A STUDY OF THE RELATi’ONSHiP-SOF SELECTED
DERMATOPHYTES USING SUBCEL‘LULAR FRACTIONS
AS ANTIGENS 1N iMMUNODIFFUSlON TECHNIQUES

Thesis for the Degree of ‘Ph. D;
MICHiGAN STATE UNIVERSITY
ALVEN LEE ROGERS '
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This is to certify that the

thesis entitled
A STUDY OF THE RELATIONSHIPS 0F SELECTED

DERMATOPHYTES USING SUBCELLULAR FRACTIONS
AS ANTIGENS IN IMMUNODIFFUSION TECHNIQUES

presented by

Alvin Lee Rogers

has been accepted towards fulfillment
of the requirements for

L94... degree in ML

EE;1&221£22§32§1;¢4QIAZEZ\\

 

Major professor

Date November 9, I967

 

0-169

ABSTRACT

A STUDY OF THE RELATIONSHIPS OF SELECTED
DERMATOPHYTES USING SUBCELLULAR FRACTIONS
AS ANTIGENS IN IMMUNODIFFUSION TECHNIQUES

by Alvin Lee Rogers

The objective of these investigations was to determine
the degree of relatedness between selected dermatophytes
using subcellular fractions as antigens in immunodiffusion
techniques.

The precipitinogenic relationships of 20 representative
dermatophytes were compared using an agar—gel slide double-
diffusion technique. The organisms included species from

the four genera of dermatophytes, Epidermophyton, Keratino-

 

myces, Microsporum, and Trichophyton, and strains within a

 

species. Two subcellular fractions separated by centrifuga—
tion from each fungus were used as antigens. Antisera were

produced in rabbits to the fractions from 3 Trichophyton

 

species.

Antigenic differences existed among the fractions of
an organism, species and groups, and in addition, among
strains within a species. No one precipitinogen was found
to be common to all dermatophytes tested.

From A to 9 precipitinogens were detected in homolo-
gous antigen—antibody systems, and in all tests, the
homologous systems reacted to give greater numbers of pre-

cipitated bands than the heterologous systems.

Alvin Lee Rogers

The trichophytons were separated into serological
groups which fall into the old colony morphological group-

ing. TrichOphyton terrestre appears to belong to a

 

separate group. The reaction with fractions from the

strains of T. mentagrOphytes indicated the possibility of

 

more than one species.

The species fl. ferrugineum was closer antigenically

 

to M. gypseum than to any Trichophyton species. 3. ajelloi

 

also appeared closer to the Microsporum species.

 

Epidermophyton floccosum appeared not to be anti-

 

genically closely related to any of the organisms investi-

gated.

A STUDY OF THE RELATIONSHIPS OF SELECTED
DERMATOPHYTES USING SUBCELLULAR FRACTIONS

AS ANTIGENS IN IMMUNODIFFUSION TECHNIQUES

By

Alvin Lee Rogers

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1967

"h

b3 ,S:

Li?)

Ut

5“,“

I"-
I.)

00

To my parents
and

Aunt Cora Clay

ii

ACKNOWLEDGMENTS

The writer wishes to eXpress his sincere appreciation
to Dr. E. S. Beneke whose invaluable guidance, interest,
patience and encouragement during the course of this
investigation has made possible its completion.

The writer also wishes to express gratitude to Drs.
C. J. Pollard, R. C. Belding, W. B. Drew, Virginia Mallman,
and S. H. Barch for their many helpful suggestions, con-
structive criticisms, and invaluable guidance during my
graduate study as well as during the writing of this thesis.

The 1964 Bessey award fund of $100 was utilized for
the purchase of rabbits for use during the course of this

work. Sincere appreciation is expressed for this award.

iii

TABLE OF CONTENTS

DEDICATION . . . . . . . . . . . . .
ACKNOWLEDGMENTS
LIST OF TABLES

LIST OF PLATES

INTRODUCTION
REVIEW OF LITERATURE . . . . . . . . . . .

Classification of the Dermatophytes-—
Variations in Systems

Agglutination and Precipitation Techniques Used
in the Identification of Fungi . . .

Immunodiffusion . . .

Immunodiffusion Applied to Identification of
Fungi . . . . . . . .

MATERIALS AND METHODS

Dermatophytes Studies
Verification of Trichophyton Species
Trichophyton Agars . . .
Tests to Separate T. mentagrophytes and
T. rubrum . . . . . . .
Pigment Production . . . . . . .
"In vitro" Hair Invasion
The Urease Test . .
Media for Growth of the Dermatophytes
Antigens . . . . . .
Preparation of Antigens . . . . . . . .
Protein Determination . . . .
Antibody Production
Rabbits Used .
Antigens Used for Production of Antisera
Injections . . . . .
Bleeding and Antiserum Preparation . . . . .
Immunodiffusion Techniques .
Electrolyte Solutions Used for Preparing
Diffusion Media . . . . . .

 

iv

Page
ii
iii
vi

vii

Page

 

 

 

 

 

Media for Diffusion Studies . . . . 56
Gels for the Preliminary Studies by the
Petri Dish Method . . . . . 56
Gels for the Preliminary Studies by the
Agar Slide Method . . . . . . . . . 56
Immunodiffusion Test Procedure . . . . . . . 57
Petri Dish Method . . . . . . . . . . . 57
Agar Slide Method . . . . . . . . . . . 58
Observations . . . . . . 59
The Influence of Antigen Dilution . . . . . 59
Photography . . . . . . . . 6O
Staining of Precipitating Lines . . . . . . 6O
PRELIMINARY RESULTS AND DISCUSSION . . . . . . . 61
Media for Growth of Dermatophytes . . . . . . . 61
Relative Sensitivities of Methods . . . . . . . 62
Reactions in Various Diffusion Media . . . . . . 68
Dilution of Antigens . . . . . . . 69
Antibody ReSponse in Individual Rabbits . . . . . 70
RESULTS . . . . . . . . . . . . . . . . 72
Verification of the Dermatophytes Used in
This Investigation . . . . . . 72
Identification of Species in the Genera
Epiderm_phyton, Keratinomyces and Microsporum . 72
Identification of Trichophyton Species . . . . 73
Use of Trichophyton Agars . . . . . . . 73
Separation of T. mentagrophytes and T. rubrum . 73
The Amount of Protein in the Antigenic Fractions
of the Dermatophytes . . . . . . . 76
Results of Precipitinogenic Studies . . . . . . 77
Homologous Antigen- Antibody Reactions . . . . 77
Precipitinogenic Relationship of the Different
Antigenic Fractions Within the Same Isolate . . 95
Precipitinogenic Relationship of the Three
Reference Strains . . . . . . . . 96
Precipitinogenic Relationship of the Different
Isolates Within a Species . . . . . . . . lOl
Precipitinogenic Relationship of Selected
Dermatophytes and the Reference Strains . . . lOA
DISCUSSION . . . . . . . . . . . . . . . 115
SUMMARY . . . . . . . . . . . . . . . . 130
LITERATURE CITED . . . . . . . . . . . . . 133

LIST OF TABLES

Table Page
1. Classification of the dermatophytes . . . . l2
2. Dermatophytes with known perfect states . . . 1A

3. Injection and bleeding schedule, supernatant
fractions . . . . . . . . . . . . 52

A. Injection and bleeding schedule, pellet fractions 53
5. Wet mycelial weights of the three Trichgphyton

Sp. grown in different media at the end of
1A days . . . . . . . . . . . . . 61

 

6. The number of precipitation lines detected in
double—diffusion tests using two sizes of
petri dishes and the agar slide method . . . 63

7. The number of precipitation lines detected in
the antigen dilution tests . . . . . . . 69

8. The number of precipitation lines detected in
individual rabbit antiserum by homologous
antigen-antibody reactions . . . . . . . 7O

9. Growth pattern of Trichophyton species on
Trichophyton Agars . . . . . . . . . 7A

 

10. The differentiation of Trichophyton rubrum
and T. mentagrophytes . . . . . . 75

 

 

11. The amount of protein in the dermatophyte
fractions as determined by the Lowry et al5
method . . . . . . . . . . . . . 78

12. The number of precipitation lines formed by
homologous and heterologous antigen—antibody
reactions of reference strains . . . . . 97

13. The number of precipitation lines formed by

homologous and heterologous antigen-
antibody reactions of the dermatophytes . . 98

Vi

Plate

II.

III.

IV.

VI.

VII.

LIST OF PLATES

Immunodiffusion reactions using homologous
antigen- antibody systems of T. mentagrgphytes
(dog) fractions in agar gel method employing
two sizes of petri dishes and a lantern
slide

 

Immunodiffusion reactions using homologous
antigen—antibody systems of T. terrestre (AlA)
fractions in agar gel method employing two
sizes of petri dishes and a lantern slide

 

Diagrams of immunodiffusion test precipitation
lines produced by homologous and heterologous
antigen-antibody systems of the fractions
within the species Trichophyton mentagro-
phytes (dog), T. terrestre (AlA) and T.
rubrum (Dar) . . . . . .

 

 

Diagrams of immunodiffusion test precipitation
lines produced by supernatant antigen
fractions from selected dermatophytes and T.
mentagrophytes (dog) supernatant and pellet
antisera. . . . . . . .

 

Diagrams of immunodiffusion test precipitation
lines produced by pellet antigen fractions
from selected dermatophytes and T. mentagro-
phytes (dog) supernatant and pellet antisera

Diagrams of immunodiffusion test precipitation
lines produced by supernatant antigen
fractions from selected dermatophytes and T.
terrestre (AlA) supernatant and pellet
antisera .

 

Diagrams of immunodiffusion test precipitation
lines produced by pellet antigen fractions
from selected dermatophytes and T. terrestre
(AlA) supernatant and pellet antisera

 

vii

Page

6A

66

80

82

8A

86

88

Plate

VIII. Diagrams of immunodiffusion test precipitation
lines produced by supernatant antigen
fractions from selected dermatophytes and T.
rubrum (Dar) supernatant and pellet antisera

IX. Diagrams of immunodiffusion test precipitation
lines produced by pellet antigen fractions
from selected dermatophytes and T. rubrum
(Dar) supernatant and pellet antisera

viii

Page

90

92

INTRODUCTION

The dermatophytes are a group of fungi which attack
the keratinized tissues, viz., the epidermis, hair and nails,
of humans and animals. Recently many of the organisms of
this group have been isolated from the soil where they live
as saprobes.

The taxonomy of this group was first based on the
clinical symptoms and the morphology of the fungi in their
pathogenic environment as illustrated by Sabouraud in his
textbook in 1910. In 193A, Emmons based his classification
on the morphology of the fungi in culture. Then, in 1957,
Georg and Camp utilized physiological characteristics under
cultural conditions to aid in the separation of species.
~There is still a certain amount of disagreement on the rela-
tionship of the organisms in this important group, for example:

1. There is the problem of considering all strains

of Trichophyton mentagrophytes as one species

 

(Emmons, 193A) or placing them into another

genus, Ctenomyces Eidam 1880 with 7 species as

 

Langeron and Vanbreuseghem did in 1952.
2. Another variable has been the placement of the

species ferrungeum in either the genus MTcrosporum

 

 

(Ajello, 1962) or Trichophyton (Langeron and Van-

 

breuseghem, 1952).

The number of genera and Species is also uncertain.
Langeron and Vanbreuseghem (1952) listed 5 genera
with 27 species and Vanbreuseghem created a new
genus (1952). Since that date 11 new species

have been described, making a total of 6 genera
and 38 species. Ajello (1962) considers A genera
and 22 species as valid, and Beneke lists A genera
and 26 species in 1966 (2 new species have been
described since this publication giving a total of
28 species). The number of genera listed by the
United Kingdon's Medical Mycology Committee (1967)

is 3 (Keratinomyces is not listed), and the number

 

of species is 26. All the keratinophilic saprobes
not proven to be parasitic were not included along
with the proven parasites. Several of the species
listed are considered to be synonymous by other
workers (Ajello e§_aT., 1963).

Sometimes separation of species by the use of

morphological and physiological characteristics

 

is difficult. Trichgphyton rubrum and T. menta—

grophytes are usually easily separated, but sub-

 

cultures of T. rubrum isolates may fail to produce
color on the reverse side as pointed out by
Weidman in 1929, while some isolates of T, menia-
grophytes produce a reddish-brown color. The
shape of the conidia may overlap or in some cases

no conidia are produced at all and the physiological

characteristics are similar. The method of "in
vitro" hair invasion is usually different. T.

mentagrOphytes invades by a wedge shaped penetra-

 

tion.

5. The asexually produced spore structures
(imperfect stage) are often not stable enough to
permit their use in accurate identification.

Seeliger (1960) reported that the application of
serology offers a valuable additional tool in the studies
of mycological phylogenetic relationships and that it could
solve some difficult taxonomic problems.

The purpose of these investigations was to determine
the degree of relationship between selected dermatophytes
using antisera produced by rabbits injected with subcellular
fractions of the dermatophytes against the subcellular

fractions as antigens in immunodiffusion experiments.

REVIEW OF LITERATURE

Classification of the Dermatophytes
Variations in Systems

 

 

Dermatophytosis, ringworm in man and animals, has been
known for centuries (Feuland, 1886; and Gates, 1939) but the
first causative agents were not reported until the nine-
teenth century. Robert Remak in 1837 (Kisch, 195A) dis-
covered hyphae in favic crusts but did not consider them as
the causative agent. It was not until two years later that
Schoenlein (1839) showed the relationship between the fungus
and the favic crusts. Remak named the fungus Achorion

schoenleinii in 18A5. During the lapse of time between the

 

discovery and the naming of the organism, Gruby (18A1) was
investigating ringworm. He recognized favus and by micro-
scopic examination diagnosed the disease and successfully
inoculated humans and animals with the fungal agent. In
18A2 he discovered the ectothrix and endothrix trichophytons

but did not name them. Microsporum audouinii, an organism

 

that produces ringworm in children, was discovered and
described by Gruby in 18A3. The clinical descriptions by
GPUby were not precise but the mycological portions were
only erroneous in that the fungi grew downward, not upward
as is described in the pilar lesion (Sabouraud, 1936). The

name TrichOphyton was introduced into the literature in 18A5

 

by Malmsten when he described Trichophyton tonsurans.

Pure cultures were first obtained in 1886 by Grawitz
and shortly thereafter by Duclaux (1886). In 1887 Verujsky
distinguished between external and mycelial Spores. Using
the external Spores, Sabouraud (1892) grouped the dermato-
phytes into divisions: (a) the large spored and (b) the
small spored organisms, each group with more than one
organism thus settling a dispute between the unicists and
the pluralists.

The first mention of "pleomorphism" among the dermato-
phytes occurred in 1896 when Bodin published his work on
"teignes tondantes" in the horse. Until this time Sabouraud
(1910) considered this phenomenon of polymorphism as
symbiosis. Sabouraud's classification, in "Les Teignes,"
published in 1910 is the oldest organized attempt to classify
the dermatophytes. He used as a basis the morphology of the
fungi in the parasitic stage as well as hair-invasion to
designate the three divisions (now genera), and morphology
of the fungi in the saprophytic phase to establish the
Species (Sabouraud, 1910). Thus, much of the fundamental
work we owe to Sabouraud and his students. Reclassification
cannot detract from his monumental work on these forms
(Emmons, 193A).

Ota and Langeron (1923) based the classification of
the dermatophytes upon cultural morphology as they grew on
Iiatural media, thus the transfer of the basis of classifica-
‘tion was from parasitism to saprophytism. In their classi-

fication a new genus Sabouraudites was substituted for

 

Microsporum, using Microsporum audouinii Gruby as the type

 

species. The new genus, Sabouraudites, includes all the

 

species of dermatophytes that produce macroconidia which

includes all the Microsporums and many of the Trichophytons,

 

thus unnatural groups result (Emmons, 193A).

In 192A Grigorakis used macroconidia, perithecia,
microconidia, aleuria, and chlamydospores as the basis for
the classifying the dermatophytes. The members of the genus

Microsporum along with all other species of the dermatophytes

 

which produced persistent macroconidia were placed into the

new genus Closterosporia. According to Emmons (193A), this

 

division did not follow natural lines as species producing
thin- and thick—walled macroconidia were placed in the same
genus.

Vuillemin (1925), Nannizzi (1927), and Guiart and
Grigorakis (1928) all modified the classification of the
dermatophytes. According to Emmons (193A) their groupings
were not along natural divisions, while Dodge (1935) stated
that they did not follow the rules of nomenclature.

After growing many of the known species of TrichOphyton
on 13 different media, Lageron and Milochevitch (1930), on
the basis of colony morphology, simplified the classification
of the dermatophytes by placing into synonymy many species
names. They used for their basis the classification of Ota

and Lageron (1923), retaining the genera: Ctenomyces Eidam,

 

1880; Sabouraudites Ota and Lageron, 1923; Epidermophyton

 

Harz, 1870; and TrichOphyton Malmsten, 18A5.

 

Ota and Kawatzure (1933) enlarged the concept of the
species, including the gypseum forms while placing into
synonymy many species that they considered to be T. rubrum
Castellani, 1909. Their reclassification was based on
cultures grown on natural media as well as animal inocula-
tions.

Dodge (1935) using Sabouraud's classification (1910)
as a basis, listed 9 genera and 118 species of dermatophytes
which he considered valid. Many of the species were based
upon clinical instead of botanical differences.

Emmons in 193A proposed and established botanical
criteria for the classification and identification of the
dermatophytes. Since at that time no perfect stages of the
dermatophytes had been found and confirmed, they were con-
sidered to be members of the "form" class Deuteromycetes,
in the order Moniliales and the family Moniliaceae (Ainsworth,
1961). The classification of these fungi therefore will
depend on the vegetative structures, mainly conidia, which
do not always show true relationships. A genus in the Fungi
Imperfecti may include species of diverse origin, as illu-

strated in Microsporum gypseum "complex" which is composed

 

of 3 perfect species (Stockdale, 1963). Still, a critical
study of the characteristics of the spores, and their
sterigmata indicates fundamental differences between groups

within a form genus.

Emmons (193A) suggested that the dermatophytes should

be classified into 3 natural genera: Trichophyton,

 

EpidermOphyton, and Microsporum. His natural grouping is

 

 

based upon the method of reproduction of the organisms in
culture, primarily considering macroconidia. He also recog-
nized the occurrence of a wide range of morphological varia-
tions among individual isolates of a Species.

Emmons described Trichophyton as having clavate macro—

 

conidia with blunt ends and broad bases which are encircled
by a collar marking the attachment to the conidiOphore. The
walls are thin and smooth, but may be constricted where the
cross—septa appear.

The Epidermophyton macroconidia are usually clavate to

 

egg—shaped, and moderately thick—walled. The species of
this genus never has microconidia.

The large thick, rough-walled macroconidia of M1232-
sporum are typically spindle-shaped with pointed tips, and
have the broad, faceted, collared base which characterizes
the microconidia and macroconidia of the dermatophytes
(Emmons, 193A).

The microconidia or conidia of the dermatophytes,
varying in shape and Size, are of little use in differen-
tiating species.

Conant e£_§T. (19AA, 195A) placed many of the species

of dermatophytes into synonymy in both editions of Manual of

 

Clinical Mycology.

 

The classification of Langeron, Milochevitch, and
Vanbreuseghem (Langeron and Vanbreuseghem, 1952) is a
modification of Langeron and Milochevitch (1930). The only

difference is the addition of the genus Langeronia Van—

 

breuseghem, 1950 and species discovered since 1930.

Vanbreuseghem described a new genus, Keratinomyces

 

Vanbreuseghem 1952, which is considered a fourth genus in
Emmon's classification. Langeron's and Vanbreuseghem's

classification would contain 6 genera which are: Ctenomyces

 

Eidam, 1880; Sabouraudites 0ta and Langeror, 1923;

 

Trichophyton Malmster, 18A5; Langeronia Vanbreuseghem, 1950;

 

 

Epidermophyton Sabouraud, 1910; and Keratinomyces Vanbreuseghem,

 

 

1952. A description of these follows.

The organisms that belong to the genus Ctenomyces

 

Eidam, 1880 have microconidia disposed in clusters or along
hyphae and have distaff—shaped macroconidia (fuseaux en
quenouille). Aerial hyphae that give rise to conidia are
branched at right angles and terminate with the formation of
Lorraine Cross configurations. The members also have Spiral—
and antler-like filaments which are regarded as equivalent

to the appendages of perithecia. The nodular bodies simulate
the commencement of ascogonium-formation.

The genus Sabouraudites Ota and Langeron, 1923 is

 

characterized by having microconidia of the Acladium type
and numerous macroconidia with lanceolate, granulose walls.

Spirals rarely appear upon ordinary media.

10

The Trichophyton Malmster, 18A5 cultures may have

 

Acladium type microconidia along aerial hyphae and if macro—
conidia are present they are cigar- or sausage-shaped, with
smooth and thin walls having obtuse extremities. The base
of the macroconidium is only slightly larger than the hypha
from which it arises. The glabrous colonies have few or no
conidia. Spirals are not seen when grown on ordinary media.

Langeronia Vanbreuseghem, 1950, is characterized by

 

having mycelium with short articulations as lateral branches
growing in opposite directions. These lateral branches
produce tertiary branches. The conidia are rare but of the
Acladium type. Arthrospores are formed and give the impres—
sion of false branches. No spirals nor macroconidia are
found.

Epidermophyton Sabouraud 1910 contains only one

 

species, E. floccosum Harz, 1870 with club-shaped macro-

 

conidia, often arranged like bunches of bananas. There are
no spirals and no microconidia.

Keratinomyces ajelloi Vanbreuseghem, 1952, the only

 

member of the genus Keratinomyces has numerous large cylindro-

 

fusiform, thick, smooth—walled macroconidia with 5 to 12
cells. Microconidia are usually abundant, pyriform to ovate
in Shape, and sessile.

