members of this ill-defined group of micro-organisms as well
as their status with respect to the genus Corynebacterium.
II. METHODS
A. Cultures
Many of the cultures were obtained through the kind
ness of several individuals throughout the country, and others
were purchased from the American Type Culture Collection. The
species designations of the cultures used in this study are the
same as those given to the author, except where those names
were in conflict with the designations given in Bergey's Manual
of Determinative Bacteriology (Breed, et al, 1948). A list of
the cultures and their sources is given in table 1.
B. Preparation of Antigen Suspensions
1. Plant and Soil Strains
These antigens were grown on Eugonagar (Baltimore
Biological Laboratory) in Pyrex Blake bottles for 1 to 3 days
at room temperature, collected in small amounts of formalinized
saline1, filtered through sterile cotton and washed once with
formalinized saline. The cells were resuspended in small amounts
of formalinized saline, and these heavy suspensions were kept
in the refrigerator as stock antigens. When tube antigens were
needed, the stock suspensions were diluted with formalinized
saline to a dilution which allowed 85$ light transmittance
using a Coleman Universal Spectrophotometer at 650 A.
2. Animal Strains
When auto-agglutinating antigens were studied, a
modification of the method of Minzel and Freeman (1950) was
10.5$ formalin and 0.85$ NaCl in distilled water.
