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Graduate Student Theses, Dissertations, & Professional Papers Graduate School
1977
Immunoserologic studies of Sporothrix schenckii
Norman R. Kufner The University of Montana
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Recommended Citation Kufner, Norman R., "Immunoserologic studies of Sporothrix schenckii" (1977). Graduate Student Theses, Dissertations, & Professional Papers. 6654. https://scholarworks.umt.edu/etd/6654
This Thesis is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. IMMUNOSEROLOGIC STUDIES OF SPOROTHRIX SCHENCKII
By
Norman R. Kufner
B.S., University of Wisconsin-Green Bay, 197U
Presented in partial fulfillment of the requirements for the degree of
Master of Sciences
UNIVERSITY OF MONTANA
1977
Approved by;
Gherman, Bo^d of Ex^^ners
^raduaTtê School /
Date
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Kufner, Norman R., M.S., August, 1977 Microbiology
Immunoserologic Studies of Sporothrix schenckii (5l pp.)
Director; John J. Taylor
Immunoserologic diagnosis of sporotrichosis in humans as well as in other mammalian species is presently based on recommended procedures leading to positive complement fixation, precipitation, agglutination and/ or skin test reactions. However, the whole cells or crude culture filtrates used as antigens in serodiagnosis routinely yield both false positive and false negative reactions in clinical practice, and the significance attached to particular serum titers is frequently equivocal. No procedure in current use is sufficiently sensitive to diagnose subclinical or atypical in fections . In this study both whole protoplasm from either mycelial or yeastlike Sporothrix schenckii cells and certain fractions of pro toplasms isolated by Sephadex G-200 chromatography were found equivalent to or somewhat more sensitive antigens in detecting sporotrichic infections in rabbits when recommended sérodiagnos tic methods were used. However, when protoplasm or protoplasmic fractions were used as antigens in indirect hemagglutination reactions with formalinized and tanned sheep erythrocytes, titers of at least 20J48 were demonstrable in infected animals wnile those in uninfected animals did not exceed 8 . The test appears to be as specific as presently recommended methods, but is far more sen sitive. The most useful protoplasmic fractions were those with relatively more protein. Two slowly-moving components demonstrated high varietal specificity in delayed skin tests by eliciting pos itive reactions in animals infected with the homologous variety but not in those infected with the heterologous variety. No antigenic differences were detected between the mycelial and the yeastlike forms of S. schenckii.
XX
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS
I would like to express ray sincere gratitude to Dr. John J.
Taylor whose enthusiasm, cheerfulness, and confidence in this project
kept me from becoraing discouraged. I could not have had a more thought
ful advisor, nor a more competent one. I thank Dr. Gary Gustafson for
lending me equipment and advise and Paul Qrndorf and Scott Mattem for
lending me and showing me how to use their Sephadex columns. Mary Beth
Baker and Kathy Kelly, of the Stella Duncan Institute, kept me supplied
with sheep erythrocytes for which I am grateful.
Especially, I want to thank my wife, Pat, for not becoming
discouraged when this work took time that I might have been able to
spend with her. Rather, she encouraged me to persevere. Her sacri
fices are greatly appreciated.