Thus, of the many classifications of the dermatophytes
that have been proposed, 2 are used most often: (1) Emmon's

classification published in 193A and brought up to date by

ll

Ajello (1962), Ajello g£_aT. (1963), Beneke (1966), and
Ingram (1967); and (2) Langeron and Milochevitch's classifi-
cation published in 1930 with a revision by Langeron and
Vanbreuseghem in 1952. The classification by Emmons is
widely used in the English speaking world and the latter is
used by many Europeans and South Americans. Both of these
classifications are based on characteristics of the dermato-
phytes in culture. Table 1 gives the two classifications.
The following are additional species not listed in the
manuals by Beneke (1966) or Langeron and Vanbreuseghem (1952):

Trichophyton kuryangei Vanbreuseghem and Rosenthal,
1961

 

TrichOphyton Vanbreuseghemii Rioux, Jarry and Juminer,
196A

 

Trichophyton gToriae Ajello and Cheng, 1967

 

Matruchot and Dassonville (1899a) were the first to
suggest that the dermatophytes might be related to the
Gymnoascaceae. They found that certain species of this
family produced asexual Spores Similar to those of some

dermatophytes. The Trichophyton species produce many spirals

 

which Matruchot and Dassonville (1899b) compared to the

peridial hyphal spirals of Ctenomyces serratus. In 1900

 

these two authors found an isolate of Trichgphyton that

 

produced clumps of hyphae which were similar to cleistothecia
but only produced conidia. Similar structures which they
called "fruit conidiens" were produced in g. serratus and

in a number of species of Trichophyton._ Based primarily

 

TABLE l.—-C1assification of the dermatOphytes.

12

 

Emmon’s Classification Modified by AJello,

1962, and Beneke, 1966

Langeron, Milochevitch, and Van—
breuseghem's Classification, 1952

 

Microsporum

g.

audouinii Gruby, 18A3

canis Bodin, 1902
cookei Ajello, 1959
distortum di Menna & Marples, 195A
'nseum IBodin) Guiart & Grigorakis, 1928
nanum (Fuentes, Aboulafia & Vidal)
Fuentes, 1956
vanbreuseghemii Georg, Ajello, Friedman
. and Brinkman, 1962
ferrugineum Ota, 1922

 

 

Epidermophyton

 

EC

floccosum (Harz) Langeron and
Milochevitch, 1930

*Keratinomyces

 

*K.

aielloi Vanbreuseghem, 1952

 

Trichophyton

 

2.

laps la

la

Ieha

IHHNS

lflhfirflafla I6

mentagrophytes (Robin) Blanchard, 1896

 

eguinum (Matruchot & Dassonville)
Gedoelst, 1902

rubrum (Castellani) Sabouraud,

verrucosum Bodin, 1902

1911

gallinae (Megnin) Silva & Benham, 1952
merninii Blanchard, 1896
simii (Pinoy) Stockdale

Mackenzie & Austwick,
tonsurans Malmsten, 18A5

1965

schoenleinii (Lebert) Langeron and
Milochevitch, 1930
violaceum Sabouraud, apudBodin,
gourvillii Catanei, 1933
concentricum Blanchard, 1896
geO‘giae Varsavsky and Ajello, 196A
terrestre Durie and Frey, 1957

 

1902

 

soudanense Joyeux, 1912
yaoundei Cochet and Doby-Dubois, 1957

Sabouraudites

 

audouinii Gruby, 18A3
langeronii Vanbreuseghem, 1950
ravalierii Vanbreuseghem, 1951
canis Bodin, 1902

|(/)|(/a|c/)|(/a

 

S. gypseum Bodin, 1907
S. gallinae Mennin, 1881
Epidermqphyton

E. floccosum Harz, 1870

Ctenomyces

O

mentagrophytes Robin, 1853
asteroides Sabouraud, 1909
granulosus Sabouraud, 1908
Lergicelor Sabouraud, 1910
interdigitalis Priestley,

guickeanum Zopf, 1890

 

1917

 

gaymvncvu

rubrur Castellani, 1909
alULUfi Salmnlratvt, 1 N19

discdides Sabouraud, 1909

' _"
.L .1

‘
. —4
_.
c
‘.

Pfl
O

IFS
.

megninii R. Blanchard, 1895

tonsurans Malmsten, 18A5
sabouraudii R. Blanchard,
sulfureum Fox, 1908
schoenleinii Lebert, 18A3

1.07))

I HI HI *3] *3

 

[*3

violaceum Bodin, 1902

H

concentricum R. Blanchard, 1895

 

IFS

ferrugineum Ota, 1921

 

w

L. soudanensis Joyenux, 1912

 

l
Indicates the species described since Langeron and Vanbreuseghem's book in

1952.

13

upon this evidence they transferred Trichophyton menta-

 

grophytes to the genus Ctenomyces.

 

 

The perfect stages for a number of the dermatophytes
have been discovered in recent years (Table 2). All the

perfect stages of Microsporum so far discovered are mem-

 

bers of the same natural genus Nanizzia and those of the

imperfect genus Trichophyton are in the genus Arthroderma

 

 

which includes the imperfect species 5. ajelloi.

Once the perfect stages of all the dermatOphytes
are found, the comparative relationships of the sexual
stages promises to provide a more sound criterion for the
validation of species and the determination of phylogenetic
relationships within the genus and within the dermatophytes.
Until these stages are found other methods including
morphological characteristics need to be used in classify-
ing the dermatOphytes difficult to separate.

In the species which produce characteristic macro-
conidia little difficulty exists in distinguishing Species.

But within the genera Microsporum and Trichophyton several

 

 

Species are in a controversial location. Microsporum

 

ferrugineum Ota (1922) does not produce macroconidia and

 

resembles the waxy form of trichophytons in culture so

some mycologists place this Species in the genus Trichophyton

 

(Langeron and Vanbreuseghem, 1952; Vanbreuseghem, 1963).

Many others prefer the genus Microsporum because of the way

 

it attacks hair "in vivo", similar to other Microsporum

 

species (Ajello, 1962; and Beneke, 1966). Microsporum

 

1A

 

<|

somoxmz Ammmpa Gav Hpmappmm .
m ma
upoxczam w came .opom EMLSHSoHpcoH .
Hmma .moapcom w comzmo ESUHmHLUmsm .
mmmfi .SOHzpmse
w oflncoxomz .mampxoOpm HHEHm
emma .wcmzo s oHHmfia msflemgcmn
Noma .wcmgo S oaamna mmHSon
:oma .oaamw< w mxm>mmpm> HHLLSMHo

 

<k1l

 

<kmkm<l

Hmma
moapcou a c0m3mo Enumcfioc: mEgopopcpp<

momfl .cmaxcfism
a cmEpoHLm .oHHom¢ .mpooo SHNDSLM .

zl

Homa .moapcoo w 20m3ma Swapoo .
mea .oampxoOQm mpm>p50cfi .

 

zflzl

mama fimamprOpm Aflmwfiscmzv mommmw .
momfi .SHSSASOSm asaso .

Hmmfi .oHHmme ficmsmwmo .

mfiNNHccmz

zkzbfl

 

:mma .Locflezh
a «mppmh .xSOHm HHEmzwomsoanm> .

BI

 

Smma .mopm a ofisdm osumossop .

mama .xoflzsmsa s IIIII.
oflmcoxomz amamoxooum Azocflmv HHEHm .
mama .opmzocmam Anabomv mopmcmmmwmpcoe .
Smma .wcmno s oaamw< msflsoam .
:oma .oHHon< w mxm>mmpm> omamsoom .H

BI

 

BENEH

 

Cowmnmozofipe

 

mmma .Eonwomzmpbcm> Hoaawdd moomEochmLom

mwma .cmExchm w :mEpoHpm

.oaammg .wnooc HHEonwomdophcm> .S
mmma .mmpcozm

AHSUH> w Sammasoo< «mopcosmv Edema .

El

mmmfi
.mfixmsowflso s psmfiso Acfloomv mmmmmxw .
mama “SASQHLD Es>asm .
mmma .oaaoha Hmsmmm .

ZEflEfl

 

ExpommOLOHz

 

mumpm poompom

opmum poompodEH

 

.moumum poomgod c30cx Spas mop>£Q0meLoQII.m mqm<e

15

langeronii Vanbreuseghem, 1950, M. rivalierii Vanbreuseghem,

 

 

1951, Species isolated primarily in Africa, and M. fer-

rugineum are considered geographic forms of M. audouinii

 

 

by Zaias et a1. (1965), but Vanbreuseghem (1963) considered

the first 2 valid species while the species ferrugineum was

 

placed in a separate genus. Representatives of the genus

Trichophyton have macroconidia either lacking, rare or are

 

not characteristic enough to separate species (Emmons, 193A;
Ajello and Georg, 1957).

Georg and Camp (1957) mentioned that with many atypical
strains and species which produced colonies resembling each
other identification on the basis of morphological criteria
alone was impossible. They reported on the effects of
vitamin and amino acids on the growth of the trichophytons
and the use of these substances as an aid to identify species.
This group of tests has been used very successfully except

for the differentiation of T. rubrum and T. mentagrophytes,

 

which can be confused with each other if T. mentagrophytes
has a red undersurface in culture or if T. rubrum is
pleomorphic. Several methods have been used to distinguish
these two Species. The growth of these two organisms on
different media, corn meal glucose agar (Bocobo and Benham,
19A9), and potato dextrose agar (Bohme and Friedrich, 1962)
results in the development of red pigment in T. rubrum but

not in T. mentagrophytes. Dyson and Landay (1963) found

 

3 out of 2A isolates of T. mentagrgphytes produced red

 

16

undersurface pigments on corn meal glucose agar and 7 of
the 2A produced red pigments on potato dextrose agar.
They concluded the two media are not valid criteria for
differentiation of these two Species. They used the "in
vitro" hair test (Vanbreuseghem, 19A9; Ajello and Georg,
1957) to distinguish the two species. In this test T.

mentagrophytes penetrates hair segments, while T, rubrum

 

does not. Sudman and Schmitt in 1965 reported potato

dextrose agar to be a valid means of differentiating the

two species as corroborated by the "in vitro" hair test.
Philpot (1967) develOped a urease test to distinguish

these two species. Trichophyton mentagrophytes produces

 

urease thus is able to utilize urea, changing the yellow
color to red throughout the medium due to the presence of
phenor red. She found 92.8% of the 20 isolates of T.

mentagrOphytes produced a red color within seven days but

 

no isolates of T. rubrum or T. mentagrophytes variety

 

erinacei were positive.

Since morphological, nutritional, and physiological
means of separation have not resolved the placement of some
of the dermatophytes, other methods need to be improved
and utilized.

hSeeliger (1960) in a review on the advances made in
the application of double-diffusion and immuno-

electrophoresis to fungi said:

17

I think that the application of serology offers a
valuable additional tool for investigating phylo-
genetic relationships among fungi. Serological test
could, indeed, help in the solution of some difficult
taxonomic problems. Last, but not least, further
studies might be rewarding to the mycologist who
tries to improve and accelerate his diagnostic work
in the study of fungi as well as of pathologic con-
ditions caused by, or associated with, these
fascinating microorganisms.

Agglutination and Precipitation Techniques
Used in the Identification of Fungi

 

 

Serological investigations using fungi have been
performed primarily as a diagnostic tool to detect mycotic
infections (De Beurman §E_il:, 1908; Kligman and De
Lamater, 1950; Conant eg_aT., 195A; Salvin, 1963; and
Campbell, 1967). The mycologists adapted many of the
serological methods used in bacteriology to the immuno-
logical study of human parasitic fungi (Seeliger, 1962).
These investigators used variations in the basic methods of
agglutination, precipitation and complement fixation (Salvin,
1963). The first report of diagnosis of thrush and sporo—
trichosis using agglutination was in the early twentieth
century (Widal e£_aT., 1910). Moses in 1916 reported comple-
ment fixing antibodies in blastomycosis in Brazil. Later,
in 1925 Balls reported on the use of the precipitin tests
in the identification of yeast. This was followed by a
group of workers using agglutination (Benham, 1931, 1935;

and Almon and Stovall, 193A), precipitin (Lamb and Lamb,

18

1935; and Stone and Garrod, 1931), and complement fixation
(Negroni, 193A; and Martin and Jones, 19A0) tests for the
diagnosis, identification and the systematic study of the
yeast-like organisms.

Chester (1937) emphasized the problems involved in
interpreting the early serological results as applied to
plant taxonomy. This concern has also been expressed by
Campbell (1960) and Seeliger (1962). The crude techniques
make the results unreliable and thus the taxonomic conclu-
sions based on such data of limited value (Seeliger, 1962).

In 1937 Jadassohn e£_aT. reported on the antigenic
analysis of four species of the dermatophytes. As antigens
they used desiccated trichophytins which were prepared by
repeatedly freezing components of the fungi in the growth
medium, removal of the mycelium, and drying the remaining
material. Guinea pigs were sensitized by injecting the
trichophytin for each strain of fungus. Then by the Schultz-
Dale method, using the sensitized guinea pig uterus and
observing the reaction "in vitro" of the uterine horns as
the different trichophytins were added to the Tyrode solu—
tion, the following antigenic formulae were worked out for

these four Species of dermatophytes.

 

 

Trichophyton quinckeanum (= T. mentagrophytes) 0:}
B
T. gypseum (= T. mentagrophytes) G A

Epidermophyton kaufman Wolf (E. floccosum) KW

 

 

Achorion schonleinii (= T. schoenleinii)

 

 

19

Sharp (19A5) used the precipitin test to study the
dermatophytes. He used 29 strains, from 1 to 6 strains

each of the following: Microsporum felineum (= M. canis),

 

 

 

M. audouinii, M. gypseum, Trichgphyton mentagrophytes, T.

rubrum, T. epilans (= T. tonsurans), and Epidermophyton

 

 

floccosum. These tests revealed common antigens throughout

 

the entire group, with T. rubrum, and T. mentagrophytes

 

exhibiting some cross reaction and M. felineum and M.

audouinii showing close relationships with clear distinc-

 

tion from the other dermatophytes. He reported no Species
Specific antigens in the members tested. In addition
Sharp studied 1A nonpathogenic fungi recovered from the skin.

He checked these against an antiserum made with Aspergillus

 

flavus. This antiserum did not precipitate any of the
dermatophytes or any of the other saprophytes used in the
precipitin tests.

Collodin particle agglutination reactions used by
Huppert (1955) resulted in extensive cross-reactions between
different species of dermatophytes. 0n the basis of absorp-
tion tests with alcohol precipitates from cell extracts,
three distinct immunological groups were defined among 15

strains of T. mentagrophytes.

 

The slide agglutination method has been used by
Tsuchiya and colleagues to produce the antigenic formulae

for a number of species in the following genera: Hansenula

 

(1957, 196A), Rhodotorula (1957), Candida (1958),

 

20

Saccharomyces (1965), Kloeckera and Hanseneaspira (1966).

 

In these tests either monOSpecific or absorbed antisera
were prepared.

Cross precipitation reactions were used by Tomomatsu
(1961) to study 11 strains of dermatophytes belonging to

the genera Trichophyton (T. mentagrophytes, T. rubrum, T.

 

sabouraudi, T. sulfureum, T. schoenleinii, T. ferrugineum,

 

 

 

T. megninii), and Microsporum (M. audouinii, M. canis, M.

 

gypseum), and Epidermophyton (T. floccosum). These fungi
were mechanically disintegrated and then fractionated to
produce crude polysaccharides for use as antigens. He
showed as in previous reports that cross reactions occurred
between all species. There was an indication of serological

differentiation of Epidermgphyton and MicrOSporum species

 

 

from Trichophyton species.

 

Ito (1965a) produced 22 purified fractions having
trichophytin activity, as shown by intracutaneous reaction,
isolated from the mycelium and culture filtrate of.Tricho-

phyton mentagrophytes var. asteroides by means of elution

 

 

column chromatography using DEAE-Sephadex A-50. These 22
fractions were analyzed by paper chromatography after
hydrolysis (1965b). The majority consisted of peptide-
containing polysaccharides or sugar-containing peptides,
except for two ribonucleic acid fractions from the mycelium,
and five simple peptide fractions and a simple polysaccharide

fraction from the culture medium. The main active antigenic

21

principles of trichophytins were found to be peptide-
polysaccharide complexes.

Any further consideration of agglutination, precipita—
tion, and complement fixation extends beyond the purpose of
this thesis. The problems and progress entailed in the
various serological procedures mentioned above as applied
to the study of mycoses in man and systematic studies have
been reviewed by Kligman and De Lamater (1950), Seeliger
(1958, 1960, 1962), Salvin (1963), Kaufman (1966), and

Campbell (1967).

Immunodiffusion

 

Immunodiffusion is the term used to replace the
phrase "agar diffusion precipitin test" and refers to
serologic tests conducted in or on semi—solid media (Crowle,
1961). Bechold (1905) was the first to use an immunodif-
fusion technique. He placed anti-goat rabbit serum with 1%
gelatin in a test tube. After gelling, goat serum was
poured onto the gel. Two heavy distinct precipitates
developed that differed from inorganic precipitating sub—
stances but no mention was made that these could be caused
by separate antigens in the goat serum.

0udin (19A6) employed the method of Simple or Single-
diffusion by placing an antigen solution over a solid immune
serum-agar in a thin—bore tube. Then he observed the forma-
tion of an antigen-antibody precipitate appearing as a sharp

band that migrated down the tube as more antigen diffused

22

into the gelled mixture. The number of bands formed was
equal to the number of antigen—antibody systems present.
0udin (19A8) reported that there is a straight line
relationship between movement of a precipitate band and the
square root of the time.
Le Beau (1952) applied the 0udin tube technique to the

study of the allergenic fractions of Alternaria species.

 

In 19A8 Ouchterlony and Elek working independently
developed the double—diffusion technique while investigating

toxins and antitoxins of Corynebacterium diphtheriae.

 

Ouchterlony poured a solution of 1% agar in saline into
petri dishes, then cut wells in the solidified agar, and
filled them with antigen and antibody solution. The gel
technique of Elek (19A8), also called the "Double diffusion
Gradient Test," differs from Ouchterlony's method in that no
holes were cut in the agar, instead two filter paper strips
impregnated with the reactants were placed on the agar at
right angles. Both techniques involve the diffusion toward
each other of antigens and antibodies at different rates
through agar which results in the formation of precipitate
in the form of a line or lines where the homologous antigens
and antibodies meet. Investigators find immunodiffusion
tests helpful in separating multiple precipitating systems
into individual components if the concentrations of the
reactants and physico-chemical factors are carefully con—
trolled. This separation is based on the assumption that

specific precipitates in semisolid media permit the

23

diffusion of unrelated antigens and antibodies. The number
of bands that develop in a multiple precipitating system
represents the minimum number of antigen-antibody systems
present because a line formed by one system may mask other
bands (Ouchterlony, 1958). "Reaction of Identity" is

formed when two identical antigens are compared by immuno-
diffusion test and develop a continuous line of precipation.
The diffusion of serologically related antigens against an
antiserum containing specific antibodies results in a spur
extending above the precipitation line and is called
"Reaction of Partial Identity." When serologically unrelated
antigens are diffused against an antiserum containing
homologous antibodies and compared, a formation results in.
which the precipitation lines cross each other and is referred
to as "Reaction of Non—Identity (Ouchterlony, 1958).

It is the difference in the rates of diffusion of
different soluble constituents which makes this test so
valuable in the study and differentiation of antigens and
antibodies (Biguet, e§_aT., 1965). Thus, the double dif-
fusion technique makes serological tests on fungi more
definitive than earlier methods (Seeliger, 1962; and
Campbell, 1967).

Immunodiffusion Applied to
Identification of Fungi

 

 

In his thesis at Bonn University, Seeliger (195A)
applied for the first time the double diffusion technique

to fungi. In 1955 he reported by use of the immunodiffusion

2A

agar gel method the following organisms were serologically

related in varying degrees: (1) Aspergillus gTaucus, A.

 

fumigatus, and A. niger; (2) strains of Madurella grisea;

 

 

(3) Monosporium clapieri and Sportrichum schenckii; (A)

 

 

Sporotrichum schenckii and polysaccharide fractions of

 

Cephalosporium recifei; (5) Cryptococcus neoformans and g.

 

 

diffluens; (6) Histoplasma capsulatum, Blastomyces derma-

 

 

titidis and Blastomyces brasiliensis (= Paracoccidioides

 

 

brasiliensis); (7) Candida albicans and Saccharomyces

 

 

 

cerevisiae; and g. albicans and g. tropicalis. Thus

 

 

immunodiffusion relationships in the above fungi are evi-
dence of the great potential of this technique for use in
studying other fungi.

Seeliger (1956) applied immunodiffusion, agglutination,
precipitation, and complement fixation tests to study
mycetoma in man caused by hyphomycetes. Diagnosis was as
successful with double diffusion as the precipitin test.

He Showed that Allescheria boydii (the perfect stage of

 

MonOSporium apiospermum), Indiella americana, and Acremoniella

 

 

 

lutzi are serologically indistinguishable. Serological

specificity was illustrated in M. apiospermum-M. boydii,

 

 

Madureng grisea, M. americana and Glenospora clapieri,

 

 

agents of maduromycosis. In this investigation he diffused
the antisera derived from humans with maduromycosis caused

by M. gpiospermum and from rabbits inoculated with M.

 

apiospermum against antigens from 21 fungal organisms not

 

causing mycetomas. Reactions occurred between the M.

25

apiospermum antisera and the antigens from T. rubrum, T.

 

mentagrophytes and the others, but the reverse antigen-

 

antibody test was negative.
A test was needed by Aschan—Aberg (1956) to Show that

the homothallic strain (Mi—A60) of Collybia velutTpes had

 

been derived from two monokaryotic, ornithine dependent,
heterothallic mutants, Since fruiting Could not be induced.
This strain, Mi-A60, also differed from the mutant strains
by producing clamp connections, large Spherical oidia and
large amounts of cellulolytic enzymes. The morphological
differences between the normal heterothallic and the Mi-A60
strains were so pronounced that although the same species,
'they appeared to be different. Aschan-Aberg used immuno-
diffusion to Show the relationship of the strains and
hypothesized the genesis of the homothallic strain.

Five serogroups of the genus Sporobolomyces were

 

described by Seeliger (1957) based on the number and inten-

sity of precipitation bands in agar gel. Sporobolomyces

 

and Rhodotorula showed no common antigens. Seeliger has

 

performed other serological studies with yeasts using the
agglutination techniques (1957).
Kaden (1957) used immunodiffusion and precipitation

tests to determine that Sporotrichum schenckii, S. beurmanni

 

 

and S. asteroides were the same species.

 

Diagnosis of histoplasmosis using precipitin reactions
in agar gel was performed by Heiner (1958) on 2026 patients;

7 with proven histoplasmosis, 257 with suspected

26

histoplasmosis, 13 with proven blastomycosis, 7 with
coccidioidomycosis, and the others with no known mycoses.
Using a modification of the Ouchterlony technique he
elicited six precipitins when histoplasmin interacted with
serum from patients with histoplasmosis. The designated
"h" band was consistently found in the serum of patients
having active histoplasmosis. The "m" precipitin band was
also present in active cases, but could be elicited by
histoplasmin skin tests in persons with a positive reaction.
Four other precipitin bands called "x", "y", "c", and "n"
were less consistently present and occurred with no definite
relation to activity of the disease. Blastomycin and
coccidioidin were tested in the immunodiffusion method with
sera from patients having proven cases of histoplasmosis
resulting in a reaction, thus demonstrating a common antigen
in these 3 organisms and a possible explanation for the cross
reaction between them in serological tests.

Pepys (1959), and Pepys S£_§T. (1959) did double-
diffusion experiments with some Species from the following

genera: Aspergillus, Cladosporium, Penicillium, Alternaria,

 

 

and Candida. They found 2 precipitin bands between anti—

serum produced using A. fumigatus and the extracts of S.

 

fumigatus. One line was common with extracts from S. flavus,

 

g. nidulans, S. niger, Cladospprium herbarium, and Penicil-

lium notatum while no affinities were shown with extracts

 

from A. terreus, Alternaria Sp. and Candida albicans.

 

 

27

ASpergillus fumigatus antiserum was absorbed with

 

extracts from each of the above fungi after which it was
diffused with the same extracts in the agar—gel inhibition
tests. Results from this test confirmed the previous
results except for A. terreus. This test Showed that A.

terreus is related to the other_§Spergillus Species.

 

Sera from patients some known to have ringworm caused

by Trichophyton rubrum and some with known aspergillosis

 

were used by Pepys et a1. (1959) in performing agar-gel

diffusion test with extracts from Aspergillus fumigatus,

 

Cladosporium herbarum, Penicillium notatum, mycelium and

 

 

cultural filtrates of T. rubrum, and T. mentagrgphytes.

 

Common antigens were shown to be present in T. rubrum, T.

mentagrophytes, Cladosporium herbarium, Penicillium notatum,

 

 

 

and Aspergillus fumigatus. The Trichophyton antigens pro-

 

duced a broad usually dense precipitin band but in some
tests 3 lines were produced. Common antigens were found

in the culture filtrates and cell sap extracts of the
trichophytons with additional bands formed between the

cell sap of T. mentagrophytes and sera over the number in
the reaction with the culture filtrate. The reverse was
true with_T, rubrum, there were more antigens in the culture

filtrate.

Extracts of Candida albicans, S. brumpti, S.

zeylanoides, S. tropicalis, and S. stellotoidea were

 

reacted with C. albicans antiserum in double diffusion

28

and immuno-electrophoresis experiments by Biquet et a1.
(1959). Five lines were identifiable with S. albicans, A

lines were shared with S. brampti, 3 lines with S. zeylanoides

 

and S. stellatoidea, and 2 lines with S. tropicalis.

 

 

The immunodiffusion technique was applied to sera of
allergic patients by Augustin and Hayward in 1959. »Serum

from a patient sensitive to Aspergillus fumigatus was

 

reacted with extracts from several Deuteromycetes. One

precipitation band was Shared by Cladosporium herbarum,

 

 

 

Aspergillus fumigatus, S. niger, and Penicillium notatum.

A faint line for A. fumigatus was the major line for S.

 

herbarum and vice versa. The major line of S. fumigatus was

 

also the major line for Paecilomyces Sp. and Penicillium

 

 

gyclopium but a minor line for S. notatum and S. herbarum.

 

Significant differences were found between S. herbarum and
S. fulvum.

Blastomyces dermatitidis and Histoplasma capsulatum

 

 

were distinguished serologically by the agar-gel method by
Ball §E_§l° (1960). They reported two precipitin bands with
histoplasmin, Heiner's "h" and "m" bands, and only one line
with blastomycin, serologically distinct from "m" and "h"
which they called "b".

‘The "h" and "m" antigens apparently specific for
detection of histoplasmosis have been separated from crude
histoplasmin and purified by means of column chromotography

on DEAE-cellulose by Greene et a1. (1960). These fractions

29

were used in immunodiffusion tests and confirmed the results
of Heiner (1958).

Later, in 1960 according to Greer and Gordon, the
New York State Department of Health discontinued the use of
the quantitative complement fixation test for diagnosing
histoplasmosis and replaced it with a modified Ouchterlony
double diffusion gel precipitin test developed by Heiner
(1958).

Agar-gel diffusion studies Showed that 12 isolates of

Trichophyton rubrum possessed an antigen not found in 20

 

isolates of T. mentagrophytes and A strains of T. menta-

 

grophytes variety nodular (Landay, 1961). Landay observed

 

spur formations in 9 of the isolates of T. mentagrophytes

 

when extracts from mycelia and culture filtrates of these
organisms were placed adjacent to T. rubrum extracts which

may indicate heterogeneity of the T. mentagrophytes species.

 

Species of T. rubrum and T. mentagrophytes were separated

 

using morphology and the hair invasion test (Ajello and
Georg, 1957). Dyson and Landay (1963) concluded that the
above immunodiffusion test results corroborated the validity
of the hair invasion test.

Histoplasmin and 5,000 sera from patients in tubercu-
losis sanatoria were used by Schubert SS_ST. (1961) in the
double diffusion method. They considered the demonstration
of a single band ("h" and/or "m") as the index of positivity

and the results obtained correlated well with those of the

3O

complement fixation test in which a titer of 1:8 or greater
was regarded as positive.

Abernathy and Heiner (1961) demonstrated positive
reaction, a Specific blastomycin band, in only 1A of 22
proven cases of blastomycosis using the immunodiffusion
technique. Correlation with the complement fixations test
was satisfactory.

Biguet SS_ST.(1961) used a combination of techniques,
including double diffusion, starch electrophoresis,
chromatography on DEAE cellulose and immunoelectrophoresis

to study the antigens of macerated Candida albicans.

 

These techniques revealed 9 antigens in S. albicans, two

specific for this species. Candida albicans was shown to be

 

related to S. stellatoidea, and S. trqpicalis with S.

 

 

zeylanoids. Only S. albicans antiserum was used in this

 

investigation. In 1962 Biguet SS_§T. used antisera for all
Species tested to confirm the previous work. This confirmed
the work by Tsuchiya SS_ST. (1958) who used the slide agglu-
tination technique to study species of the genus Candida.
Tran Van Ky SS_ST. (1963) carried out electrophoretic
studies on an antigenic extract of S. albicans containing
glycoproteins with a few protein bonds. Double diffusion
in agar gel was used to show 7 fractions, 5 of which were
identical with other Candida Sp. in the antigenic analysis
of the extract. The results confirmed the work of Biguet

et a1. (1961, 1962).

31

Schubert and Hampson (1962) concluded from their
research that the agar gel method could not serve as an
inclusive screening test for coccidioidomycosis as it is
less sensitive than the complement-fixation test.

Huppert and Bailey (1963) obtained sera from patients
in a hospital for immunodiffusion, complement—fixation, and
precipitin tests; using as antigen a toluene lysate from

pooled growths of strains of Coccidioides immitis. The

 

results between the immunodiffusion and the complement-
fixation tests correlated well in 10 proven coccidioido-
mycosis cases. They were the first to attempt to relate the
bands formed in agar gel with antibody demonstrated in the
precipitin and complement—fixation test described by Smith
23.31- (1950). Thus, they recommended the employment of the
immunodiffusion and the precipitin methods for routine
serological testing of coccidioidomycosis.

Rowe, Newcomer and Wright (1963) studied the effect
of temperature, pH and enzymes on the soluble antigens
derived from the culture filtrates in which different growth

phases of Coccidioides immitis were grown. They were exam-

 

ined by the double diffusion technique against sera of
patients with coccidioidomycosis. High temperature and
extreme changes of pH did influence the formation of precipi-
tin lines although the enzymes used had no apparent effect.
Evidence was obtained which indicated antigenic differences

between various strains and different growth phases.

32

Rowe, Newcomer and Landau (1963) studied the effect
different cultural factors have on the development of anti-
gens by S. immitis using the immunodiffusion technique.
Factors influencing an increase in the amount of antigen
were type of media used (such as brain heart infusion
medium supplemented with dextrose), a rise in temperature
from 30°C to 37°C, and the strain used.

The sera of asthmatics were examined in the immuno-
diffusion technique by Feinberg and Temple (1963) for the
precipitating antibodies to 1A different fungus extracts.
They found Aspergillus giganteus and S. fumigatus shared

one precipitin, but S. giganteus had two lines not shared

 

with S. fumigatus. Aspergillus giganteus and S. terreus

 

had one common line as did S. terreus and S. nidulans.

McMillen and Devroe (1963) used the agar-gel method
to study well documented cases of histoplasmosis. Data
presented suggested that immunodiffusion reactions when
used in conjunction with complement-fixation titers were
useful in differentiating active from inactive infections.
The "h" and "m" bands were demonstrable shortly after
infection and throughout the active stage of the disease
‘Which parallelled the rise, peak and drop in complement-
fixation titers. Busey and Hinton (1965) reported similar
results.

Goldin and McMillen (1963) developed a micro—method

f0Pthe agar-gel precipitation test for histoplasmosis which

33

simplified and gives more rapid reactions than the Petri-
dish procedure used by Heiner (1958).

Seeliger and SchrSter (1963) used agglutination,
precipitation, agar-gel diffusion and complement fixation
tests with whole cell antigens and crude polysaccharide
extracts from all recognized species of the genus

Trichosporon to Show that they can be subdivided into A

 

serological groups.

Using sera obtained from rabbits injected by three
different methods with antigenic solution of Hansenula
wingei, Brookbank and Heisler (1963) failed to demonstrate
specificity for mating type in this yeast in the double
diffusion technique. There was a common band with M.
anomalie, and M. saturnus. This was the only component in
common.

Kind and Meyer (1963) established species Specificity
for Actinomyces israelii and S. SSKTS using the Ouchterlony
agar-gel diffusion method: Actinomyces naeslundii cross-
reacted with both of these species, suggesting a transitional
form with antigenic relationship to the other 2 species used
in the test. These 3 actinomycetes Showed no cross reactions
With any anaerobic diphtheroids used in the experiment.

Employing the immunodiffusion technique Torheim (1963)
studied antigens extracted from and antisera produced by 12

strains of Geotrichum and related fungi. Three distinct

 

serological groups were discovered. The first serological

gPOLug included Geotrichum candidum, S, asteroides, S.

3A

javanense, S. matalense, S. pulmoneum, S. versiforme,

 

 

 

 

Endomyces lactis, and S. magnusii. He suggested that the

 

first 7 strains are in reality the same species since they

are serologically identical. Endomyces manusii did not

 

Show identical reaction with the others but did cross react
with them. The second serological group contained S.

amycelicum, S. hirtum and Trichosporon cutaneum which were

 

 

not serologically identical but cross-react with each other.

Serogroup 3 included only Endomycqpsis selenospora.

 

Markowitz (196A) found 3 serologically active fractions,
11, IV, and V, after fractionizing on a DEAE-cellulose
column, a mixture of polysaccharides obtained from culture

filtrate of Histqplasma capsulatum grown in the yeast phase.

 

With immunoelectrophoresis Markowitz showed fractions 11, IV,
and V each contained at least 2 antigenic components.

Taschdjian SS_ST. (196A) suggested that immunodiffu-
sion test may be of value as an aid in the diagnosis of
chronic systemic candidiasis.

A case of mycetoma caused by Allescheria boydii was

 

serologically identified by Baxter §£_2l- (1965) using the
immunodiffusion technique with the patient's serum and
extracts of the fungus.

Franke (1965) applied immunodiffusion and immuno-
electrophoresis to a study of species of myxomycetes in
the order Physarales. He observed strong affinities among

the following: Physarum gyrosum, S. tenerum, S. pusillum,

 

E, oblonga, and Fuligg septica; Physarum sp. and Cienkowskia

 

35

reticulata; Diderma effusum, and S. pusillum; S. pglycephalum,
and S. flavicomum; Fuligo septica and many of the 22 species
tested. The results supported the close relationship of
Didymium nigripes and S. iridis. Fuligo cinerea and E.
septica were related only in the absorbed antisera tests.

Two isolates of S. septica gave identical results but the
results of the u isolates of Physarum pusillum reacted
different serologically.

La Borde and Abernathy (1965) separated the "h" and
"m" antigens from crude histOplasmin by DEAE ion exchange
column to study their immunological and physical-chemical
characteristics. The "m" antigen produced 3 bands in the
immunodiffusion test while the "h" antigen only produced
one band. No common lines were formed between these two
antigens in immunodiffusion or immunoelectrophoresis.

In 1965 Wiggins and Schubert disagreed with the prog—
nostic scheme proposed by McMillen and Devroe (1963) due to
no demonstrable "h" band in immunodiffusion test using sera
from a number of histoplasmosis cases considered to be
Clinically active.

Huppert and Bailey (1965a and 1965b) described a
Tmathod for preparing an antigen solution-toluene lysate
“”tich correlates in the immunodiffusion test with the results

Obtained in the standard tube precipitin test and complement
fixation test for detecting coccidioidomycosis. This anti-
ger1 solution contained the original toluene lysate coccidioi-
dirl initially described by Pappagianis e§_al. (1961), and a

Se<30nd lysate preparation which contained only the tube

36

precipitin component, the heat labile complement component
having been deliberately destroyed by heat.

Burrell et_§£. (1966) studied serologically three
species of the genus Phytophthora by means of gel diffusion
and immunofluorescence. Species-Specific sera were obtained
and proved efficient for the identification of S. cactorum,
g. cinnamoni, and S. erythroseptica.

Sawaki, g£_al. (1966) reported two antigen—antibody
systems in coccidioidomycosis demonstrated by immunodiffu-
sion and immunoelectrophoresis, through the use of 1131-
coccidioidin. Sera which were positive in the complement-
fixation test but negative in the tube precipitin test
produced one line of precipitation in the immunodiffusion
test. The same was true in the immunoelectrophoresis and
appeared in the y2-globulin region. The sera positive to
both precipitin and complement fixation tests yielded 2
different lines in both immunodiffusion and immunoelectro-
phoresis with l arch in the y2—globulin area and the other
in the yl—globulin region. By the use of radioimmuno-
(electrophoresis, they demonstrated that the antibody
activity in Ig-G globulin parallels the CF titer and the
IIg-M antibody parallels the TP (= tube precipitin test)
:Peactivity. There was antibody activity with the Ig-A
églobulin but it did not reflect either CF or TP activity.

Walters gt_al. (1966) studied “04 sera from 50
pertients with chronic histoplasmosis in the Ouchterlony

teSt using a concentrated histoplasmin as antigen and

37

observed only "h" and "m" precipitin bands. In the
immunoelectrophoresis tests they found u precipitin bands,
1, 2, 3, and U, in 7 combinations.

Immunodiffusion technique was used to diagnose 21
cases of paracoccidioidomycosis in a study with pooled
filtrates from 3 strains of Paracoccidioides brasiliensis
in the yeast and mycelial phases as antigens (Restrepo,
1966). Sixteen of these cases were positive using the agar
gel method to only 15 for the complement fixation test.
Two cases were negative with both tests. One of the two
negative cases produced the causitive organism, g.

brasiliensis when cultures were made. Both tests produced

 

negative results in 3 clinically cured patients. The

above antigenic solution was tested with sera from patients

with culture-confirmed cases of sporotrichosis, cocidioido-

mycosis, and blastomycosis, resulting in no cross reactions.
Common antigens were shown in Trichophyton rubrum,

T. mentagrophytes, Hormodendrum sp., and Penicillium sp.

 

 

using the agar gel diffusion test by Reyes and Friedman
(1966). These investigators found specific antibodies
developed in sera of animals immunized with dermatophytes
as well as an antibody directed against a common antigen
found in dermatophytes, Hormodendrum sp., and Penicillium
sp.

Grappel et_al. (1967) produced antisera in rabbits

with autoclaved mycelial suspensions of Microsporum

 

quinckeanum (= Trichophyton mentagrophytes) which were

 

38

reacted with three neutral polysaccharides, galactomannans I,
galactomannans II and glucans, isolated from five species of

dermatophytes, M. quinckeanum (= T. mentagrophytes), T.

 

 

granulosum (= T. mentagrophytes), T. interdigitale (= T.

 

 

 

mentagrophytes), T. rubrum, and T. schoenleinii by the

 

immunodiffusion method. Significant differences were found
among the glucans and galactomannans II, but not among the
galactomannans I of these species.

Georg E£_il- (1967) evaluated the immunodiffusion
technique for diagnosing actinomycosis and found it to be
good but cross reactions occurred between sporotrichosis,
nocardiosis, tuberculosis, hydradenitis supurative leukemia,
and hepatoma.

Ray (1967) used a modification of the agar—gel
precipitin-inhibition technique (Ray and Kadull, 196“) to

detect Coccidioides immitis antibodies in sera from human

 

clinical cases and experimental animals infected with this
organism. He had more consistent and reliable results than
with complement fixation and double immunodiffusion tests.

Using the agar gel diffusion technique Landay gS_ST.
(1967) reacted antiSpherule and antiarthrospore from

Qggccidioides immitis and anti-HistOplasma capsulatum sera

 

‘With extracts of spherule, arthrospores, and coccidioidin.
The antispherule sera formed multiple precipitin bands with
eXtracts of spherules and of arthrospores while the anti-
arthrospore serum failed to precipitate with the spherule

extract and formed a single band in the presence of an

39

arthrospore extract solution. When these extracts were
tested with different antisera and anti-Histoplasma, it was
found that the spherule preparation formed bands only in
combination with antipurified spherule serum, whereas the
arthrospore extract precipitated with anti—purified Spherule,
antiarthrospore, and anti-Histoplasma capsulatum sera, 1
band with antiserum from rabbits with coccidioidomycosis but
no bands in the presence of antiarthrospore serum.
Coccidioidin formed 2 bands in the presence of any of these
antisera. The conclusion was made that the extract from

the Spherule phase of S. immitis differed from the extracts
of the arthrospore and mycelial phases.

Okada and Yamamura (1964) prepared antisera in rabbits
against microsomal and unsedimented fractions isolated from
adult chicken livers. The reactions of these antisera with
several microsomal extracts and unsedimented fractions
prepared from livers in different stages of development
were studied by immunodiffusion technique. Adult liver
microsomes showed 5-6 components which are predominant in
the deoxycholate (DOC) extract of microsomes. Three of these
components were highly "liver-Specific.” One was detected
in the 9-day old embryonic liver and the number progressively
increased to 3 at the time of hatching. Common components
occurred in the microsomal and unsedimented fractions and
appeared as early as the 6th day in development. Two of
these "common components" were associated with esterase

activity. Unsedimented fraction of 6-day embryonic liver

NO

contained u components specific for this fraction. These
components showed complete or partial identity with those
seen in the unsedimented fraction of adult liver.

A similar investigation was done by Okada and Sato
(1963) with chicken kidney. They found 3 highly specific
kidney components in the DOC extract of the microsomes
and 2 components common with all fractions tested. The
sera were absorbed with extracts of liver and lung to
remove the antibodies that would cross react.

Immunodiffusion techniques have been used very little
in investigating the dermatophytes. This technique was
applied to the study of the relationship of these organisms
to each other using some subcellular fractions. The possible
use of microsomal fractions as highly specific components of
the dermatophytes as was found in the chicken liver by
Okada and Yamamura (196“) was investigated in this research.

Thus from a review of the literature it is apparent
that investigators are using immunodiffusion primarily to
diagnose mycotic infections, while only a few (Biguet 22
figs, 1959; Seeliger, 1960; Torhiem, 1963; and Franke, 1965)
have commented on the potential taxonomic value of this

tmechnique as an aid in clarification of the phylogenic

Iwalationship of fungi.

MATERIALS AND METHODS

I. Dermatophytes Studied
Twenty cultures of dermatophytes which included at
least one species from each genus and 2 or more strains
(isolates) of the species to be used in preparing antisera,
the reference strains, were selected for these investiga-
tions. Stock cultures were grown on Sabouraud's glucose

agar. The species of dermatophytes investigated and their

sources are listed on the following page.

II. Verification of Trichophyton Species
MacrOSCOpic and microscopic characteristics are used
to separate the typical species of the dermatophytes in the
genera Epidermophyton, Keratinomyges, and Microsporum.
Some members of the genus Trichophyton are difficult to
separate using these characteristics, thus other means are
used, including nutritional requirements, pigment production,

hair invasion and urease production.