1X1
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS
Page
ABSTRACT...... il
ACKNOWLEDGMENTS...... ill
TABLE OF C O N T E N T S ...... Iv
LIST OF T A B L E S ...... vii
LIST OF F I G U R E S ...... viii
Chapter
I . INTRODUCTION ...... 1
II. MATERIALS AND METHODS ...... 6
Source of F u n g i ...... 6
Growth of F u n g i ...... 6
Mycelial f o r m ...... 6
Yeastlike f o r m ...... 6
Preparation of Antigens ...... 6
Whole mycelial cells ...... 6
Whole myc'.-'l ial protoplasm...... 7
Mycelial cell walls ...... 8
Whole yeastlike cell protoplasm ...... 8
Protein and Carbohydrate Determinations ...... 8
Sephadex G-200 Chromatography of Whole Protoplasms ...... 8
Sephadex G-200 chromatography of yeastlike cell whole protoplasm ...... 8
Sephadex G-200 chromatography of mycelial cell whole protoplasm...... 9 iv
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Ammonium Sulfate Precipitatio*, ...... 9
Artificial Infection of Rabbits ...... 10
Histological Examination of Lesions in Infected R a b b i t s ...... 10
Serology...... 10
Tube agglutination t e s t ...... 10
Slide latex agglutination t e s t ...... 11
Indirect hemagglutination test with untanned sheep erythrocytes ...... 11
Indirect hemagglutination test with tanned sheep erythrocytes ...... 11
Immunodiffusion ...... 11
Leukocyte Adheranee Inhibition Test ...... 11
Skin Testing...... 12
III. R E S U L T S ...... 13
Purity of Cultures...... 13
Protein and Carbohydrate Content of Whole Protoplasms ...... 13
Sephadex G-200 Chromatography of Whole Protoplasms...... 13
Artificial Infection of Rabbits ...... 17
Histology of Spon-trichic L e s i o n s ...... 17
Serology...... 18
Tube agglutination...... 1 8
Slide latex agglutination...... 18
Indirect hemagglutination with untanned sheep erythrocytes ...... 18
Indire' t hemagglutination with tanned sheep erythrocytes ...... 21
Immunodiffusion ...... 26
V
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Leukocyte Adherance Inhibition Test ...... 26
Skin Testing ...... 26
IV. DISCUSSION...... 32
Artificial Infection of Rabbits ...... 32
Host Resistance to S^. s c h e n c k i i ...... 33
Skin Testing...... 33
Immediate-type and delayed skin hypersensitivity...... 3U
Prior research in skin testing for sporotrichosis ...... 3U
Skin testing results of the present study ...... 36
Serological Testing ...... 36
Tube agglutination with yeastlike cells ...... 36
Soluble antigens used previously in serological testing ...... 38
Whole protoplasm as a n t i g e n ...... 38
Slide latex agglutination t e s t ...... 39
Indirect hemagglutination ...... 39
Indirect hemagglutination with tanned sheep erythrocytes ...... UO
Similar Antigenicity with Yeastlike and Mycelial Cell ...... U2
Similar Antigenicity with S. schenckii and schenckii var. l u r i e i ...... h2
Protein Important for Antigenicity ...... U3
Directions for the F u t u r e ...... U3
V. S U M M A R Y ...... UU
REFERENCES CITED ...... h i
VI
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES
Table Page
1. Tube Agglutination Titers with Mycelial and Yeastlike Whole Cells and Mycelial Cell Walls of S^. schenckii ...... 19
2. Slide Latex Agglutination Test with %rcelial Whole Protoplasm and a Sephadex Fraction of Mycelial Protoplasm of schenckii...... 20
3. Indirect Hemagglutination Titers with Mycelial Whole Protoplasm of schenckii Using Untanned Sheep Erythrocytes ...... 22
ii. Indirect Hemagglutination Titers with Mycelial Whole Protoplasm from schenckii, and a Sephadex Fraction, Using Tanned Sheep Erythrocytes .... 23
5* Indirect Hemagglutination Titers with Mycelial Whole Protoplasm from S^. schenckii var. luriei Using Tanned Sheep Erythrocytes ...... 2k
6 . Indirect Hemagglutination Titers with Whole Protoplasm and Sephadex Fractions of Yeastlike Cell Protoplasm of S. schenckii Using Tanned Sheep Erythrocytes...... 2S
7. Immunodiffusion with Mycelial Whole Protoplasm and Sephadex Fractionation Tubes 37 to Li of Whole Protoplasm of S_. s c h e n c k i i ...... 27
8 . Immunodiffusion with Sephadex Fractions of Whole Protoplasm of Yeastlike schenckii and a Homologous A n t i s e r u m ...... 28
9. Skin Testing Rabbits with Various Components of Mycelial Form schenckii...... 30
10. Skin Tests of Rabbits with Whole Protoplasm and Sephadex Fractions of Protoplasm of Yeastlike S. schenckii ...... 31
Vll
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES
Figure Page
1 . Comparison of Absorbance at 280 nm of Protoplasms of S^. schenckii Fractionated by Sephadex G-200 Chromatography ...... 1U
2. Protein Content of Sephadex Fractions of Whole Protoplasm of Yeastlike S. schenckii ...... l5
-3. Carbohydrate Content of Sephadex Fractions of Whole Protoplasm of Yeastlike S. schenckii...... 16
vxii
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER I
INTRODUCTION
Sporotrichosis is an infection caused by Sporothrix schenckii
Hektoen and Perkins 1900, following traumatic implantation of spores
into the skin, or rarely, following inhalation of spores. In 75^ of
reported cases (3 , 8 , 37 ) it takes the form of a cutaneous or subcuta
neous nodular lesion which spreads to adjacent lymphatics. The lesions,
most often found on hands, arms, or legs (ii) suppurate, ulcerate, or
drain. Secondary spread to bone, articular surfaces, and muscle is
not uncommon; occasionally the central nervous system, lungs, and/or
genitourinary tract are involved (32).