A. Trichgphyton Agars

 

Mycelial inocula about one—eighth of an inch in
Cliameter were removed from each Sabouraud's glucose agar
Cnllture of Trichgphyton studied. These were imbedded in
Serven nutritional media used for the differentiation of

lfinghophyton species (Bacto-Trichophyton Agars, Difco, as

 

Ml

 

|-—'
1%”
L:H
Q4

moohvton floc
geron and M. L

2. Keratinomyces afielloi
Vanbreuseghem, 195?

 

3. T41

ospwwru"i ferwuigirmvxm
a, 1933

 

r‘
._
L,

‘\
\.-

q
V

U. Microsporum gypseum (Bodin)
Guiart and Grigorakis, 1938

5. Trichophyton equi d“
Dassonville) Gedce

 

 

' .. 1 H _ l
6. Trichoanton gall-rae ('egn-n)
“1‘...“ p... '- 1"”‘3
Silva ans Henna", 14?;

7. Tfi‘i‘fliOt‘h"to, Ite.fi;iriii
slarlmlard, .Cib

 

 

 

 

 

C. mentaxroynytes var.
‘, _ r', \
Indven-Imflhiirt, 1311
9. mertar“orhvtes VEF.
I"‘1C!l-..JL ‘I‘lli’e’ 19.51
I : V‘ ,-. ,. ,-
13. 1r-ch mp .t n mentagr rqjtes v3“.
erinac e; smith e: 1rp.es, -ab3
________

 

11. Trichophyton rubrum (Castellani)
Bab F341, 1911

12. inicntrn,tcn rubrum (Caste11ani)
Sabouraud, 1911

 

13. Tr ivc h-ochiton schte lein
Lange “a1 and ILiiOCik

 

l“. Tricho onhyton soudarense
Joye eux, 1912

 

15° TrichQPhyton terrestre
Durie and Frey, 1)j/

 

(D

16. Txichc phyton terresti
Durie and Frey, 1957

 

l7. Trichophyton terre
Durie and Frey,

 

st: e
1957

18. Trichophyton tonsurans
Malmsten, 1835'

 

19. Trichophyton verrucosum
Bodin, 1902

 

20. Trichophyton violaceum
Sabouraud, apud Bodin, 1902

 

Specimens of all the above are deposited in the collection of fungi (E. S.

142

Tin a corporis, Ulbrich Dermatology Clinic,
Detroit, Michigan, identified by
E. S. Beneke

Collection at the Section of Dermatology,
Department of Medicine, University of
Chicago, identified by T. Benedek.

Co11ectinn at the Department 0 .erna’clozv,
cghc 1 or ”eiicihe, University cf Michigan

Soil, Sad Paulo, Brasil, 1961, identified

by E. S. Beneke

Collection at the Department of Dermatology,
School of Medicine, University of Michigan

on data unknown

0
( )
*_.J
H
(b

.-A-

china State University
1 f ed by E. S. Beneke

7 .
#UTB N.C.D.C.‘ T’nea beiis ide tified b?
J. Pislnar, c1«nito, (‘arwiia
fi': é \‘ l‘ i {‘1 "“n‘a 'x " “0“”0‘A”
kfifl.‘ . .co..¢.../. 11.3?” if] d. ”eatery“: ,
ixentified by 3.3rH31e flew Zealand.

1 nea corneris, isolated by G. Dardas,
‘ .' ‘ ' i. L. Steers, 1305

y E. S. Beneke,
rs, 1)6§.

ecticn at Derartment of Der!natolcgy,
chool of Medicine, University of Michigan

Departnent of Jermatoiouy,
' ity of chnigan
'ck, En :1and.

Soil isolate, G. Orr,
ty of California, Los Ange es.

#XZBS N.C
Univers

H 0

Soil, Sa identified by

A. L.

Brasil,

Pailo, .
gwer 1961

m<m

Tinea capitis,U1brich Dermatology Clinic,
De roit, identified by E. S. Beneke

Collection at the e
Department of Me i

ction Of Dermaiology,

cine, Lnilers ity of Pn1n.;w

Collection at Department of Dermatology,
School of Medicine, University of Michigan

Beneke),

at Michigan State University, East Lansing, Michigan.

1National Communicable Disease Center,

Mycology Unit,

Atlanta, Georgia.

“3

formulated by Georg and Camp, 1957). The cultures were
incubated at 2A°C for three weeks and checked for amount of
growth on each medium.
B. Tests to Separate T.
mentagrophytes and T. rubrum

l. Pigment production.——Petri dishes containing corn
meal dextrose agar formulated by Bocobo and Benham (1949),
potato dextrose agar (Difco) and Sabouraud's glucose agar
(Difco) were inoculated with small pieces of mycelia from
each isolate of T. mentagrophytes and T. rubrum. After
incubation at 2A°C for 3 to U weeks the presence or absence
of undersurface pigment was noted.

2. "In vitro" hair invasion (Ajello & Georg, l957.--
The medium contained 25 ml of sterile distilled water, 5
drops of 10% yeast extract and an assortment of human and
horse hairs placed in petri dishes. Mycelial transfers were

made from the isolates of T. mentagrophytes and T. rubrum

 

into this medium and incubated at 2A°C. These cultures
were agitated for one minute each day for 21 days. At the
end of this time hair segments were removed from the medium
with sterile forceps, placed in a drop of lactophenol
cotton blue mounting fluid and examined under the micro-
scope to determine whether or not they had been perforated.

3. The Urease test (Philpot, l967).--Ureasé is known

 

to be present in T. mentagrophytes but not in T. rubrum,

 

thus the urease test was performed on these isolates used

in this investigation. Christensen's medium was modified

MA

as suggested by Philpot (1967). The medium contained the
following: peptone, l g; NaCl, 5 g; KH2POA’ 2 g; glucose,
5 g; agar, 20 g; distilled water to make 1 liter. These
ingredients were dissolved by heat and 6 ml of phenol red
solution (0.2% in 50% alcohol) was added. After the medium
was autoclaved at 115°C for 15 minutes and cooled to 50°C,
100 m1 of a 20% aqueous solution of urea, sterilized by
filtration, was added. Fifteen ml of the medium was dis—
pensed aseptically into sterile % oz bottles, and slanted.
Small pieces of mycelium from the T. rubrum and T. menia-

grophytes strains were transferred to these slants, incubated

 

at 24°C, and observed daily for 21 days. The development of
a deep red color throughout the medium was regarded as the
end point of the reaction.

All of the tests to separate the Trichophytons were

 

done in at least duplicate.

111. Media For Growth Of The Dermatophytes

 

Preliminary studies were performed to determine which
of several media would yield the largest amount of hyphal
wet weight in the shortest time in order to supply large
quantities of the organisms for the microsomes to be used

as an antigenic fraction. Trichophyton mentagrophytes (dog),

 

T. terrestre (A14) and T. rubrum (Dar) were the organisms

 

selected for this test. Sabouraud's glucose broth was the
medium selected for the growth of the fungi used in the

major portion of the research. The formula for this medium

“5

and those for other media used in the preliminary studies

are as follows:

Sabouraud's Glucose Broth

Glucose no.0 g
Neopeptone 10.0 g
Distilled water q.s. 1000.0 ml

Ingredients were added to the distilled water,
dissolved, and the pH adjusted to 5.6. The medium was
dispensed and autoclaved at 121°C, 15 pounds pressure for

15 minutes.

Schaffer, Molomut and Center's Medium (1959)

Sucrose 40.00 g
Ammonium sulfate 2.00 g
Ammonium nitrate 1.30 g
Ammonium citrate 1.00 g
Potassium phosphate, monobasic 0.15 g
Potassium phosphate, dibasic 0.15 g
Ammonium phosphate, dibasic 0.20 g
Citric acid 1.00 g
Potassium citrate 0.u0 g
Magnesium sulfate 0.24 g
Calcium carbonate 0.80 g
Trace elements, stock solution* 1.00 ml
Distilled water q.s. 1000.00 m1

*Formula for Trace Elements, Stock Solution:

Copper sulfate u.
Manganese sulfate 5
Potassium dichromate 2
Zinc sulfate 5.
Ferric sulfate 1
Distilled water q.s. 1000

000000
BOQOQOQOQOQ

The ingredients were added to the distilled water,
heated to dissolve, then filtered to clarify. The pH was

adjusted to 6.5, then the medium was dispensed into

M6

cusntainers and autoclaved at 121°C, 15 pounds pressure for

12 minutes .

Bulmer's Modification of Czapek's Medium (1965)

Sodium nitrate 2.00 g
Potassium phosphate 1.00 g
Magnesium sulfate 0.50 g
Potassium chloride 0.50 g
Ferric sulfate 0.01 g
Sucrose 30.00 g
Glucose 5.00 g
Peptone 5.00 g
Yeast extract 1.00 g
Distilled water q.s. 1000.00 ml

The ingredients were dissolved in the distilled water,
‘trle‘pH adjusted to 5.6, the medium diSpensed into containers

arrd.autoc1aved at 121°C, 15 pounds pressure for 15 minutes.

IV. Antigens

 

A. Preparation of Antigens

Strains of the dermatophytes used for antigen produc-
ticnu were grown on Sabouraud's glucose agar, the medium
useci for stock cultures, in 50 m1 test tubes for two weeks
at 255°C. The fungal mat was removed with as little medium
as p<>ssible and blended for one minute in a 360 m1 Monel
meteil. semi—micro Waring Blendor with 25 m1 of Sabouraud's
glu€<>serbroth or the medium in which it was to be grown.
Ten rnl of the blended fungus, the inoculum, was pipetted
intc> 2000 m1 Erlenmeyer flask containing 500 m1 Of the
SterfiJlized test medium and placed on a reciprocating shaker

Whicrllfiad a stroke of one inch and gave 90 two-inch

147

excursions per minute. Incubation temperature was 2U°C.
The flasks containing the different media were covered
with pieces of aluminum foil four inches square and auto-
claved before inoculations were made.

Materials and equipment used in the preparation of
the antigens were sterilized and procedures were performed
under as aseptic conditions as possible.

At the end of the 1A day incubation period the fungi
xvere vacuum filtered using No. 500, 12% cm. diameter
ESargent filter papers, 142 mm diameter Buchner funnels, and
:filtering flasks. The mycelia were washed with sterile
ciistilled water at least six times, until no medium color
Iwemained in the filtrate. Two additional washes were
Eippflied if the fungus produced a water soluble pigment.
Mhashing was to remove as much medium as possible. The mass
cxf hyphae was weighed and the weights recorded.

The 20 different organisms were processed in the
fxbllowing manner. Eighteen grams of a fungus, 10 grams of
UL). 110 Superbrite glass beads from Minnesota Mining and
[Warmifacturing Company, and 50 ml of a homogenizing medium
werue blended at 0°C in a 360 m1 Monel metal semi-micro
Warring Blendor equipped with a jacket for 30 minutes or
Untj;1 only small fragments of hyphae could be detected
miCI‘Oscopically. The temperature was kept at near 0°C by
filliJig the jacket with crushed ice and slowly running

water“ through the ice which was replaced as it melted.

48

The homogenizing medium was composed of 0.25M sucrose,
0.05M 2-amino—2 hydroxymethyl-l, 3-propanediol (TRIS), and
0.01M MgCl adjusted to pH 7.8 (Pollard, et_aT. 1966).

This homogenate was vacuum filtered through 4 layers
of Kleenex tissue in a cold Buchner funnel into a filtering
flask in crushed ice. The filtrate was centrifuged at 0°C
in an International Centrifuge (Model HR—l) at 20,000 times
Egravity for 20 minutes to remove most organelles. The
:supernatant solution was decanted and the pellet was dis—
czarded. The supernatant solution was centrifuged at
104,000 times gravity for 4 hours in a Beckman Ultracentri-
:fuge Model L-2 at 0°C. The resulting supernatant solution
vvas decanted and used as one antigenic fraction, referred
tCD in this thesis as "supernatant antigen." This fraction
vvas stored at —10°C. The pellet was rinsed twice with
rusmogenizing solution at 0°C, resuspended in 25 ml of the
saame cold solution and centrifuged for 1% hours at 104,000
tximes gravity. At the end of this centrifugation the
Slxpernatant solution was discarded, the pellet rinsed twice
deth cold homogenizing solution and suspended in a small
anubunt of the same solution. This fraction was used as
thE: second antigenic fraction and will be referred to as
'WMEllet antigen."

Sterility tests were conducted by placing 1 ml
quavl’ltities of each fraction into test tubes containing
eithfirr Sabouraud's glucose agar to detect fungus growth or

nutriGnt agar to detect bacterial growth. If inocula in

49

either medium showed growth during a two week period, the
antigenic fraction was discarded and the fractionation was
repeated with that fungus. The antigenic fractions were

diSpensed into 3 ml vials and stored at —10°C. In the

cases of Trichophyton mentagrOphytes (Dog), T. terrestre

(414), and T. rubrum (Dar.), the reference strains, several
repeats of the fractionation procedure were necessary to
liave sufficient quantity of fractions to perform the desired
:studies so the like antigenic fractions for each of the
:isolates were pooled before any experiments were begun.

!tfter pooling, sterility tests were conducted and the frac-

tlicms were dispensed and stored at —10°C.

B. Protein Determination

The amount of protein in each of the dermatophyte
f‘Pactions for all organisms was determined using the method
017 Lowry et_aT. (1951). Crystalline bovine plasma albumin
‘W61s used as the standard. The tests were run in duplicate,

ufiSing freshly made reagents, except for the Folin phenol

r'eagent. The determinations were performed at the beginning

Cf? the investigation.

V. Antibody Production

5:;_,Rabbits Used

German checker rabbits, both bucks and does, approxi-
mately six months old were used for production of antisera.

Tktbee rabbits were injected with each antigenic fraction to

50

produce antibodies. The rabbits were watered and fed on

commercial pellets. Weights were taken at the beginning

and at the end of the studies. An increase in weight in

each rabbit was noted.

B. Antigens Used for Production
of Antisera

Supernatant and pellet antigens were used from

_3?ichqphyton mentagrophytes (dog), T. terrestre (414), and

I, rubrum (Dar), reference strains, to produce the antisera

usxed in these investigations. Supernatant antigens were

irrjected into rabbits as follows: rabbits numbered 47, 48

arici 49 received T. mentagrophytes (dog); numbers 53, 54, and
555 received T. terrestre (414), and rabbits numbered 59,

6C) , and 61 received T. rubrum (Dar). The pellet antigens
wealre injected into the following rabbits, numbers 50, 51,
arici 52 received T. mentagrophytes (dog), numbers 56, 57,

muci 58 received T. terrestre (414), and numbers 62, 63, and

64 .received T, rubrum (Dar).

C. Injections (Tables 3 and 4)

Two weeks before injections each rabbit was bled from
the: Inarginal vein of an ear to obtain control sera (pre-
inJ’ection sera).

Antigenic solutions injected were emulsified with an
equfil. volume of incomplete Freund's adjuvant (Difco) since
thifis Inaterial is known to enhance antibody production

(GOI”Z:rnski, 1963). Complete Freund's adjuvant (Difco) was

51

not used as it contains killed Mycobacteria which might have

 

produced antibodies that would cross react with the dermato-

phytes.

Details of the injections of antigens are given in
Tables 3 and 4. The first injection, containing 1% m1 of
antigenic solution and 1% m1 of incomplete Freund's adjuvant
for each rabbit, was given in the subscapular muscle. Two
days later each rabbit received an intramuscular injection
consisting of 1 ml of antigenic solution emulsified with 1 m1
of incomplete adjuvant, in the right thigh. The third injec-
tion of the same concentration was given in the left thigh
two days after the second injection. The fourth injection
with the same concentration as the second was given intraperi-
toneally 6 weeks later and was considered the first booster.
Every 2 weeks for eight more weeks similar booster injections
were given in alternating sides of the abdomen. Leskovitz
and Waksman (1960) reported that intramuscular and subcutan-
eous injections gave the highest circulating antibody titers
in their experiments. When booster injections were given
intraperitoneally, the level of antibodies was doubled.

Preliminary work included intravenous injections, but
tiince these caused anaphylactic death in three rabbits the

“Nithod was discontinued.

ELL_ Bleeding and Antiserum
€31§paration

 

Rabbits were bled from the marginal ear vein. Xylol

‘WEis applied to the area around the vein which encouraged

52

TABLE 3.--Injection and bleeding schedule for rabbits injected with super-
natant fraction TrichOphyton mentagrophytes (dog) was injected into rabbits
numbered 47, 48, andi49; T. terrestre (414) was injected into rabbits
numbered 53, 54, and 55; and T; rubrum (Dar) was injected into rabbits
numbered 59, 60, and 61.

 

 

 

Injection

 

Bleeding from

 

Month Day Marginal vein
Quantity Site of an ear
1 l. 50 cc Control
sera
l4 2 x 1.5 emulsion (1.5 ml intramuscular
antigenic fraction/1.5 subscapular
ml adjuvant) muscle
16 2 x l emulsion (1 ml anti— intramuscular
- genic fraction/l m1 right thigh
adjuvant)
18 2 x 1 emulsion (1 ml intramuscular
antigenic fraction/1 ml left thigh
adjuvant) ‘
2 46 50 cc
60 2 x l emulsion (1 ml intraperitoneal
antigenic fraction/l ml right side of
adjuvant) lower adbomen
3 68 50 cc
74 2 x 1 emulsion (1 m1 intraperitoneal
- antigenic fraction/1 ml left side of
adjuvant) lower abdomen
82 50 cc
88 2 x l emulsion (1 m1 intraperitoneal
>~antigenic fraction/1 ml right side of
adjuvant) lower abdomen
4 96 50 cc
102 2 x l emulsion (1 ml intraperitoneal
antigenic fraction/1 m1 left side of
adjuvant) lower abdomen
110 50 cc
116 2 x l emulsion (1 m1 intraperitoneal
antigenic fraction/l ml right side of
adjuvant) lower abdomen
5 124 50 cc

53

TAIlLE 4.--Injection and bleeding schedule for rabbits injected with
pejllet fraction. Trichophyton mentagrophytes (dog) was injected into
ratflaits numbered 50, 51, and 52; T. terrestre (414) was injected into
ratflaits numbered 56, 57, and 58; and T. rubrum (Dar) was injected into
ratflaits numbered 62, 63, and 64. -

 

 

 

 

- Injection Bleeding from
Montdi Day Marginal vein
Quantity Site of an ear
1 - 2 , 50 cc Control
sera
15 2 x 1.5 emulsion (1.5 m1 intramuscular
antigenic fraction/1.5 subscapular
m1 adjuvant) muscle
17 2 x l emulsion (1 m1 intramuscular
antigenic fraction/l ml right thigh
adjuvant)
19 2 x 1 emulsion (1 ml intramuscular
antigenic fraction/1 ml left thigh
adjuvant)
2 47 50 cc
61 2 x 1 emulsion (1 ml intraperitoneal
antigenic fraction/l ml right side of
adjuvant) lower abdomen
3 69 50 cc
75 2 x l emulsion (1 ml intraperitoneal
antigenic fraction/l ml ‘ left side of
adjuvant) lower abdomen
83 50 cc
89 2 x 1 emulsion (1 m1 intraperitoneal
antigenic fraction/l ml right side of
adjuvant) lower abdomen
4 97 50 cc
1133 2 x 1 emulsion (1 m1 intraperitoneal
antigenic fraction/l m1 left side of
adjuvant) lower abdomen
11.1 50 cc
1137 2 x 1 emulsion (1 m1 intraperitoneal
antigenic fraction/l ml right side of
adjuvant) lower abdomen
5 125 50 cc

54

dilation of the veins and faster bleeding. Vaseline was

spread over the vein and area under the ear so the blood
would not clot. A razor blade was used to cut the vein
as near the tip of the ear as possible. Fifty ml of blood
was collected into sterilized, 50 ml centrifuge tubes.
The collected blood was allowed to set for about 30

Ininutes at room temperature to encourage clotting. The

clot was "ringed" from the sides of the tube and refrigerated
at 4°C overnight to allow full contraction of the clot.

(The tube was removed, allowed to reach room temperature, and

tdie serum decanted. The clot was then centrifuged and any

euiditional serum was poured off. Merthiolate from a 1%

stzock solution was added to the serum to make a final con—

cenitration of 1:10,000. The serum was then frozen and the

ClJDt discarded. The bleeding schedule is given in Tables

3 éand 4.

VI. Immunodiffusion Techniques

A- Electrolyte Solutions Used
Mreparing Diffusion Media

 

The electrolyte solution used to prepare the medium
enu3113yed for all final investigations was 0.15M phosphate-
SaJJirmeat pH 7.2. The formula for this buffer and those

for' others used in preliminary experiments follows:

55

Phosphate—Saline, 0.15M, pH 7.2

Monopotassium phosphate solution, 0.15M 150 ml
Disodium phosphate solution, 0.15M 350 ml
Sodium chloride solution, 0.15M 500 m1

Phosphate-Saline, 0.15M, pH 7.4

Monopotassium phosphate solution, 0.15M 150 ml
Disodium phosphate solution, 0.15M 350 ml
Sodium chloride solution, 0.15M 500 ml

Barbital, Ionicity 0.15,* pH 7.4

Sodium barbital 6.98 g
Sodium chloride 6.00 g
l N hydrochloric acid 2.70 ml
Distilled water q.s. 1000.00 m1
Ethylenediamine-Acetic Acid (EDTA)
Ionicity 0.15,* pH 7.4
Ethylenediamine tetraacetic acid 15.80 g
Glacial acetic acid 23.80 g
Distilled water q.s. 1000.00 ml
Tris, Ionicity 0.15,* pH 7.4
2—Amino-2-hydroxymethyl—l, 3-propanediol 9.30 g
l N hydrochloric acid 74.00 g
Sodium chloride 7.00 g
Distilled water q.s. 1000.00 m1
Isotonic Sodium Chloride, Ionicity
0.15,* pH variable
Sodium chloride 8.80 g
Distilled water q.s. 1000.00 ml

*
Ionicity values (based on electric conductivity tests)
taken from p. 302 in Immunodiffusion by A. J. Crowle (1961).

 

56

B. Media for Diffusion Studies

Different diffusion media were used in preliminary

tests with the petri dish and glass slide methods. Oxoid

Ingar No. 3* was the gelling agent. To 200 ml of an

elxectrolyte solution contained in a flask, 2 ml of a 1%
scnlution of merthiolate, a weighed quantity of agar and a
nuigmetic stirrer were added, and the flask placed on a hot

pfilate. When the agar had dissolved and boiled for one

Inixdute, it was immediately put in petri dishes or placed

Ori slides.
l. Gels for the Preliminary Studies by the Petri

IDiJSh Method.-—Gels containing 1%, 1.5% and 2% agar were

 

 

IDINEpared as above in 0.15 M phosphate-saline buffer, pH

7 ~22 and 0.15M phosphate-saline buffer, pH 7.4 for studying

a-I’11:igen-antibody systems by the petri dish method.
2. Gels for the Preliminary Studies by the Agar

§i§g§de Method.-—Preliminary studies to determine the best

 

C1iffusion medium for the development of precipitation bands

‘VEBIe conducted on slides covered with agar media by varying

‘tllee agar concentration, pH, ionic strength, and the

buffering system. Electrolyte solutions used to prepare

1J% , 1.5% and 2% agar media, with ionic strength of 0.15

‘Wfire: sodium chloride barbital, tris hydroxymethyl and

aJrlinomethane (TRIA), ethylenediamine tetraacetic acid

*
Oxoid Agar No. 3, Consolidated Laboratories, Inc.,
Cl'licago Heights, Illinois.

57

(EDTA), and phosphate-saline. Other media used were 1%,

1.5% and 2.0% agar gel prepared in 0.15M phosphate-saline,

pPI 7.2.

C. Immunodiffusion Test Procedure

In preliminary eXperiments the petri dish and agar
slgide methods were used. The later method was employed in
true final studies of precipitinogenic relationships.
Seflsouraud's glucose broth, incomplete Freund's adjuvant,
thus homogenizing medium, and the preinjection rabbit serum
Seelrved as controls. The reactants were supernatant and
FHEILlet antigens from the dermatOphytes and rabbit antisera.

1. Petri Dish Method.--Glass petri dishes, 100 x 15
HUTI, and 60 x 10 mm were washed with "7X"* cleaning liquid
allci put through one rinse of tap water, five rinses of
ddistilled water and a final rnise in 95% ethanol. Twenty—
ffiifve m1 of diffusion medium was used for large plates and 10
"t1 for the smaller plates. They were placed in a portable
humidity chamber at 24°C for 6-12 hours. With plexiglas
templates as guides, circular reactant wells were cut with
cOpper cylinders or with a Feinberg agar gel cutter #1803**
111 the small plates and the agar discs were removed with a

16-—gauge needle. Wells employed were 12mm in diameter,

 

 

*
Linbro Chemical Company, New Haven, Connecticut.

**
Feinberg agar gel cutter #1803, Colsolidated Labora-

t(Dries, Inc., Chicago Heights, Illinois.

58

pfilaced 13 mm apart, or 10 mm in diameter, placed 12 mm apart
iri the large dishes. and in the small dishes the wells were
7 2mm in diameter, placed 10 mm apart. The bottoms of the
““3118 were sealed with 0.3% agar, prepared with the same
scplution used to produce the diffusion medium and the plates
vuere placed in the chamber for one hour or longer.

The wells were filled using sterile disposable Pasteur
pijdettes. The dishes were incubated for 10 days in a moist
cruanmer at a constant temperature of 24°C. The wells of the
resictants were refilled the first 4 days and at no time
dul?1ng this period were the wells empty. Reactions were
ob S erved daily .

2. Agar Slide Method.——Lantern slide glass plates,

 

1C3. 1 X 8.2 X 0.1 cm or 4 X 3% inches, were washed with
"IX—uJAX", rinsed in tapwater, five distilled water rinses,
811C1 left in 95% ethanol until used. They were then wiped
Wifitri a lintless soft cloth, placed on a level surface and
ccavtered with a uniform 2.0 mm thick layer formed by pipet-
ting 16.5 ml of diffusion medium onto each slide. After
SCHLiinfying these agar blanks were placed into moist
Chaxnbers at 24°C for 6—12 hours.
Circular reactant wells were cut with cork borers.

In Ibreliminary studies several different well sizes and
diStLances between wells were used. Wells used were 5 mm
well}; placed 6 mm apart, or 6 mm wells placed at distances
Of 6, 7, and 8 mm apart. The final investigations were all

perkarmed with a 6 mm central well surrounded by six 6 mm

59

““3115 with 6 mm distances between all wells. Agar discs
unexe removed from the wells with a glass pipette attached
tc: a rubber hose connecting to a filter and then to a
vacuum line .

The reactants were added by means of sterile dispos-
eflale pipettes. Antisera were added 4 hours before the
axrtigens to allow for the difference in rates of diffusion
of“the two substances. Slides were incubated in moist
cheunbers at 24°C for 48 hours after the addition of the
antxigens. Observations were made at 8 hour intervals for
at least 48 hours.

3. Observations.-—The petri dishes and the lantern
Sigicjes were illuminated from the sides with light from day-
liigkmg fluorescent, and 4 watt tubes in 2 adjustable

ilfilldmination lamps. They were placed into openings in card-
bCDagrds cut to fit the dishes and slides. The cardboard
WEiS mounted on the rods of a Bausch and Lomb Photomicro-
gr11Efliic camera Model L apparatus fitted with a macrostage,
8i§ innches from the black base, with the lights 4% inches
beHeath the cardboard. Precipitation lines were recorded
by Cirnwing with pencil while viewing the dishes and slides
undfir'these conditions.

4. The Influence of Antigen Dilution.--Preliminary
tEStss were conducted by the agar slide method with dif-
fererit dilutions of the antigens against undiluted homolo-
gous Eintisera. The supernatant and pellet antigens of

EElEEEERhyton mentagrophytes (dog), and T. terrestre (414)

 

60

were used as follows: undiluted, and diluted 1:2, 1:4,
1 : 8 and 1:16. The dilutions were made using the stock
antigenic fractions and the homogenizing solutions.

5. Photography.——A11 petri dishes and lantern slides

 

were photographed under the same conditions as for viewing.
Pictures were taken of the dishes at the end of one week,
and at 10 days, while the lantern slides were photographed
at 24 and 48 hours. Sometimes additional pictures were
taken of the slides after staining.

6. Staining of Precipitating Lines.-—After the slides

 

INeI?e photographed, they were washed with physiological
Satline, pH 7.2 for 24 hours and then placed in a bath of
Ciisstilled water for 24 hours. The surface of each slide was
C<D\rered with a moistened piece of Whatman #1 filter paper,
W11j_ch prevented the agar from curling as it dried, and left
tCD dry overnight. After moistening the filter paper, it
“Vafs gently removed. The plates were then immersed in
C3IPCD‘w1e's triple stain for proteins (1961) for one minute.
3338? background stain was removed with a 1%% acetic acid.
The3n the plates were rinsed in distilled water, drained,
dPiJed, marked with a diamond point glass marker, and

seDarated with a piece of tissue paper before storage.

PRELIMINARY RESULTS AND DISCUSSION

I. Media for Growth of Dermatophytes

The weights of the vacuum filtered, wet mycelia for
the dermatophytes grown in 3 different media are given in
Table 5. The weights of the mycelia grown in Sabouraud's
g11u30se broth was approximately 3 times greater than the
vnxights of the mycelia grown in Schaffer, Molomut, and
(knrter's medium and 2 times greater than that grown in
Bulnuer's modification of Czapek's medium at the end of 14
days.. The variations might be due to the amount of glucose
in tlie different media, 40 grams in Sabouraud's, 5 grams in
Bulnmer's and no glucose in Schaffer g£_aT. medium. The

latex~ two had sucrose, which may not be as readily utilized

by tile organisms.

TABIJE 5.—-Weight of the wet mycelium for the three Tricho—
REXEggg_sp. grown in different media at the end of 14 days.

‘

Weights of Vacuum Filtered Mycelium*

 

 

 

Media
T.menta ro- T.terrestre T.rubrum
phytes (dog) _ (414) — (Dar)
___~___
Sabouraud's Glucose 13.9 15.2 21.1
IVDth
Schaffer, Molomut and 4.9 5.3 7.0
eriter's Medium
Burl-mer's Modification of 9.0 11.8 13.8
fledium

 

_*
fl Weights given are averages of mycelia from three
EiSLis of each type of medium.

61

62

Schaffer, Molomut and Center's medium is a synthetic
medium with an inorganic nitrogen source compared to the
organic sources in the other two media. Any residue of
‘this inorganic source of nitrogen would have no influence
cud antibody formation when injected into rabbits, while the
cxrganic sources might, but since in this medium and in
Ehilmer's modification of Czepek's there was so much less
IH37C€lial growth by weight than in Sabouraud's glucose broth,
true medium containing the greatest amount of mycelial growth
avais selected for the antigenic relationship studies. In
true immunodiffusion studies when Sabouraud's glucose broth
“was used as a control reactant, no reactions occurred

bertween the antisera and this medium.

II. Relative Sensitivities of Methods

Table 6 presents a comparison of the relative sensi-
ti‘vities of the petri dish and agar slide methods for
Stlldying the precipitation lines formed by homologous
antigen~antibody systems of T. mentagrophytes (dog) and T.
E§Ifibestre (414).

In repeated tests performed by the agar slide method
8 precipitation lines were detected (Plate 1, Fig. 1) in
th”3 reaction area between the T. mentagrophytes (dog)
SuDfiBrnatant antigens and its antiserum, 4 lines between T.

{Eiflflgagrophytes (dog) pellet antigens and the homologous

 

antxiserum, while between T. terrestre (414) supernatant

anttigen,homologous system 9 lines were detected (Plate II,

63

TABLE 6.--The number of precipitation lines detected in
double-diffusion tests using two sizes of petri dishes and
the agar slide method.

 

Lines Formed by the Homologous
Antigen-Antibody System

 

 

 

 

T.mentagrophytes T.terrestre
Methods (EOEI (414)
Super- Pellet Super- Pellet
natant natant
fraction fraction
Petri Dish 1.0% Agar* 3 2 5 2
(1(30X15mm) 1.5% Agar 3 2 5 2
2.0% Agar 3 2 5 2
Pet:I~i Dish 1.0% Agar 5 4 7 3
(60x10mm) 1.5% Agar 5 u 7 3
2.0% Agar 5 4 7 3
Agar Slide 1.0% Agar 8 u 9 5
1.5% Agar 8 4 9 5
2.0% Agar 8 4 9 5

*
The diffusion media used in all methods were pre-
PaIVEd.with 0.15M phosphate-saline at pH 7.2.

Fiég. l), and 5 lines in the homologous antigen-antibody

SyStzehlof T, terrestre (414) pellet.

 

In tests conducted by the petri dish method using
largfi? petri dishes, 100 X 15 mm, the homologous antigen

antitnody systems of T. mentagrophytes (dog) (Plate I, Fig.

 

2) gerve the following: supernatant antigen formed 3 pre—
cipituation lines and pellet, 2 lines; While the homologous

SySteflns of T, terrestre (414) supernatant antigen produced

 

5 linfis, and the pellet, 2 lines (Plate II, Fig. 2).

64

Plate I

Immunodiffusion reactions using homologous antigen-antibody

systems of T. mentagrophytes (dog) fractions in agar gel

 

method employing two sizes of petri dishes and a lantern

slide.

Fig. l.-—Lantern slide

 

 

a = T. mentagrophytes (dog) supernatant
fraction antiserum

b = T. mentagrOphytes (dog) pellet antiserum

c = T. mentagrgphytes (dog) supernatant

 

fraction

Fig. 2.--Petri dish (100 x 15 mm)

 

 

a = T. mentagrophytes (dog) supernatant
fraction antiserum

b = T. mentagrophytes (dog) pellet antiserum

c =1T. mentagrophytes (dog) supernatant

fraction

 

Fig. 3.--Petri dish (60 x 10 mm)

 

 

 

a = T. mentagrophytes (dog) supernatant
fraction antiserum

b = T. mentagrophytes (dog) pellet antiserum

c = T. mentagrophytes (dog) supernatant
fraction

d = T. mentagrophytes (dog) pellet fraction

 

65

Plate I

 

66

Plate II

Immunodiffusion reactions using homologous antigen-antibody

systems of T. terrestre (414) fractions in agar gel method

 

employing two sizes of petri dishes and a lantern slide.

Fig. l.--Lantern slide

 

 

a = T. terrestre (414) supernatant fraction
antiserum

b = T. terrestre (414) pellet antiserum

c = T. terrestre (414) supernatant fraction

 

Fig. 2.--Petri dish (100 x 15 mm)

 

 

a = T. terrestre (414) supernatant fraction
antiserum

b = T. terrestre (414) pellet antiserum

c = T. terrestre (414) supernatant fraction

 

Fig. 3.--Petri dish (60 x 10 mm)

 

 

 

a = T. terrestre (414) supernatant fraction
antiserum

b = T. terrestre (414) pellet antiserum

c = T. terrestre (414) supernatant fraction

d = T. terrestre (414) pellet fraction

 

67

Plate II

 

68

The results of the reactions in small petri dishes

for the homologous antigen—antibody were as follows: T.

mentagrophytes (dog) (Plate 1, Fig. 3) supernatant antigen

;produced 5 lines, and the pellet, 4 lines, while in T.

‘terrestre (414) (Plate 11, Fig. 3) supernatant antigen

 

(developed 7 lines, but the pellet fraction, only 3 lines.
Varying the method by replenishing the antiserum
Ireactant depots on the agar slides and petri dishes served

t:o intensify the lines of precipitation that developed, and

(lid not increase the number of lines.

In addition to being the most sensitive, the agar
slide method was the simplest method of the three to use,

t:he reactions were more easily observed, photographed, and

11he amount of antigenic fraction was less. Thus the agar

Silide method was employed for the relationship study.

III. Reactions in Various Diffusion Media
An identical number of lines formed in all diffusion

Theadia for a given fraction. The number of lines are as

fOllows: 8 lines from the homologous antigen—antibody

S37Stems of T. mentagrophytes (dog) supernatant fraction, 4

:Lildes from T. mentagrophytes (dog) pellet, 9 lines from T,

Efiigggggggg (414) supernatant fraction, and 5 lines formed in

g:- terrestre (414) pellet.
A 1% agar gel prepared with 0.15M phosphate-saline,

pli '7.2, was selected as the medium for studying the antigenic

relationships of the dermatophytes.

69

IV. Dilution of Antigens

After selecting the diffusion medium and the method
for studying the precipitinogenic relationships, tests were
performed to determine the necessity of diluting the
antigenic solutions.

At various dilutions of the supernatant and pellet
antigens of T. mentagrophytes (dog), T. terrestre (414),
and T, rubrum (Dar), with their undiluted homologous anti-
sera, it was found that the undiluted antigen fractions
{Droduced the greatest number of lines, as indicated in
'Table 7. Therefore the undiluted antigenic fractions and
“the undiluted antisera were used in the precipitinogenic
:relationships study of the dermatophytes.

CPABLE 7.——The number of precipitating lines detected in

tnie antigen dilution tests using homologous antigen-
antibody systems in the agar slide method.*

Lines Formed by the Homologous Antigen-
Antibody System

 

Di lutions of
**
Antigens T. mentagpoghltes (dog) T. terrestre (414)

 

 

 

Supernatant Pellet Supernatant Pellet
Fraction Fraction
Undiluted 8 4 9 5
Diluted 1:2 8 3 9 4
Diluted 1:4 6 2 8 4
-Dj—1uted 1:8 5 2 6 1
Diluted 1:16 4 1 4 1

\

x
All tests were conducted in 1.0% agar prepared with
O-315M phosphate saline at pH 7.2 and incubated at 24°C.

**
Diluted with homogenizing solution

70
V. Antibody Response in Individual
Rabbits
Tests of the reactivity of antisera collected from
rabbits showed some variation in the precipitin response of
.individual rabbits to injections of the same antigenic

Irreparations. The results of these tests are presented

irl Table 8.

TUXBIE 8.—-The number of precipitating lines detected in
irndividual rabbit antiserum by homologous antigen—antibody
reactions.*

 

Arnsigenic # Rabbits Maximum Number of Precipi-
Frwaction Injected tation Lines Detected in
Individual Rabbit Antiserum

 

T. igentagrophytes (dog)

 

 

ESupernatant 3 8 (47)** 7 (48) 8 (49)

Pellet 3 u (50) 3 (51) 4 (52)
T. 1:errestre (414)

ESupernatant 3 9 (53) 9 (54) 9 (55)

Pellet 3 5 (56) 5 (57) 5 (58)
T. rugbrum (Dar)

Ehdpernatant 3 7 (59) 7 (6O) 6 (61)

Phellet 3 4 (62) 11(63) 2 (64)

x
All tests were conducted on agar slides in 1.0% agar
SESSEtPed.with 0.15M phosphate-saline, pH 7.2, incubated at

i6*
rabbit;,

Number in parenthesis is the number assigned to the

CDf the 6 antigenic fractions that were each injected
JJMXD 3 rabbits, only the fractions prepared from T. terrestre

superm-Eitant fraction and pellet elicited identical

71

precipitin responses in all rabbits. The other antigenic
fractions elicited identical responses in only 2 rabbits.
The antisera employed in the study of precipitino-
genic relationships were those which contained the greatest
llumber of detectable precipitins in the preliminary tests

(Df homologous antigen-antibody systems.

RESULTS

I. Verification of the Dermatophytes
Used in This Investigation

 

A. Identification of Species in
the Genera Epidermophyton,
Keratinomyces and Microspgrum
Dermatophytes are usually identified by macroscopic

and microscopic characteristics. The species in the genera

Epidermophyton, Keratinomyces, and Microsporum produced

 

 

characteristic macroconidia except for a few Species in the

genus Microsporum (M. audouinii, M. ferrugineum, and M.

 

 

 

rivalieri). The differences in the macroconidia were used

 

to separate the species, M. floccosum, M. ajelloi, and M.

 

gypseum.

Epidermophyton floccosum produced many large, clavate,

 

smooth, thin—walled macroconidia in clusters and singly on
hyphae. No microconidia were produced.

The thick, smooth-walled macroconidia of M. ajelloi
were cylindrofusiform, and had 5 to 12 cells. Microconidia
were abundant and pyriform to ovate in shape.

Thin, echinulate walls were present in the ellipsoid,
3 to 9-celled, macroconidia of M. gypseum. Sessile and
stalked, clavate microconidia were numerous.

Since M, ferrugineum does not produce macroconidia it

 

was identified by colony morphology and its reactions on the

72

73

'Trichophyton Agars. The deep yellow to orange-colored

colony was slow-growing, heaped, glabrous and waxy, with

Inany deep furrows. The growth of this organism on the

'TrichOphyton Agars showed no specific nutrient require—

Inents. At times a non—pigmented isolate develops resemb-

ling T. verrucosum. This requires the use of the

'Trichophyton Agars for differentiation of the organisms.

I3. Identification of Trichophyton

Species
l.
ssgdecies of the genus Trichophyton may be differentiated

Use of Trichophyton Agars.——A number of the

<>r1 the basis of colony morphology, however others cannot be

cij_stinguished readily on this basis. Some of the species

aixée more readily distinguished on a physiological basis

t>37 the use of the Trichophyton Agars.
The growth characteristics on Trichophyton Agars

£231? the Trichophyton sp. used in this investigation are

g;ixlen in Table 9. All cultures of the Trichophytons used

11811 characteristic colonies and the usual nutritional

grrywth requirement (Table 9) for the species to which it
vuas assigned by the individual who did the original

iderrtification.
Separation of T. mentagrophytes and T. rubrum.--

Table 10 contains the

2.

a. Pigment Production:

data.<3n the undersurface pigment production for T.

EEKEEflgyophytes and T. rubrum. The 2 T. rubrum isolates

74

TABLE 9.-—Growth pattern of Trichophyton species on Tricho—

phyton Agars.x

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

o o
4—3 4—3
'o cu m
+4 p a
O 4—3 4—3
<1: H HG.)
+ ‘z 2::
O H
+e+ + +-mr+ +«4 E ET)
. o c c o c :3 :«4
Organism c: :24: 2H CH4: ma H H43
+4 war; ++E ++E~4 C13 C c1m
Q) (DU) (DCU (DCUUJ HO O OH
m we mod raw—{O mo 5 Em
m as m: an: «H4 E
O OH 0E4 OE—‘H OZ <1: 41+
Microsporum ferrugineum 4+ 4+
Trichophyton quinum
T. gallinae 4+ 4+
T. megninii 0 4+
T. mentagrophytes (dog) 4+ 4+ 4+
T. mentagrophytes (455) 4+ 4+ 4+
T. mentagropgytes (536) 4+ 4+ 4+
T. rubrum (Dar) 4+ 4+
T. rubrum (J.H.) 4+ 4+
T. schoenleinii 4+ 4+ 4+ 4+
T. verrucosum 0 +1 0 4+
T, soudanense 4+ 4+ 4+
T. terrestre (414) 4+ 4+ 4+
T, terrestre (285) 4+ 4+ 4+
T, terrestre (Brasil) 4+ 4+ 4+
T. tonsurans : to 1+ 4+
T. Violaceum : 4+

 

 

1* .
A 4+ indicates good growth, while a 0 indicates no
growth. Spaces left blank are not critical in differentiation

'usually occurring on these media.

of Species, with growth

75

TABLE 10.-—The differentiation of Trichophyton rubrum and
T. mentagrophytes.

 

 

 

Undersurface Pigment Color

 

 

 

 

 

f.1
H
m (U +313
- m m<v
o aim
:50) HQ) (D 05: B
mcn wen m L.o o
:40 Q)O c>o p44 ®+J
23:4 Est-o 43:4 w—Im m L.
04-) C143 «54: :><U (Ur/JO
LDN L.x +>x > o:sa
Organism com oo oo Cc: ecoo
UDQ L>Q n.o Hi4 :DCJU
Tl. mentagrophytes Brown—red White Whlte + 5 days
(DogI
.__. mentagrophytes Brown-red Lt. Tan White + 6 days
(455)
jTL- mentagrophytes Yellow Yellow Yellow + 7 days*
var. erinacei
(536)
El. rubrum Wine Red Wine Red Red — -
(Dar)
2:. rubrum Wine Red Wine Red Red — —
(J.H.)

 

*
The color of the medium was light red on the 7th day,
deep red on the 12th day.

pIWDduced a wine red pigment in all the media used, including
Sarnouraud's glucose agar, corn meal agar, and potato
dexnzrose agar. Trichophyton mentaggophytes (dog) and E-
EEEflgagrophytes (455) both produced a red pigment in
Sat>Ouraud's glucose agar but no red pigment in any other

meetia. The third strain, 3. mentagrophytes (536), developed

 

a 3531low undersurface color in all these media.