Schenck, in 1898 (35), was the first to report a case of sporo
trichosis in sufficient detail to be recognized conclusively- Hektoen
and Perkins (ii8 ) later named the etiologic agent Sporothrix schenckii,
the presently accepted name. (However, because of a mistake made in
1921 (U8 ), some call it Sporotrichum schenckii.) It is presently
thought that the perfect stage of Sporothrix schenckii is Ophiostoma
stenoceras (Robak) Melin and Nannf (27, 28, U5).
Usually introduced to a host through a break in the skin by
a scratch or puncture wound, S. schenckii has a widespread saprophytic
existence particularly on vegetation. Implantation of the fungus into
the host has been effected by splinters, thorns, rough bark, the bar
berry shrub, sphagnum moss, and bites of wild and domestic animals.
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birds, and insects. The incubation period varies from three days to
three weeks {hit h8 ).
Organisms grown on Sabouraud glucose agar at room temperature
produce colonies which are white and wrinkled at first and become
leathery and smooth with longer incubation. Pigmentation varies from
cream to black among different strains. Slide cultures or mounts show
branching, septate hyphae 2>im in diameter and pyriform to spherical
conidia borne laterally or sympodially on the tips of conidiophores
resulting in a floral-like arrangement. Conidia are 2-h >im by 2-6 pm.
When grown at 37° C on media rich in proteins and vitamins, the myceli
al phase can be converted to a yeastlike form resembling that found in
tissues. Yeastill e colonies grown on brain-heart infusion agar or
Francis glucose-cystine-blood agar at 37° C appear soft and white or
cream in color with an irregular surface similar to colonies of some
yeasts. A slide mount of the yeastlike phase shows round, oval, and
fusiform budding cells called "cigar bodies", 1-3 pm by 3-10 pm.
Cells grown on synthetic media at 37° C and those recovered from in
fected animals are similar when stained and observed microscopically
(h) • A variant of S. schenckii, called S_. schenckii var. luriei,
which has been isolated from humans in Africa is not as delicate and
possesses larger conl lia. In tissues it reproduces both by budd'.ng
and by transverse septation (1, 32).
D. H. Howard demonstrated that the conversion of the mycelial
to the yeastlike phase in tissue culture involves the production of
short hyphal branches from conidia which then form yeastlike cells
(19). Taylor (39) demonstrated that the short mycelial fragments
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described by Howard are actually sympodulae which bear secondary sym-
podulospores.
Sporotrichosis affects all age groups, races, and both sexes
without preference. A predominance of reported disease in males be
tween the ages of 25 and 50 is attributed to the increased likelihood
of this group to sustain cutaneous injuries in their work and play.
The infection occurs not only in man, but also in horses, mules, don
keys, camels, cattle, dogs, fowl, rats, and mice. Transmission of the
disease from man to man is very rare (U, 1+8 ).
In 1912, DeBeurman and Gougerot (13) were the first to observe
that disseminated sporotrichosis was coincident with malnutrition or
other debilitating conditions. This is still a common finding (26, 1+1,
1+2 ). In immunologically normal hosts the infection is limited to a
localized cutaneous or Ijrmphocutaneous one. Man's natural resistance
to infection is apparently high, although the organism is universally
present in the soil (32).
Diagnosis uf sporotrichosis is seldom possible by a direct ex
amination of pus aspirated from an unruptured lesion because the fungus
is seldom seen in sufficient numbers for identification. When streaked
on Sabouraud glucose agar containing cycloheximide and chloramphenicol,
the pus produces identifiable colonies in 3 to 5 days (1+). In many
cases, however, cultural identification is not possible; for example,
when the agent is sparse, non-viable or overgrown by contaminants.
Sometimes accurate identification takes 2 weeks or longer. Fluor
escent antibody techniques have been investigated to facilitate iden
tification of the agent in clinical specimens (22), but have not
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gained wide acceptance.
The common chancriform-lymphoglandular type of infection is so
indicative of sporotrichosis that the clinical picture is often suffic
ient evidence for diagnosis by a trained observer. It can be confused,
though, with certain bacterial infections, such as syphilis, or fungal
infections, such as chromomycoois, blastomycosis, mycetoma, paracocid-
iomycosis, or granulomatous trichophytosis (32, U8 ).
Many serological procedures have been investigated since Norden
(31 ), in 19$1, made the first significant contribution to the serolog
ical diagnosis of sporotrichosis. These include immunodiffusion, slide
latex agglutination, an immunocytoadherance test (33 ), indirect fluor
escent antibody tests, and others (5, 21, 3U). Tests studied to detect
cell mediated reactions include skin tests (29, 3 U, 38) and an ^ vitro
lymphocyte blastogenesis test (34). The leukocyte adherance inhibition
test has recently been reported to give very accurate diagnosis of sev
eral dermatophytic infections (lS, 43), but it has not apparently been
investigated as a tool for diagnosis of subcutaneous fungal infections.
Common antigens used in these diagnostic techniques inurude
whole yeastlike cells (21, 29, 34) as particulate antigens and a cul
ture filtrate of both mycelial and yeastlike cells as soluble antigens
(5, 21). Other antigens investigated include the supernatant fluid
following disruption of yeastlike cells (34), in complement fixation
and precipitin tests, and many individually treated fractions of
yeastlike cells used in skin testing (29). A recent study in Japan
(36) demonstrates tiiat a peptide-polysaccharide fraction obtained from
a crude polysaccharide extract of mycelial cells possesses antigenicity.
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The polysaccharide portion of the fraction is effective in precipitin
tests and immediate-type skin reactions in guinea pigs whereas the pep
tide moiety is able to evoke delayed or cell mediated skin reactions.
Presently, the most widely used and respected serological meth
ods used in clinical laboratories are complement fixation, precipita
tion, agglutination (21), and slide latex agglutination tests (23, 2h)•
Although these procedures are of adjunct value, they are not capable of
conclusive diagnosis. It is, therefore, desirable to uncover an anti
gen from the organism, schenckii, or to modify an existing technique
using presently available antigens, that will allow more accurate and
specific diagnosis of sporotrichosis, especially atypical and inappar-
ent infections.
The purposes of this investigation were to use rabbits as
models and (a) study comparatively the infections and immune responses
in animals intramuscularly infected with schenckii or schenckii
var. luriei, (b) fractionate cells of mycelial and yeastlike
schenckii and determine the usefulness of those fractions in skin tests
and serological techniques, and (c) compare and/or contrast the anti
genicity of yeastlike and mycelial cells of schenckii and its vari
ety luriei.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER II
MATERIALS AND METHODS
Source of Fungi
Sporothrix schenckii, University of Montana (UM) k2^, and
Sporothrix schenckii var. luriei, UM U30, were obtained from Dr. J. J.
Taylor, University of Montana, who received them from the Center for
Disease Control. They had been recovered from human sporotrichic le
sions .
Growth of Fungi
Mycelial form. Both schenckii and S_. schenckii var, luriei
were inoculated into 100 ml of Sabouraud glucose broth containing 1 .0%
yeast extract in a ^00-ml volumetric flask and shaken for 7 days at 2$^
C. Resulting cultures were then streaked on brain heart infusion agar
plates and incubated for 7 days to insure the absence of bacterial or
fungal contamination.