76

b. In Vitro Hair Test: Hair invasion has been

 

used to differentiate Trichophyton rubrum and T. mentagro-

 

 

phytes. Microscopically, T. mentagrophytes showed a wedge

 

shaped or irregular clear area in the hair, apparently
from protsolysis. Mycelium and spores were observed cling-
ing to the outside of the hair shaft. The three isolates

of T, mentagrophytes invaded the hair in 5 replications

 

taut one strain, T. mentagrophytes (455) (var. gganulosum)

 

 

vvas always slower.

Trichophyton rubrum did not invade the hair shaft.

 

Ddicroscopically, mycelial elements and spores of T. rubrum
czould be seen clinging to the outside of the hair shaft
t>ut no wedge shaped or irregularly digested areas were
c>bserved (see Table 10).

c. The Urease Test: Trichophyton mentagrophytes

 

 

ijsolates possess the enzyme urease (Rosenthal, 1965) and
arne able to split urea in a modification of Christensen's
”Radium. Thus it was possible to differentiate T. menta-

Efiflophytes from T, rubrum. The T. mentagrophytes strains

 

STNJWed the presence of urease while the T. rubrum isolates
gawng no indication of this enzyme at the end of 21 days as
indicated in Table 10.

II. The Amount of Protein in the Antigenic
Fractions of the Dermatophytes

 

 

The amount of protein in the 40 different antigenic

fr’actions was determined using Lowry method (1950). The

77

tests were performed before the immunodiffusion studies
wermebegun. The results are shown in Table 11 for the
sulpernatant and pellet antigens. The range in milligrams

per'nfllliliter for the supernatant fractions was 0.725 to

3.'700, while the range for the pellet fractions was 0.350

tc> 1.950.

111. Results of Precipitinogenic Studies

The reactions of the homologous and heterologous

arrtigen—antibody systems, using fractions of a dermato-

pfiyte isolate, were compared by two procedures. In the first

IDIRDcedure the antigenic fraction was placed in the center
‘veell and diffused against the antisera of the 2 fractions
(ssupernatant and pellet) from an isolate placed in 6 equi-

<ii;stant depots (3 adjacent wells per antiserum) surrounding

‘tkie antigen fraction well (Plate III). In the second

I>rnacedure the antiserum was placed in the center well and
Ciiffused against the antigenic fractions from an isolate in

-3 <>f the 6 equidistant wells, alternated with fractions

frrfiom other organisms (Plate IV through IX). The latter pro-

‘363ciure was followed in testing the relationships of antigen

fI“a.ctions for all organisms used in these investigations.
?‘
£\ Homologous Antigen-

A\‘
14Eigibody Reactions

In immunodiffusion investigations of 6 fractions, 2

ffirwom each of 3 species of Trichophyton, employing homologous

78

TABLE ll.--The amount of protein eXpressed in mg/ml in the
dermatophytes fractions as determined by the Lowry et al.
(1951) method. ‘

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

()rganism Supernatant Pellet
T. mentagrOphytes (dog) 1.850 1.000
T, nuentagrophytes (455) 2.380 1.950
T, nuentagrOphytes (536) 1.600 0.750
T. Inlbrum (Dar) 1.475 0.850
T. Iflibrum (J.H.) 1.705 1.010
T. terrestre (414) 1.842 1.322
2- :gerrestre (285) 2.225 1.825
2- 35errestre (Brasil) 3.050 0.890
T. equinum 2.100 1.475
2- ggallinae 1.340 1.040
T menginii 0.725 0.350
1- ghoenleinii 1.275 0.625
T. §55udanense 3.700 0.700
I Lonsurans 2.275 1.500
E 2;;Prucosum 1.550 0.810
2 X_}31aceum 2.900 0.735
E- floccosum 1.175 0.475
E- _a.lelloi 2.025 0.875
M' Eélzyugineum 2.150 0.550
.41- EXEseum 1.800 0.475

/

 

79

fraction antigens and antisera from 4 to 9 precipitation

lines were observed (Tables 12, and Plates 111 through IX).

80

Plate III

Diagrams of immunodiffusion test precipitation lines pro-
duced by homologous and heterologous antigen—antibody

systems of the fractions within the species Trichophyton

 

mentagrgphytes (dog), T. terrestre (414), and T. rubrum

 

 

 

 

 

 

(Dar)
a = T. mentagrophytes (dog) supernatant fraction
antiserum

b = T. mentagrophytes (dog) pellet antiserum
ls = T. mentagrophytes (dog) supernatant antigens
1p = T. mentagrophytes (dog) pellet antigens

c = T. terrestre (414) supernatant fraction antiserum
d = T. terrestre (414) pellet antiserum
6s = T. terrestre (414) supernatant antigens
6p = T. terrestre (414) pellet antigens

e = T. rubrum (Dar) supernatant fraction antiserum
f = T. rubrum (Dar) pellet antiserum

4s = T. rubrum (Dar) supernatant antigens

4p = T. rubrum (Dar) pellet antigens

81

 

82

Plate IV

Diagrams of immunodiffusion test precipitation lines

produced by supernatant antigen fractions from selected

dermatophytes and T, mentagrOphytes (dog) supernatant

 

and pellet antisera.

G‘

HHH
mwommqmmzwmw

NHHHHHHH
ommqmmzw

I3$3:WFmifiifiwafiflHwflhflfiflflwflfifififlflI

T, mentagrophytes (dog) supernatant fraction-
antiserum

2.

T.

mentagrophytes (dog) pellet antiserum

mentagrophytes (dog) supernatant antigen
mentagrophytes (455) supernatant antigen
mentagrophytes (536) supernatant antigen
{ubrum (Dar) supernatant antigen
rubrum (JH) supernatant antigen
terrestre (414) supernatant antigen
terrestre (285) supernatant antigen
terrestre (Brasil) supernatant antigen
equinum supernatant antigen
gallinae supernatant antigen
' supernatant antigen

schoenleinii supernatant antigen
soudanense supernatant antigen
tonsurans supernatant antigen
verrucosum supernatant antigen
Violaceum supernatant antigen
floccOsum supernatant antigen

ajelloi supernatant antigen
ferrugineum supernatant antigen
gypseum supernatant antigen

 

 

 

 

 

 

 

 

 

 

 

 

 

83

 

84

Plate V

Diagrams of immunodiffusion test precipitation lines pro-
duced by pellet antigen fractions from selected dermato-

phytes and T, mentagrophytes (dog) supernatant and pellet

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

antisera.
a = T. mentagrophytes (dog) supernatant fraction
antiserum.

b = T. mentagrophytes (dog) pellet antiserum.
l = T. mentagrophytes (dog) pellet
2 = T. mentagrophytes (455) pellet
3 = T. megtagrophytes (536) pellet
4 = T. rubrum (Dar) pellet

5 = T. rubrum (JH) pellet

6 = T. terrestre (414) pellet

7 = T. terrestre (285) pellet

8 = T. terrestre (Brasil) pellet

9 = . eguinUm pellet
10 = T. gallinae pellet

11 = T. megninii pellet

12 = T. schoenleinii pellet

13 = T. sbudanense pellet
14 = T. tonsurans pellet

15 = T. verrucosgm pellet

16 = T. Violaceum pellet

17 = E. floccosum pellet

18 = M; ajellEIpellet

19 = M. ferrugineum pellet
20 = M. gypseum pellet

85

 

sea L

0

©\@/ K

@\\©/®

('k \ J

0.“...0

® ,

@
omoo

WV® T.

86

Plate VI

Diagrams of immunodiffusion test precipitation lines pro-

duced by supernatant antigen fractions from selected

dermatOphytes and T. terrestre (414) supernatant and

 

pellet antisera.

\OCDNO‘xUTJEUUNF-J Q10

HP4F’
N H O
Ianameldaeleleelane

RJH+4F4H+4FJH
ouaahqouncn»

lia‘ta'fa'ia'fa'fa 4943

terrestre (414) supernatant fraction antiserum
terrestre (414) pellet antiserum

 

 

mentagrophytes (dog) supernatant antigen
mentagrophytes (455) supernatant antigen
mentagrophytes (536) supernatant antigen
rubrum (Daf) supernatant antigen

rubrum (JH) supernatant antigen
terrestre (414) supernatant antigen
terrestre (285) supernatant antigen
terrestre (Brasil) supernatant antigen
equinum supernatant antigen

gallinae supernatant antigen

me ninii supernatant antigen
schenleinii supernatant antigen
soudanense supernatant antigen

tonsurans supernatant antigen

verrucosum supernatant antigen

Violaceum supernatant antigen

floccosum supernatant antigen

ajelloi supernatant antigen

ferrugineum supernatant antigen

gypseum supernatant antigen

 

 

 

 

 

 

 

 

 

 

 

 

88

Plate VII

Diagrams of immunodiffusion test precipitation lines pro—

duced by pellet antigen fractions from selected dermatOphytes

and T. terrestre (414) supernatant and pellet antisera.

 

CLO

MHHHHI—‘t—‘i—‘I—JHH
oxooo-xlochwmwoxoooxloxmcwmr-J

lahd

Islaelml BIHI 6| en :3! {aural .3. e1 :31 rah-3| relegate

terrestre (414) supernatant fraction antiserum
terrestre (414) pellet antiserum

 

mentagrophytes (dog) pellet
mentagrophytes (455) pellet
mentagrophytes (536) pellet
rubrum7(Dar)'pe11et

rubrum (JH) pellet
terrestre (414) pellet
terrestre (285) pellet
terrestre (Brasil) pellet

eguinum pellet
gallinae pellet

megninii pellet
schoenleinii pellet

soudanense pellet
tonsurans pellet
verrucosum pellet
Violaceum pellet
floccosum pellet

ajelloi pellet

ferrugineum pellet

gyseum pellet

 

 

 

 

 

 

 

 

 

 

 

 

 

89

@
peeve
o a .

@

@

D

C

B

A

 

90
Plate VIII

Diagrams of immunodiffusion test precipitation lines pro—
duced by supernatant antigen fractions from selected

dermatOphytes and T. rubrum (Dar) supernatant and pellet

 

 

 

 

 

 

 

 

 

 

 

 

 

antisera.
e = T. rubrum (Dar) supernatant fraction antiserum
f = T, rubrum (Dar) pellet antiserum.
l = T, mentagrophytes (dog) supernatant antigen
2 = T. mentagrgphytes (455) supernatant antigen
3 = T. mentagrophytes (536) supernatant antigen
4 = T. rubrum (Dar) supernatant antigen
5 = T. rubrum (JH) supernatant antigen
6 = T. terrestre (414) supernatant antigen
7 = T. terrestre (285) supernatant antigen
8 = T. terrestre (Brasil) supernatant antigen
9 = T. eguinum supernatant antigen
10 = T. galligge supernatant antigen
11 = T, megninii supernatant antigen
12 = T. schoenleinii supernatant antigen
13 = T. soudanense supernatant antigen
14,= T. tonsurans supernatant antigen
15 = T. verrucosum supernatant antigen
16 a T. Violaceum supernatant antigen
17 i M. floccosum supernatant antigen
18 = M. ajelloi supernatant antigen
19 = M. ferrugineum supernatant antigen
20 = M. gypseum supernatant antigen

 

0

@
me»). Ale 0 o ; ®
NA @V® a? ray/ 0

l

9

866
.>. B
o®e

m

 

92
Plate IX

Diagrams of immunodiffusion test precipitation lines pro-
duced by pellet antigen fractions from selected dermatophytes

and T. rubrum (Dar) supernatant and pellet antisera.

 

 

 

 

 

 

 

 

 

 

 

 

 

e = T. rubrum (Dar) supernatant fraction antiserum.
f = T. rubrum (Dar) pellet antiserum
1 = T. mentagrophytes (dog) pellet
2 = T. mentagrophytes (455) pellet
3 = T. mentagrophytes (536) pellet
4 = T. rubrum (Dar) pellet
5 = T. rubrum (JH) pellet
6 = T. terrestre (414) pellet
7 = T. terrestre (285) pellet
8 = T. terrestre (Brasil) pellet
9 = T. eguinum pellet
10 = T. gallinae pellet
11 = T. megninii pellet
12 = T. schoenleinii pellet
13 = T. soudanense pellet
14 = T. tonsurans pellet
15 = T. verrucosum_pe11et
16 = T. Violaceum pellet
17 = M. floccosum pellet
18 = M. ajelloi pellet
19 = M. ferrugineum pellet
20 = M. gypseum pellet

93

 

® ®
0 @ G
0 ® ® ® _ ®
owe ww/e . Km/e K . sewm/e

®

® ® ©\ 7]: ® 6

W9 .. ape/UM F swim/Mo . exam/We
o e

0 0 ® ® «70 0
o©® . may E a@o _.. WQo

94

Reactions of the homologous antigen-antibody system of

T. mentagrophytes (dog) supernatant developed 8 lines

 

(Plate III,A and Plate IV, A through H) in all agar gel
slides except for one that had 7 lines (Plate IV, G). This

was evidence that the T. mentagrophytes (dog) fraction

 

possessed a minimum of 8 precipitonogens.

The pellet fraction of T. mentagrophytes (dog) reacted

 

with the homologous antiserum to produce 4 lines of precipi-
tation (Plate III, B and Plate V, I through P).

Trichgphyton terrestre (414) supernatant fraction

 

formed 9 lines of precipitation with the homologous anti—
:serum (Plate III, C and Plate VI, A through H). Five lines
(Pflate 111, D and Plate VII, 1 through P) were produced with
true homologous pellet fraction antigen-antiserum system of

2:. terrestre (414) which was originally isolated from the

 

hair of a pig in England.

The least reactive homologous antigen—antibody super—
Ifiertant system, T. rubrum (Dar) fraction, produced 7 lines
(Plate III, E and Plate VIII, A through H). The pellet
fr’action of T. rubrum developed 4 precipitation lines when
reacted with T. rubrum (Dar) pellet antiserum (Plate 111, F
ar1c1 Plate IX, I through P).

Most of the lines of precipitation occurring between
re%ic3tants in homologous systems of the supernatant fractions
WEEIVE of greater intensity than the lines in homologous

S57Stems of the pellet fractions. Replenishing reactant wells

95

enhanced the intensity but did not increase the number of
bands observed.

The low intensity precipitation lines were not dis-
tinguishable in photographs and some of the moderate
intensity lines were lost. Thus diagrams were made in
place of photographs to show the precipitation lines as

they appeared on the original agar gel slides (Plates III

through IX).

B. Precipitinogenic Relationship
9: the Different Antigenic Fractions
Within the Same Isolate

The number of precipitation lines formed in the
heterologous fraction-antiserum systems within an isolate
are given in Tables 12 and 13.

Trichophyton mentagrophytes (dog) supernatant when
ciiffused against T. mentagrophytes (dog) pellet antiserum
13roduced 3 lines which were identical with 3 of the 8 lines
jproduced by the homologous antigen-antibody system (Plate
3111, A). The pellet fraction of T, mentagrophytes (dog)
Iwaacted with the supernatant antiserum of the same isolate
tC) develop 3 lines which were identical with 3 of the 4
Itines formed in the homologous pellet antigen-antibody
sbfstem for this organism (Plate III, B).

Four precipitation bands (Plate III, C) were observed

WTlen T, terrestre (414) pellet antiserum was reacted with

 

tfle supernatant fraction in the agar gel slide. These lines

Wfire common with 4 lines in the supernatant homologous

96

antigen-antibody system. The supernatant antiserum developed
4 lines when reacted with the pellet fraction of T. terrestre
(414). These were lines of common identity with 4 of the 5
lines that were observed when the pellet homologous system
was tested (Plate 111, D).

The heterologous antigen-antibody systems of T. rubrum
(Dar) produced 3 lines when the pellet antiserum was reacted
with the supernatant fraction, and 3 lines when the super—
natant antiserum diffused against the pellet antigenic fraction

of this strain. The latter 3 lines appeared to be common

 

with 3 of the 4 lines in the homologous pellet system
(Plate 111, E and F).

To cOnclude briefly, the number of lines produced in
the homologous antigen-antibody systems indicated the super—
natant fractions contained a greater number of antigenic
substances than the pellet fractions. Except for one line,
the number of lines produced by the pellet homologous systems
were identical to lines in the supernatant, hence at least
one antigen in the different pellet fractions was not found
in the supernatant fractions.

C. Precipitinogenic Relationship
of the Three Reference Strains

The number of precipitation lines formed in the
homologous and heterologous reactions of the reference

strains are summarized in Table 12.

97

'TABLE 12.-gThe number of precipitation lines formed by
Inomologous and heterologous antigen—antibody reactions of
reference strains.

 

 

 

 

 

 

Antisera
m 2?
(1) H
4—3 A :7
m p 1,
.C +3 (U 4—3 4—3
Q. s O c: o c
Antigenic 8 .3 ‘J 3 .5 3
FM 29,. a s a a s :3 a s
+360 p a) a x. m L :4 o
cc: m H p 0; r4 a o H
gm: 0. e1 :3 Q H q; a H
M :3 (1) $4 :3 <1) 43 .‘3 0)
m 9. a: Q m CL
BI BI 50
31. mentagrophytes (dog)
supernatant 8 3 5 2 4 2
pellet 3 4 2 2 3 3
21. rubrum (Dar)
supernatant 5 2 7 3 3 2
pellet 2 2 3 4 2 2
El. terrestre (414)
supernatant 4 2 3 2 9 4
pellet 2 2 2 2 4 5

When T. mentagrophytes (dog) supernatant antiserum was

Iweacted with the supernatant fraction of T, terrestre (414),

 

4 lines were developed which were identical with 4 precipita—

tionlines formed with the T, mentagrophytes supernatant

 

alltigen-antibody system (Plate IV, B). In the other hetero-
1-<>gous systems between antisera produced by the fractions of
tilis organism after injection into rabbits and the fractions
‘Df T, terrestre (414) only 2 bands formed and these were

lires of identity (Plate IV, J; v, B; and v, J).

98

TABLE l3---The number of precipitation lines formed by homologous and heterologous
antibody-antibody reactions.

Antisera

 

Antigenic

Fractions

mentagrpphytes
1

 

supernatant
pellet
rubrum
‘supernatant.

pellet
terrestre
supernatant
pellet

8

'1‘
(do
T.
I-

 

“T. mentagrophytes (dog)
supernatant
pellet

 

km
NW

 

“pernat nt

(0CD wm

NM
NC.- wt:
NM WM

:1»
MO
mm

‘7\

“T. mentagrophytes (53
supernatant
pellet
”T. rubrum (Dar.)
supernatant
pellet
*T. rubrum (J. H.)
- supernatant
pellet
*“T. terrestre (s14)
- supernatant
pellet 2
terrestre (285)
supernatant
pellet
”*T. terrestre (Brasil)
— supernatant 4
pellet 2
*T. equinum
supernatant
pellet
”T. gallinae
supernatant
pellet
fl"1‘. megninii +
'- supernatant 5(+l)
pellet 3
“T. verrucosum
supernatant . 5 1
pellet
"'T.‘schoen1einii
supernatant

 

NU} m—q

R)l'\) DOW

mm

CL.) MN

NLA) WIL-

N|\) MN LAJLA)

O

I: w

|'\ N
NLA) WON bofl

Us)

NW

Mn
1:\
U‘It:

I!

I»:
J
f\) [U M r\)
{U
H H m
L“ (1) cm
U1!» U7 1:

(\JKAJ
y

H mm m
H out» mrx mm
H Ul pow mm mm
HH but» R)“J mm
D—‘w rum our: Hui
HH mm Low HH

mm
mm
f\.)|'\)

3.:

2
2 2(+;)*
“'"T. soudanense
supernatant
pellet
“‘"T. Violaceum:
supernatant
pellet
“‘"T. tonsurans
supernatant
pellet
. floccosum
supernatant
pellet
. ajelloi
supernatant
pellet
21. ferru ineum
supernatant
pellet
. gypseum
supernatant
pellet

’ Ectothrix species of Tricho h ton.
'* Saprophytic species of Tricho h ton.
*" Endothrix species of TrIcRophyton.

+ One line more in one of the three plates used in the tests.

NUT

l\)
mm M1:
l\.)

L‘

['8‘ I47
mm
HH HH out» mm
Hm tow mm
mm HH out» mm
woo woo Hm mu) Ho.)
mm but» HH mm mm

[3'
H: mm H: Hw WN

MN

MI: MI: mm
mm

NJ:

NM

HH
mm

I

99

Five precipitation lines developed in the reaction of

T. mentagrophytes (dog) supernatant with T. rubrum (Dar)

 

supernatant. These lines were common with 5 of the 8 lines
in the homologous T. mentagrophytes (dog) supernatant test
(Plate IV, A) while the pellet fraction of T. rubrum (Dar)
produced 2 lines when reacted with this antiserum (Plate IV,
I). The pellet antiserum of T. mentagrophytes (dog) diffused
against the supernatant and pellet fractions of T. rubrum
produced 2 bands identical with 2 bands in the T. mentagro-
phytes (dog) homologous system (Plate IV, I and Plate V, A).
The supernatant fraction of T, mentagrophytes pro-
duced 4 lines, 2 common and 2 partially identical lines in
the homologous system of T. terrestre (414) when double dif-
fusion tests were performed with T. terrestre (414) super-
natant antiserum (Plate VI, B), and only 3 lines (2 common
lines and 1 partially common) were developed when the latter
supernatant antiserum was diffused against the T. mentagro—
phytes (dog) pellet (Plate VII, B). Trichoppyton terrestre
(414) antiserum produced by the pellet reacted with the T.
{Baptagrophytes (dog) supernatant by producing 1 common and l
Kmartially common line with those in the homologous T.
PEirrestre (414) pellet system (Plate VI, J), but 3 lines
(2’ common and 1 partial identical line) were formed by the
P6311et fraction of T. mentagrophytes (dog) and this anti—

SEErum (Plate VII, J).

lOO

Trichophyton terrestre (414) supernatant antiserum dif—
fused against T. rubrum (Dar) supernatant developed 1 common
and 2 partially identical lines with the homologous T. pep:
restre (414) supernatant system (Plate VI, A). Two precipi-
tation lines were produced by the reaction of T. rubrum (Dar)
pellet with the latter antiserum (Plate VII, A), T, rubrum
(Dar) pellet diffused against T. terrestre (414) pellet anti—
serum (Plate VII, I and P) and T. terrestre (414) pellet anti-
serum with T. rubrum (Dar) supernatant (Plate VI, I). One
line in each of the last 3 reactions was of partial identity
and the other 1 was common to lines in the homologous system
to which each was compared.