Yeastlike form. One ml of a 7-day-old broth culture of mycelial-
form S. schenckii was spread over the surface of blood-glucose agar
plates and incubated for 7 days at 37° C under about S% GOg*
Preparation of Antigens
Whole mycelial cells. Broth cultures of S. schenckii and S.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 7
schenckii var. luriei were harvested, and washed twice, by centrifu
gation with sterile distilled water. Packed cells were resuspended
(1 ;10) in sterile distilled water and stored at -20° C until later rup
tured for removal of protoplasm. Cells of similar suspensions were
killed and stored 1:100 (v/v) in 1 :10,000 (w/v) merthiolate and kept
refrigerated at 1+° G to be used in tube agglutination and skin tests.
Whole yeastlike cells. Yeastlike cells were carefully brushed
from the surface of blood-agar plates flushed with sterile distilled
water and washed 3 times by centrifugation with sterile water. The
final pellet was resuspended 1 :10 (v/v) in sterile distilled water and
stored at -20° C until processed to recover protoplasm, or stored 1:100
(v/v) in 1 :10,000 (w/v) merthiolate until used in agglutination and
skin tests.
Whole mycelial protoplasm. %rcelial form cells of S_. schenckii
and S_. schenckii var. luriei were centrifuged and resuspended in 3 vol
umes of sterile distilled water in a chilled food blender. Fragmenta
tion of hyphae was effected by blending at high speed for 10 min. One
volume of glass pavement marking beads size "10A ” was then added and the
suspension was ground for 1 h. The suspension was sedimented by centri
fugation, the supernatant fluid was removed, evaporated in dialysis
tubing to about 3 mg protein/ml as determined by spectrophotometrie ab
sorbance at 260 and 280 nm (ij-i;), and stored at -20° C to be used later
in various serological tests, skin tests, and a leukocyte adherance
inhibition test.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 8
Mycelial cell walls. After mycelial cells were fractured by a
food blender and the protoplasm fraction removed, cell walls were re
moved from the glass pavement marking beads by differential sedimen
tation. They were washed by centrifugation, the sediment was resus
pended 1:10 (v/v) in distilled water and stored at -20° C to be used in
tube agglutination and skin tests.
Whole yeastlike cell protoplasm. Viable whole yeastlike cells
of 2 " schenckii were centrifuged and resuspended 1 (v/v) in distilled
water and subjected to explosive decomposition twice in a Sorvall-Ribi
refrigerated cell fractionator at U0,000 psi. Cell fragments were
stained with lactophenol blue and observed microscopically to determine
adequate breakage. The fragmented cells were sedimented by centrifu
gation and the supernatant fluid was removed and stored at -20° G until
needed for various serological and skin tests.
Protein and Carbohydrate Determinations
Whole protoplasms were analyzed for their protein content by
Coleman’s (11) adaptation of the method of Lowry et Bovine serum
albumin (BSA) was used as the protein standard. The method of Herbert
et ai. (17) was employed to determine carbohydrate content of proto
plasms. Glucose was the carbohydrate standard used.
Sephadex G-200 Chromatography of Whole Protoplasms
Sephadex G-200 chromatography of yeastlike cell whole proto
plasm. Whole protoplasm from yeastlike cells was lyophilized and re
hydrated with 1 ml of aqueous 10% sucrose to contain 13 mg protein/ml
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 9
(7). The suspension was carefully layered onto a 90 cm by 1.7 cm
Sephadex G-200 column with a void volume of about 100 ml. Samples were
eluted with phosphate buffered saline (PBS), pH 6.h to facilitate later
adsorption to sheep erythrocytes, and 1-ml fractions were collected.
Protein content of fractions was initially determined by spectrophoto-
metric absorbance at 260 and 280 nm (hh)• Later, protein and carbohy
drate determinations were made by the same procedures used for whole
protoplasms.