The supernatant T. rubrum (Dar) antiserum produced 5
precipitation bands with the antigens in the supernatant
fraction of T. mentagrophytes (dog). Four of these lines were
continuous with 4 lines in the T. rubrum (Dar) homologous
antigen-antibody system indicating that there is a minimum of
4 identical antigens in the supernatant fractions of these 2
organisms. The fifth line shows partial identity with a
.fifth line in the homologous system of T. rubrum (Dar) (Plate
IIIII, A). At least 2 antigens were common in T, rubrum
Emellet fraction with T. mentagrophytes (dog) supernatant and
Ipeellet fractions as indicated by continuous precipitation
lines (Plate VIII, 1; IX, A; and IX, I).

When T. rubrum (Dar) supernatant antiserum was dif-

fhdsed against T. terrestre (414) supernatant fraction, 3

 

EDrecipitation lines appeared in the agar medium which were

lOl

continuous with lines in the homologous system of T. rubrum
(Dar) (Plate VIII, B). Two identical antigens were
evident in reactions of T. rubrum (Dar) supernatant anti-

serum with T. terrestre pellet (Plate IX, B), T. rubrum

 

(Dar) pellet antiserum with T. terrestre supernatant
Plate VIII, J), and the latter antiserum with T. terrestre
(414) pellet (Plate IX, J).

To conclude briefly, the results after crossing the
fractions of the reference strains with the reference anti-
sera indicate a number of homologous antigens in these
strains. Trichophyton rubrum and T, mentagrophytes show
a closer relationship than T. terrestre.

D. Precipitinogenic Relationship

of the Different Isolates Within
a Species

In an effort to establish that the number of lines
produced by any isolates of a species against a specific
antiserum are characteristic of that species, 3 isolates
(strains) of Trichophyton mentagrophytes, 3 isolates of T.

Eerrestre, and 2 isolates of T, rubrum were used for the

 

inmmnodiffusion studies.

Trichophyton mentagrpphytes strains were selected
fTrom 3 widely different environments. The dog strain was
fYrom tinea on a dog in the Michigan State University
Veterinary Clinic while the "455" strain was obtained from
51 tinea pedis case in Toronto, Canada, and the third

Eitrain "536" was from tinea on a hedgehog in New Zealand.

102

The number of lines produced by the homologous antigen-
antibody systems of T. mentagrophytes (dog) was 8 for the
supernatant and 4 for the pellet (Plate IV, A; Plate V, I;
and Table 13). When the 2 antisera produced by this
isolate after injection into rabbits were diffused against
the fractions of the other 2 isolates, the only difference
was found in the T. mentagrpphytes (536) strain (Plate IV,

B, D, and J, L; Plate V, J and L). The supernatant fraction
of this strain produced 1 less line with antiserum of T.

mentagrophytes (dog) supernatant (Plate IV, B and Table 13)

 

as did the pellet fraction when diffused against the pellet
antiserum of T. mentagrophytes (dog) (Plate V, J and Table
14). Trichophyton mentagrophytes (455) fractions produced
the same number of lines as the homologous systems of T.

mentagrophytes (dog) with 1 line being of partial identity

 

in the supernatants (Plate IV, D).

The strains of T. terrestre were also from widely

 

different locations. Trichophyton terrestre strain "414"
Iuas isolated from the skin of a pig in England, while
strain "285" was isolated from the soil in California and
strain "Brasil" was isolated from the Brasilian soil in 1961
"by the author (1963).

Homologous antigen-antibody systems developed 9 pre-
cipitation lines with the supernatant fraction and 5 lines

Vvith the pellet fractions from T. terrestre (414). In the

 

Zimmunodiffusion test with T. terrestre (414) fraction

éantisera with fractions from strains "285" and "Brasil,"

103

a difference of 1 line occurred (total of 8 lines) with the
supernatant fraction systems in all 3 times repeated

(Table 13, Plate VI, B and D), and in all cases, the pellet
systems of these isolates produced 5 lines (Table 13, Plate
VII, J and L). Precipitation lines numbered 4 (Plate VI,

J; Plate VII, B and D) in all of the agar gel tests in which
pellet diffused against supernatant antigen-antibody Systems
for the strains of T. terrestre, except when the "Brasil"
strain supernatant fraction was reacted with T. terrestre
(414) pellet antiserum, only 3 lines were produced (Plate
VI, L).

The 2 isolates of T. rubrum were form human cases of
ringworm of the body, tinea corporis, isolated in Michigan.
Diffusion of supernatant fraction T. rubrum (Dar) antiserum
against the supernatant fraction of T. rubrum (J.H.) strain
developed 6 lines, one line less than was produced by the
homologous T. rubrum (Dar) supernatant antigen-antibody
system (Plate VIII, D and Table 13). The heterologous
systems of supernatant and pellet antigens produced 3 lines
of precipitation (Plate VIII, L; IX, D; and Table 13) while
‘the pellet antigens of T. rubrum (J.H.) and the pellet
antiserum of T. rubrum (Dar) produced 4 lines (Plate IX, L;
and Table 13).

The precipitation lines observed in the reactions of
lleterologous systems above were all common with lines in
tflle respective reference strains antigen-antibody systems

e'Xcept when T. mentagrophytes (455) supernatant was diffused

104

against T, mentagrOphytes (dog) supernatant antiserum, 1

line of partial identity was produced.

To summarize, the fractions from the isolates of T.

terrestre were identical in reactions with T. terrestre

 

(414) antisera except for 1 less line in the reactions of
the supernatant fractions. The same was true for fractions
from the isolates of T. rubrum. There was one more line
between the reference strain supernatant reactants. However,
in T. mentagrpphytes reactions, strain 455 supernatant
fraction reacted like the homologous dog strain with 1 line
being of partial identity and T. mentagrpphytes (536)
differed by lacking 1 line in the supernatant reaction and
1 line in the pellet fraction.

E. Prechitinogenic Relationsth

5? Selected Dermatophytes and the
Reference Strains

Strains were selected from the various dermatophytes
to compare reactions between different isolates within a
Species, species in different genera, and species in the
(different groups (based on "in vivo" hair invasion) of the
ngdchgphyton genus when reacted with the reference strains
armisera.

The reactions of fractions from strains belonging to
tune species used in the production of the reference strains
axltisera with heterologous Species antisera produced some
\Nariation in the number of precipitation lines between

Stzrains of a species (Table 13).

105

Trichophyton mentagrophytes (455) supernatant produced
6 lines, of which 1 line showed partial identity (Plate VIII, D)
while T, mentagpophytes (536) developed 5 lines (Plate VIII, B)
when reacted against T. rubrum (Dar) supernatant antiserum.
The supernatant fractions of these 2 strains when crossed with
the pellet antiserum of T. rubrum (Dar) showed 1 line differ-
ence in the reactions. Two lines appeared with the 536 strain
(Plate VIII, J) and 3 lines with the 455 strain (Plate VIII, L).
The pellet fractions of these 2 strains reacted with the
supernatant and pellet antiserum of T. rubrum (Dar) to produce
2 lines (Plate IX, B, J and L) except in the reaction of T.

mentagrophytes (455)Vnth T. rubrum (Dar) supernatant antiserum

 

(Plate IX, D) in which 3 lines were produced. When T. mentagro—
phytes (455 and 536) were reacted with T. terrestre (414) anti-
sera, only the supernatant reactants produced the same number
of'lines which was 4 (Plate VI, B and D) but the lines differed
iri that strain 455 had 1 line that was of partial identity and
stzrain 536 had 2 partially identical lines. In all other
Iweactions (supernatant against pellet antiserum, pellet against
SLlpernatant antiserum, and pellet against pellet antiserum) T.
mggltagrgphytes (536) produced 3 lines with 1 of the 3 being of
patrtial identity (Plates VI, J; VII, B and J) while T. TEEEE’
ETTDphytes (455) produced 2 lines (Plates VI, L; VII, D and L).
The reactions of the fractions of T. rubrum (J.H.) with

the? antisera of T. terrestre (414) and T. mentagrophytes (dog)

 

Welfie as follows: Supernatant fraction with T. mentagrophytes

(dcug) supernatant antiserum, 5 lines developed (Plate IV, D)

106

and with T. terrestre (414) supernatant antiserum 3 lines

 

developed (l of partial identity) (Plate v1, D). With
both reference strains, the reactions of supernatant anti-
sera with T. rubrum (J.H.) pellet fraction (Plates V, D and
VII, D), pellet antisera with supernatant fraction (Plates
IV, L and VI, L), and pellet antisera with pellet fraction
(Plates V, L and VII, L) deve1Oped 2 lines each.

Similar reactions were observed between the 2 strains

of T. terrestre that were not used as reference strains when

 

reacted with T. mentagrOphytes (dog) antisera. Four lines
appeared between the supernatant reactants (Plate IV, B and
D). The reactions which remain all produced 2 lines of
precipitation between reactants of each strain (Plate IV,
J axmd L; VII, B and D, J and L). However, the reactions
of T; terrestre (Brasil) differed by 1 line in the tests
pmnfiformed with T. rubrum (Dar).antisera. The lines that
weree produced are: 3 lines (1 line between T, terrestre
(2855) and antiserum is of partial identity) with the super-
natéint reactants (Plate VII, B and D), 2 lines with super—
nateuit antisera against pellet fraction (Plate IX, B and D)
l liJle with pellet reactants (Plate IX, J and L) and 1 line
withl supernatant T. terrestre (Brasil) diffused against
T. ruibrum pellet antiserum (Plate VIII, L), while T, terrestre
(2855) developed 2 lines (Plate VIII, J).

In the above reactions all the lines are common with
the Iwaference strain lines on either side of the non—

referwence strains in the agar slides unless otherwise stated.

107

For convenience the Trichophytons are placed into
groups based on colony morphology or on the method of TM
ngg hair invasion. At present, the most frequently used
system of classification is based on TM Klzp_hair invasion
(Emmons ep_gT., 1963) and has been used as a basis for the
organization of the results. The divisions and examples
are:

l. Ectothrix species of Trichophyton invade the hair
folicle, penetrate the hair, grow to the keratogenous zone,
then the mycelium breaks out onto the surface of the hair
and fragments into arthrospores. Examples are: T, meppef

ggophytes, T. equinum, T. rubrum, T. verrucosum, T. gallinae,

and T, megninii.
2. Endothrix species of Trichophyton invade the hair

irl the same manner described for the ectothrix, however, the
Hmrcelium remains inside and breaks into chains of large
eurthrospores. Examples are: T. tonsurans, T; schoenleinii,

T. 'violaceum, and T. soudanense.

 

3. Ectothrix—endothrix type of invasion is a combina-
tixon of both. An example is: T. gTMTT.

4. Hair not invaded. An example is: T. concentricum.

5. SaprOphytic in soil. Examples are: T. terrestre,
E- georgiae, and T. gloriae.

Two of the reference strains used belong to the ecto-

thrfiix species were reacted with antisera produced by using

thEE reference strains.

 

108

The number of precipitation lines formed by the
fractions from the ectothrix species in reactions with
reference antisera is shown in Table 13. The ectothrix
Species included T. mentagrophytes, T. rubrum, T. equinum,

T. gallinae, and T. megninii. All of the species possessed
1 or more antigens related to those of the reference
strains.

The reactions of the supernatant fractions of the
ectothrix species varied from 8 to 7 in the reference ecto-
thrix strains T. mentagrophytes (dog) and T. rubrum (Dar)
homologous antigen-antibody systems to 4 lines in the
heterologous system of T. equinum and T, mentagrophytes (dog)
antiserum (Plate IV, A). When this antigenic fraction of
T. eguinum was diffused against T, rubrum (Dar) supernatant
anisiserum, 5 instead of 4 lines were produced (Plate VIII, A).
Su;>ernatant fractions of T. gallinae and T, megninii (Plate
IV, E» produced 6 lines when diffused against T, mentagro-
2h§gtes (dog) antiserum and 5 lines when diffused against T,
rutirum (Dar) antiserum (VIII, E) while T. verrucosum pro-
Chuzed.5 bands with each of these reference antisera (IV, F
anCI H; VIII, F and H). The 2 reference ectothrix strains
PPCKiuced 4 lines in homologous pellet systems while the
pelJlet fractions of the other ectothrix species produced the
fOllowing when reacted with these 2 reference antisera: T.
megrrinii produced 3 lines (Plate V, M; IX, M), T. eguinum
(Plate v, 1; IX, I), and T. gallinae (Plate v, M; and IX, M)

PPOCiUced 2 lines each and T. verrucosum (Plate IV, N, P; IX,

109

N and P) produced only 1 line with each of the ectothrix
reference strains. When the supernatant fractions of these
ectothrix Species were diffused against the 2 reference
ectothrix species pellet antisera and when the pellet frac-
tions were diffused with the supernatant antisera, the
following number of lines resulted: T. megninii, 3 lines
(Plate IV, M, V, E; IX, E), T. gallinae, 2 lines (Plate IV,
M, V, and E; VIII, M; IX, E) T. eguinum, 2 lines (Plate

IV, I: V, A; VIII, 1; IV, A), and T. verrucosum, 1 line

 

(IV, N and P; V, E and H; VIII, N and P; IX, E and H).
Reactions of the ectothrix filtrates with the reference

saprOphytic strain, T. terrestre (414), antisera are as

 

followsz' T. gallinae developed 4 lines (VI, E), T. eguinum

(VI, A), T. megninii (VI, E), and T. verrucosum (VI, F and H),

 

produced 3 lines when the reactants were supernatant in
origin; T. gallinae (Plate VI, M; VII, E and M), developed 3
lines, T. megninii (VI, M; and VII, E and M), 2 lines, while

T. eguinum (Plate VI, 1; VII, A and I), T. verrucosum (Plate

 

VI, N and P; VII, F, H, N and P), produced 1 line each when
the supernatant and pellet fractions diffused against T.

terrestre (414) pellet antiserum, and supernatant antiserum

 

with pellet fractions.
The endothrix species used in this investigation were

T. schoenleinii, T. soudanense, T. tonsurans, and T. Violaceum.

 

 

 

 

Each fraction of these species produced at least 1 line when
reacted with the reference antisera (Table 13). Reactions

of the fractions from the different endothrix Trichophyton

 

110

species with the reference ectothrix species antisera
produced the following precipitation lines: 5 lines

developed for T. schoenleinii (Plate IV, E and F), T.

 

soudanense (Plate IV, G), and T. Violaceum (Plate IV, F),

 

 

while T. tonsurans (IV, A and H) had 7 lines when the

 

supernatant fractions were diffused against T, mentagro-
phytes (dog) supernatant antiserum; supernatant fractions

against T. rubrum (Dar) supernatant antiserum T. soudanense

 

(Plate VIII, G) produced 6 lines, T. Violaceum (Plate VIII,

 

F) developed 5 lines, T. schoenleinii (Plate VIII, E and F)

 

exhibited 4 lines and T. tonsurans (Plate VIII, A and H)

 

showed 3 lines. Pellet fractions with supernatant antisera,
supernatant fractions against pellet antisera, and pellet
fractions with pellet antisera showed the following: T.

tonsurans (Plate IV, I: V, A and 1: VIII, 1; IX, A and I)

 

produced 3 lines while T. schoenleinii (Plate IV, M; V, E, M

 

and H; VIII, E and M), T. soudanense (Plate IV, 0; V, G and

 

0; VIII, 0; IX, G and O), and T. Violaceum (IV, N; V, F and

 

N; and VIII, 0, G and O) deve10ped 2 lines each.
As the-fractions of the endothrix species were diffused

against the antisera of T. terrestre (414), 2 species, T.

 

schoenleinii (Plate VI, E and F), and T. soudanense (Plate

 

 

VI, G) developed 4 lines, and 2 species, T. tonsurans (Plate

 

VI, A and H) and T. Violaceum (Plate VI, F) produced 3 lines

 

when the reactants were supernatant. Two precipitation lines
were observed in each reaction of pellet reactants, pellet

fractions with supernatant antisera, and pellet antisera with

lll

supernatant fractions for T. schoenleinii (Plate VI, M;

 

VII, E, M and N), T. soudanense (VI, 0; VII, G and O), and

 

T. tonsurans (VI, I; VII, A, H and I), while T. Violaceum

 

 

(VI, N; VII, N) produced only 1 line when the pellet
fraction was diffused against the supernatant antiserum of

T. terrestre (414) (Plate VII, F).

 

It should be pointed out that the number of lines pro—
duced by the pellet fractions of the reference strains and
strains which belong to the same species produced 1 more

line than any of the above Trichqphyton species with the

 

exception of T. mentagrophytes (536) strain.

 

The immunodiffusion results indicate that there may be

a closer relationship among species of the Trichpphyton as

 

grouped by colony morphology rather than TM Vivo hair invasion.

Epidermophyton floccosum.--The number of precipitation

 

lines formed by the fractions of M, floccosum and the

 

reference strains are shown in Table 13. The supernatant

fraction of M. floccosum produced 3 identical precipitation

 

lines with T. mentagrophytes (dog) (Plate IV, 0), and T.

 

terrestre (414) (Plate VI, C) supernatant antisera but only 1

 

identical line and l of partial identity with T. rubrum

(Dar) supernatant antiserum (Plate VIII, C). Of the 3
remaining reactions with fractions of this organism with each
reference strain, each produced a precipitation line of
identity (Plate IV, K; V, C and K; Plate VI, K; Plate VII, C
and K), except with T. rubrum (Dar) antisera which developed
a line of partial identity in each reaction Plate VIII, K;

IX, 0 and K).

112

Keratinomyces gjelloi.-—In the immunodiffusion tests

 

of M. ajelloi supernatant fraction against the reference
antisera the following precipitation bands developed: 4

with T. mentagrophytes (dog) supernatant antiserum

 

(Plate IV, C), 3 with T. rubrum (Dar) supernatant antiserum

3 with T. terrestre (414) supernatant antiserum (Plate VI,

 

C), 3 with T. terrestre (414) pellet antiserum (Plate VI, K),

 

2 with T. rubrum (Dar) pellet antiserum (Plate VIII, K) and

l with T. mentagroppytes (dog) pellet antiserum (Plate IV,

 

K). The pellet fraction reactions resulted in the following:

1 line with both antisera of T, mentagrophytes (dog) (Plate

 

V, C and K), 2 lines with both antisera of T. rubrum (Dar)
(Plate IX, C and K). All lines were continuous with lines
resulting from reactions between reactants of reference

strains except the partial identity lines in T. terrestre

 

(414) immunodiffusion tests, the second line from the outside
was always of partial identity. These results are summarized
in Table 13.

The 2 species M. floccosum and M. ajelloi, from 2

 

different genera indicate no close relationship in these
immunodiffusion tests to any other species except in 2

instances. Trichophyton mentagrophytes (dog) supernatant

 

antiserum produced the same identical lines when diffused

against the supernatants of M. ferrugineum and M. floccosum

 

 

supernatants and M. ajelloi supernatant fraction produced
the same number of lines but not all identical with M,
gypseum. These reactions were not evident with the other anti-

sera.

113

Microsporum ferrugineum.—-Reactions of the 2 fractions

 

of M. ferrugineum with the reference antisera resulted in a

 

difference of 1 line in the 12 double diffusion reactions

(Table 13). When T. rubrum (Dar) antiserum was diffused

 

against M. ferrugineum supernatant, 4 lines developed
(Plate VIII, G), while only 3 lines develOped with T.

mentagrophytes, and T. terrestre (414) supernatant antisera

 

 

(Plate IV, G; VI, G). The lines were continuous with lines
in the homologous antigen antibody systems and indicated
common antigens. Two lines of identity appeared between

each of the other reactions of the fractions of M, ferrpgineum

 

and the reference antisera (Plates IV, O; V, G and 0; VI, 0;
VII, G and 0; VIII, 0; IX, G and 0).

Microsporum gypseum.-—The supernatant fractions of M.

 

gypseum reacted similarly with all supernatant antisera by
producing 4 precipitating lines between reactants (Table 13).
The outer line in each reaction was a line of partial
identity while the other 3 were lines of identity (Plate IV,
C and G; VI C and G; VIII, C and G). Lines of precipitation
numbered 2 when the M. gypseum fractions were diffused against
the antisera of T. rubrum (Dar) and T. terrestre (414)
(Plates VIII, K and 0; IX, C, G, K and 0; VI, K and O; and
VII, C, G, K and O) in the 6 possible combinations left.
Reactions of these fractions with T, mentagrophytes (dog)
antisera produced 1 line each (Plate IV, K, O; V, C, G, K

and O). The outer precipitation line was a partial identity

and the others were lines of identity between the following

114

reactions: M. gypseum supernatant and T. terrestre (414)

 

pellet antisera (Plate VI, K and O) and M. gypseum pellet

and T. terrestre (414) supernatant antisera (Plate VII, C

 

and G) and T. terrestre (414) pellet antiserum (VII, K and

 

0).

Thus not only did the fractions from the 2 Microsporum

 

Species differ in the number of precipitation lines pro-
duced when diffused against the 6 reference antisera but
the lines of identity were not always the same when com-
pared to the reference strain reactions (Plate IV, G and 0;
VI, G and 0; VIII, G). The number and arrangement of lines
indicated a closer relationship of these 2 species to each
other than to any of the genera or species studied.
Keratinomyces ajelloi appears to be a close relative to M.

gypseum and M. ferrugineum.

 

DISCUSSION

Many variable factors must be considered in the
system employed in immunodiffusion techniques. The utili-
zation of fractions separated by centrifugation in dif-
fusion reactions and for eliciting antibodies in rabbits
is not ideal. Not only Should one expect the various
antigens present in the fraction to exist in varying con-
centrations, but also, the fraction preparation itself to
contain a wide variety of fragments from subcellular parts,
and metabolic products of the fungal cells. These sub-
stances may have varying affinities for combining with the
determinant groups of different antigens, thus altering
antigenic specificity or completely blocking the antigen.

In this investigation no attempt beyond centrifuga-
tion was made to purify the fractions used to produce anti-
bodies in rabbits.

The method employed to elicit antibodies is another
factor that may alter immunodiffusion results. A form of
hyperimmunization was adopted due to the low antigenicity
of the fungi used. Crowle (1961) reported that "hyperim-
munization tends to induce production of a range of anti—
bodies which make the antiserum likely to cross-react

even with distantly related antigens."

llS

116

There was no direct evidence to show that the form of
hyperimmunization employed caused non-specificity of the
antibodies. However, each of the 40 fractions of the 20
dermatophytes reacted with each reference serum in various
degrees as illustrated by the variation in the number of
precipitation lines formed between reactants. The reactions
of homologous antigen-antibody systems of reference strains
gave evidence of specificity in both fractions. The results
suggest that the antibodies possessed at least moderate
Specificities.

The variation of individual animal in response to
injections of antigens is another factor influencing the
quality of antisera, and, consequently affecting the immuno-
diffusion results. In this study, there was some variation
in the number of antigens that were detectable when a
fraction was diffused against antisera obtained from dif—
ferent rabbits that had been injected with identical fil—
trates. The antiserum for each rabbit was tested individ—
ually and only those which developed the largest number
of precipitation bands in homologous reactions were used
for investigating the precipitinogenic relationships.

During incubation periods precipitation lines are
formed at varying rates and have different relative inten-
sities. A dense line developing at a rapid rate may some-
times mask the low intensity, slower developing line. This
limitation was minimized with observations made at 8 hour

intervals during incubation of the agar diffusion slides.

117

It is well to realize that the aforementioned short—
comings are inherent in immunodiffusion studies when
interpreting results.

As shown earlier, the results of immunodiffusion
studies of 40 fractions from 20 dermatophytes with the 6
reference antisera suggest that antigenic differences
exist among various strains of dermatophytes. These
variances not only exist among species but also among
strains within a species. However, a definite pattern of
antigenic relationships was noted within species.

The observation of identical precipitinogens in M.

floccosum, M. ajelloi, Microsporum Sp., and Trichophyton

 

 

sp., substantiates the results reported by many authors of
cross reactions among the dermatOphytes in serological
tests (Jadassohn gp_gT., 1937; Huppert, 1955; Tomomatsu,
1961; Reyes and Friedman, 1966).

Jadassohn gp_gT. (1937) established by use of the

Schultz-Dale method that Achorion Quinckeanum, Trichophyton

 

 

gypseum (both now synonyms of T, mentagrophytes), Epider-

 

mpphyton Kaufman (= M. floccosum), and Achorion Schoen-

 

 

leinii (= Trichophyton schoenleinii) had 1 antigen in
common, the first three had 1 in common, and the first two
had 1 in common, while each had 1 specific antigen. The
precipitation lines in this work showed these 3 species to

have 3 common antigens when T. mentagrophytes (dog) super-

 

natant antiserum was diffused against the supernatant

fraction of the 3 species, 2 when T. rubrum (Dar) antiserum

118

was used, and only 1 when T. terrestre antiserum was used.

 

This difference is probably due to the presence of a greater
number of similar antigens in the fraction used to produce
the T. mentagrophytes (dog) antiserum reacted with the
supernatant fractions of the other species.

The number of lines observed in this investigation
were greater than those previously reported in the litera-
ture when dermatophytes were used in the agar gel immuno—
diffusion technique. Landay (1961), and Dyson and Landay
(1963) reported only 2 lines in homologous antigen-antibody

reactions using extracts of T. mentagrophytes and extracts

 

from 21 other isolates of this species as antigens. These
two precipitation lines were identical or partially identi-
cal with two of the three lines produced by the T. rubrum
homologous system. The one line difference, occurring

with the T. rubrum system, was considered to have resulted
from the action of a species specific antigen. In the
present investigation the number of lines observed in
supernatant homologous antigen-antibody systems for T.

mentagrophytes was 8 and 7 for T. rubrum while the number

 

of lines that were identical was five. Reyes and Friedman
(1966) detected four lines when T, rubrum and T, mentagro—
phytes were diffused against T, rubrum antiserum, with all
four lines common. No species specific line was detected.

The results of precipitation lines of the Trichophyton

 

species are recorded, compared and discussed by groups

based on TM vivo hair invasion. This follows the arrangement

119

in current reference books (Emmons gp_gT., 1963; Ajello
gp_§T., 1963; Rebell ep_§T., 1964; and Beneke, 1966).
Grouping the dermatophytes in this manner is not supported
by evidence in this work. It must be remembered that the
grouping of the species on the clinical characteristic

of Tp_xTyg hair invasion was not originally intended to

show a phylogenitic relationship of these Trichophyton sp.

 

In another method of grouping, the species of

Trichophyton have been separated on colony morphology,

 

using color, surface texture, surface profile, etc. for
differentiation. Tomomatsu (1961) using the precipitation
test, an antigen dilution technique, showed no major

antigenic differences among the five groups of Trichophyton

 

(Gypseum, Rubrum, Crateriform, Faviform, and Rosaceum
groups), but reported a closer antigenic relationship among

these fungi than with the members of the genera Microsporum

 

and Epidermophyton.

 

The results of this study indicate the fractions from
the species that have been grouped together on colony

characteristics as the "Faviform group," T. schoenleinii,

 

T. verrucosum and T. Violaceum, show a close relationship

 

 

to each other based on the number of lines, including the
number of identical lines produced with the reference

antisera. Trichophyton soudanense fractions also reacted

 

in a similar manner to the fractions of the species of the
"Faviform group" to the reference antisera, thus a close

serological relationship exists between these four species.

120

Trichophyton gallinae and T. megninii fractions

 

(diffused against the reference antisera resulted in lines
showing a close serological relationship to each other.

The lines were not all identical lines, indicating that they
are not the same organisms, but the occurrence of identical
lines and partially identical lines with the total number

of lines separate the two organisms apart from other species

in the genus Trichophyton. These two organisms have been

 

placed in the "Rosaceum group" of the Trichpphyton genus.

 

Only one species has been placed in the Crateriform

group," T. tonsurans. This organism displayed a close

 

serological relationship to T. mentagrophytes but less so

 

to T. rubrum.
The "Rubrum group" contains one species, T. rubrum.
Serologically this organism appeared closely related to T.

mentagrophytes while the number and arrangement of lines

 

varied with the different reference strains.
Two species have been placed in the "Gypseum group,"

IT, mentagrophytes and T. eguinum. The results of this

 

linvestigation indicates the two organisms are not very
czlose serologically. The supernatant fraction of T. equinum
EDroduced lines closest in number and arrangements to T.
ITubrum fractions with the reference antisera.

Fractions of Trichophyton terrestre, a saprophytic

 

 

Especies, reacted with reference antisera to produce a
rudmber of lines indicating a closer relationship to T.

rmentagrophytes (dog) than to T. rubrum (Dar). However, T.

 

121

13errestre antiserum reacted with the supernatant of M.

 

gypseum to produce the same number of lines as the super-

natant of T. mentagrophytes (dog). Thus, this species

does not appear to belong to any of the designated groups.
On the basis of the present results it would appear

from the serological VieWpoint that the morphological

groupings might have a better basis than the clinical basis

for groupings of the Trichpphyton sp.

 

Tomomatsu (1961) found that the extracts and antisera

of the species in the genera Microsporum and Epidermophyton

 

 

produced stronger reactions with each other than with the

species of the genus Trichophyton. The three species of

 

Microsporum showed no considerable antigenic differences.
He obtained stronger reactions when homologous antigen-
antibody systems were used. Observations in the present
investigations indicate by the number of identical and

partially identical lines formed, that the Trichophyton

 

Species are related closer to each other than with species

of other genera tested. In addition, Microsporum

 

,Terrugineum fractions reacted with the reference antisera to

Ibroduce fewer lines than fractions from the species of the

fTTdchophyton genus and showed a closer resemblance to but

riot identical with the reactions to fractions of M, gypseum.
The relationship of M. ferrugineum to the genus

Tflicrosporum rather than Trichophyton was also shown by

 

IBiguet et a1. (1964) using immunoelectrophoresis techniques.

'They found this species closer to three other species of this

122

ggenus (M. audouinii, M. laggeroni, and M. canis) than to

 

‘the Species used in this investigation, M. gypseum. This

'would explain why the two species, M, gypseum and M,

ferrugineum, were not closer in the present investigations.

 

Fractions of M. ajelloi and M. floccosum developed
only a few, seldom identical lines when reacted with the
reference antisera. Thus these two species are not closely
related serologically to any of the Trichophyton species
studied but M. ajelloi seems closer related to the

Microsporum species.

When tests were performed with the antisera of a
strain diffused against the supernatant fraction of the
same strain, all the lines which appeared between the pellet
antiserum and the supernatant fraction were identical with
some of the lines between the supernatant reactants. Thus,
the identical lines resulted from identical antibodies from
the two antisera reacting with the same antigens in the
fraction. Using each reference antiserum separately, only
one precipitation line of the 4 or 5 formed during the
ciiffusion of pellet antiserum against pellet fraction was
riot identical with the lines formed between supernatant
Eintiserum and the pellet fraction within a reference strain.
UThis indicates that the 2 antisera (supernatant and pellet
IProm each reference strain) possessed 3 or 4 similar anti-
tbodies (depending on antiserum). Since both antisera

:formed by a reference strain contained identical antibodies,

123

cane can suggest 3 possibilities for this. The first is that
ijhe 2 fractions contain some like antigenic make up; and
second that not all of the supernatant antigenic material
'was removed during the separation procedure by centrifuga-
tion, thus a pellet fraction was contaminated with super—
natant material, since the supernatant contained soluble
substances and complete removal is hard to obtain; and
third, a combination of the previous 2 possibilities may be
the explanation.

Thus a certain number of identical antibodies was
produced when the fractions of a strain with some identical
antigens were injected into rabbits.

In the comparison of the fractions from the different
strains within a species with the homologous antigen—
antibody system, one precipitation line difference occurred
when the supernatant fraction diffused against the reference
antiserum of that species. The additional line appeared
between the supernatant homologous systems in each reference
strain ET. terrestre (414), T. rubrum (Dar), and T, mentagro-
Ighytes (dog)] and T, mentagrophytes (455) supernatant against

3:. mentagrophytes (dog) antiserum. These two T, mentagro-
EDhytes strains resembled each other morphologically even
Tlhough they were isolated from different sources. The (dog)
EStrain was from a dog ringworm, and strain (455) was from a
(Ease of human tinea pedis. However these dermatophytes are
riot specific on animal or human in their pathogenicity. In

61 comparison of pellet fractions with different isolates of

124

a :species to the reference antiserum of the same species it was
dijscovered that all the pellet isolates of a species produced
true same number of precipitation lines, 1 detectable line more
trian any other type of pellet reaction except T. mentagro-
Efiaytes (536). This 1 additional line between pellet reactants
vvithin a species appeared not to be identical with any precipi—
tzation lines that occurred in the reactions between supernatant
zantisera and the pellet fractions. Thus there is a component
:in.the pellet that is specific to the species if T. mentagro-
Ighytes (536) is not considered or considered another species.
Okada and Yamamura (1964) detected 3 components which
yvere indicated as "highly liver" specific in the (DOC) extracts
(bf microsomes from chicken livers and 4 components in the un—
ssedimented fraction. Some of the components of the unsedi-
rnented fraction (equivalent to the supernatant fraction in the
IDresent work) were common with some of the components in the
rnicrosomal fraction (equivalent to the pellet fraction in
TZhis study). Okada and Sato (1963) found somewhat the same
r’esults when working with chicken kidney fractions.
Since there was 1 less line in the supernatant
r‘63actions for each of the strains in the species to which

tlfle reference strain belongs except in T. mentagrophytes

 

C L155) which is the same variety (var. gpanulosum) as the T.

 

Elggntagrophytes (dog), and since no supernatant fractions
CDI‘ other species of the dermatophytes produced as many
iIdentical or partially identical lines as the homologous

Supernatant systems it appears that these lines might be due

125

to differences produced in the strains within the species.
The difference in the number of lines using the pellet
reactants was the same for all the strains of a species

except T. mentagppppytes (536) and never as many pre—

 

cipitation lines appeared to form between pellet fraction
of the other species of dermatophytes and the pellet
reference antisera. It is possible that this additional
line might be species specific. Many more strains must
be tested to determine this possibility.

If the additional line is species specific then T.

mentagrophytes would be divided into at least 2 species

 

based upon this present work, since the pellet reaction

between T. mentagrqphytes (dog) pellet antiserum and T.

 

mentagrophytes (536) pellet produced 3 lines and the homolo—

 

gous reaction produced 4 lines as did the reaction with the
strain 455. This indicates 2 immunological types of T.

mentagroppytes as has been reported by Landay (1961), and

 

Dyson and Landay (1963) in the 24 isolates that they used
in agar gel immunodiffusion test.
Huppert (1955) established 3 distinct immunological

groups among 15 strains of T. mentagrgphytes used. Neither

 

Landay nor Huppert worked with T. mentagrophytes var.

 

erinacei (= 536) which was first reported in 1963 by Smith
and Marples.

Grappel gp_gT. (1967) reported significant differences
Sin the serological reactions of the neutral polysaccharides

Zisolated from M. qginckeanum (= T. mentagrophytes),

 

 

126

T. gpanulosum (= T. mentagrophytes), T. interdigital (= T.

 

 

 

mentagrophytes), T. rubrum, and T. schoenleinii, and anti-

 

 

serum to autoclaved antigens of M. quickeanum in immuno-

 

diffussion and complement fixation tests. They worked with
3 species thought to be varieties of one Species.
Ajello and Cheng (1967) reported cleistothecia were

produced by crossing strains of T. mentagrpphytes var.

 

granulosum [the same as strains (455) and (dog) in the
present work]. Upon back crossing the parent strains

they found that the organism is heterothallic and the in-
compatibility is controlled by a single locus with 2 alleles

"A" and "a". When 21 strains of T, mentagrophytes var.

 

gpanulosum were crossed with the "A" and "a" strains only

 

4 produced cleistothecia. Thus, are the 17 strains that
did not produce cleistothecia incompatible due to another
factor or are they members of a different species?

With the 2 immunological groups of T. mentagrophytes

 

reported by Landay (1961); the 3 distinct immunological

groups established by Huppert (1955); the significant dif-
ferences in reactivity of the neutral polysaccharides of 3
species reported by Grappel ep_gT. (1967), considered to be

morphologically one species, T. mentagrophytes, by most

 

mycologists; the obtaining of only 4 cultures with cleisto-

thecia from the many strains of T. mentagrophytes var.

 

granulosum when crossed by Ajello and Cheng (1967); and the

 

differences between strain 455 (T. mentagrophytes var.

 

127

gpanulosum) and strain 536 (T. mentagroppytes var. erinacei)

 

 

in the present immunodiffusion study; there is probably
sufficient evidence to indicate that the present species,

T. mentagrophytes, is a complex of species as was found for

 

the imperfect species, Microsporum gypseum.

 

In the present investigation the fractions from the
reference strains which possessed the largest amount of pro-
tein per ml determined by the Lowery 32 MT. (1951) method,
produced the highest intensity lines. Protein is not the
only type of organic compound that will elicit antibody
formation and cause antigen—antibody reactions. Grappel
SE 21' (1967) showed that neutral polysaccharides are anti-
genic components with significant differences in the sero-
logical reactions among the glucans and galactomannans II
but not‘among the galactomannans I in the 5 species of

Trichophytons used. The polysaccharide determination was

 

not investigated in the present study.
Ito (1965) isolated 22 purified fractions having
trichophytin activity from the mycelium and culture filtrate

of T. mentagrophytes var. asteroides. The majority of these

 

 

fractions consisted of peptide—containing polysaccharides.
Two ribonucleic acid fractions were from the mycelium while
5 simple peptides fractions and a simple polysaccharide
fraction were from the culture medium. Ito found the inten-
sity of the antigenic activity of the fractions were of the

following order; peptide-containing polysaccharide >

 

128

>

sugar-containing peptide _ simple peptide ribonucleic

acid > simple polysaccharide.

The number of precipitation lines in this study was
not as great as the number of different protein fractions
detected by Shechter ep MT. (1967) when the nondialysable
proteins from culture filtrates and mycelial mats were
fractionated by disc electrOphoresis. The number of
fractions varied from 9 to 15 and the fractions identities
varied. They found a high number of homologous fractions
present:th. gypseum and M. pngp, and in T. mentagrophytes

and T. tonsurans. Similar results were obtained in the

 

present studies for the latter 2 organisms. Homologous

fractions in T. mentagrophytes, T. tonsurans and T. rubrum

 

 

were few as was the case for the lines in my results. They
obtained only 2 homologous fractions in the 6 organisms
when using the culture filtrates and no fraction was
homologous for all 6 organisms in extracts from the dif-
ferent mycelia. This corresponds to my results as no 1
identical antigen was evident in all organisms studied.
Even though the number of lines differs in the present
work from the number of fractions reported by Shechter pp
MT. (1967), similar results were obtained in that species
showed specific identities of protein fractions and anti-
gens, and that homologous protein fractions and homologous
antigens occurred in different species and species of

different genera.

‘__.-

129

The sensitivity in detecting small amounts of protein
is probably greater with disc electrophoresis than with
the agar gel immunodiffusion technique. The rabbit's
antibody producing mechanism might be able to detect the
smaller quantities of proteins and produce antibodies in
response to the antigens but the detection of the
antigen-antibody reaction in the gel would be difficult if
the antibodies and antigens are of very low concentrations.

Even though the perfect stages are being discovered
for many of the dermatophytes, the recognition of species
is still difficult as reported by Pore and Plunket (1967).
They found the distinctive appendages of Arthroderma (the
perfect genus for species of Keratinomyces and Trichophyton
for which the perfect stages have been found) were of
little value for determination of species, so they con-
sidered the concept of biological species. However, in
their discussion of crosses, the possibility of incompat—
ibility factors within a species was not discussed. Incom-
patibility factors are known to exist in strains within
species of the fungi: Schizophyllum commune, Pleurotus

ostreatup, Sordaria fimicola and others (K. Esser, 1966).

 

 

When incompatibility factors are discovered some of these
biological species in the dermatophytes may become only
strains. Thus immunodiffusion, disc electrophoresis,
immunoelectrophoresis and other techniques will still be
needed to supply the necessary information of the true

relationships.

.7 "I‘I
p

SUMMARY

Twenty strains of the dermatophytes including strains
within a species and species from the 4 different genera
were grown in Sabouraud's—glucose broth for two weeks,
filtered and rinsed. The mycelium was macerated, filtered

and the filtrate centrifugred at 20,000 X g for 20 minutes.

 

Hi
The supernatant was separated into two fractions by centri-
fugation at 104,000 X g for 4 hours. The supernatant and
pellet fractions from the reference strains, Trichophyton g

mentagrophytes (dog), T. terrestre (414), and T. rubrum

 

 

(Dar.) were used to produce antisera in rabbits by a
series of intramuscular and intraperitoneal injections.

The precipitinogenic relationships of the 20 repre—
sentatives were compared by using the 2 fractions of each
organism and the reference strains antisera in the agar-gel
slide immunodiffusion technique.

Antigenic similarities and dissimilarities existed
between supernatant and pellet fractions within a strain,
the fractions of the strains of a species, the fractions
of the reference strains and the fractions of the 12 other
dermatophytes species.

The supernatant homologous antigen-antibody reactions
produced more lines than the pellet homologous reactions.
Thus, more detectable antigenic substances were in the

Supernatant fractions.

130

131

The supernatant and pellet fractions of a strain
possessed some homologous antigens, with all but one antigen
in the pellet being identical to antigens in the supernatant.

The supernatant fractions of the strains within a
species, produced one less line than the homologous antigen-
antibody system except in T. mentagrophytes (455). The
fractions of this strain or isolate acted identically to
the fractions of the reference T. mentagrophytes (dog)
strain and these two strains are of the same variety

granulosum. These is the possibility of a "strain antigen"

 

producing the additional line between homologous supernatant
systems.

The pellet fractions of the different organisms within
a species did not differ except in one case, the strain 536

T. mentagrophytes var. erinacei. The pellet homologous

 

reactions produced one line more than in reactions with
heterologous pellet fractions. Thus there is the possibility

of a "species antigen." Since the pellet fraction of

 

T. mentagrophytes var. erinacei did not indicate the presence
of such an antigen, it could be considered a separate species.

The separation of the trichophytons into groups
according to their colony morphology (a separation little
used at present), Gypseum-, Crateriform—, Faviform-,
IRosaceum-, and Rubrum- groups (with T. terrestre in a group
:for saprophytic species) was suggested by the degree of

hcnnogeneity of the reactions by the members of these groups.

PF“

132

Trichophyton soudanense was closest antigentically

 

to T. Violaceum, T. schoenleinii, and T. verrucosum,

 

 

 

members of the faviform group.

Microsporum ferrugineum showed a closer relationship

 

 

to M. gypseum than to any species of the Trichophytons

studied, while M. floccosum and M. ajelloi differed in

 

their reaction with each other and the other organisms in
the immunodiffusion tests. Keratinomyces gjelloi appeared
closer related serologically to M. gypseum.

The supernatant reference antiserum of T. Mgppgy

O
grophytes (dog) was the only one to demonstrate a homologous

 

antigen in all the dermatophytes tested except M. ajelloi.

While both T. mentagrophytes (dog) and T. rubrum (Dar)

 

supernatant antisera showed one common antigen with all

the trichophytons

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