Sephadex G-200 chromatography of mycelial cell whole proto
plasm. Four ml of mycelial cell whole protoplasm containing about 3
mg protein/ml were chromatographed on a 90 cm by 2.8 cm Sephadex G-200
chromatography column with a void volume of 88 ml and a bed volume of
approximately 300 ml. The absorbance at 260 and 280 nm of 3.7 ml frac
tions eluted with PBS, pH 7.2, was recorded. Fractions were stored at
-20° G until used as serological and skin test antigens.
Ammonium Sulfate Precipitation
Seveial fractionations of mycelial cell whole protoplasm were
conducted with the Sephadex G-200 column and fractions within the early
peak with high absorbance at 280 nm (see Figure 1) were pooled, ammon
ium sulfate was added to saturation, and the mixture was allowed to
stand overnight at C. The precipitate was then collected by fil
tering the mixture through a prechilled filter, and the precipitate was
washed several times with 10 ml of cold saturated ammonium sulfate sol
ution. The membrane was allowed to dry following each wash and finally
the precipitate was dissolved in sterile distilled water. The dissolved
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 0
material was stored at -20° C until used as a skin test antigen.
Artificial Infection of Rabbits
Four New Zealand white rabbits were injected intramuscularly
into the thigh at weekly intervals with 1 ml of 1:10 (v/v) suspension
of whole cells in PBS, pH 7.2. Two rabbits received weekly injections
of schenckii and 2 received schenckii var. luriei. Control rab
bits were not injected.
Histological Examination of Lfc.-sions in Infected Rabbits
Nodules uhich formed in response to intramuscular injections of
viable organisms into the • .gii of rabbits were imbedded in paraffin
and cut into $ yufi sections. The sections were mounted on albumen-
coated slides and then stained by the periodic acid-Schiff (PAS) method
and counterstained with eosinate and Wright's polychrome for 9 seconds
each. Sections were then observed microscopically. The nature of the
cellular reaction and of the cells involved in the reaction were noted.
Serology
Sera from artificially infected rabbits, 2 infected with
schenckii and 2 with schenckii var. luriei, and several uninfected
control rabbits were titrated by various serological methods. In all
titrations, titer is expressed as the reciprocal of the highest dilu
tion just showing positive agglutination.
Tube agglutination test. Whole mycelial cells, mycelial cell
walls, and whole yeast cells of schenckii were used as antigens ac
cording to the method of Welsh and Dolan (h^).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 1
Slide latex agglutination test. Mycelial cell whole protoplasm
and its major Sephadex fractions, those with maximum absorbance at 280
nm (see Figure 1 ), were employed in the slide latex agglutination test
of Kaufman (2U).
Indirect hemagglutination test with untanned sheep erythrocytes.
Sheep erythrocytes were formalinized by the method of Czismas (12) and
then titrated by the method of Pant e^ ad. (30) using whole mycelial
protoplasm of schenckii.
Indirect hemagglutination test with tanned sheep erythrocytes.
Sheep erythrocytes were formalinized by the method of Czismas (12) and
then tanned by the method of Kagan and Norman (20). Titration of
whole protoplasm and various Sephadex fractions of whole protoplasm
was performed in the same way as the above titration with untanned
sheep erythrocytes (30).
Immunodiffusion. Agar fc>r immunodiffusion was 0.3^ ion agar in
0 .87$^ NaCl containing 1:10,0)0 (w/v) merthiolate. Either 12 ml were
poured into a disposable pias ,ic petri dish and a couple of drops put
in each well to seal it, or ml were layered onto a microscope slide
precoated with 1.0% ion agar .hat had been allowed to dry. Undiluted
antiserum was placed in the center well and various antigens in the
peripheral wells. Petri dishus were incubated for 7 days and micro
scope slides for 1 day in a moist chamber.
Leukocyte Adherance Inhibition Test
Whole mycelial cells and the respective whole protoplasm were
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12
tested as antigens by the methods of Halliday and Miller (15) and
Walters £t (i;3). Instead of Dextran DR^ (Macrodex, 6% in $% dex
trose, Pharmacia, Sweden), S% dextran of M, W. 500,000 daltons in S%
dextrose was used as a gradient to separate leukocytes from blood.
Skin Testing
Whole protoplasm from S^. schenckii and schenckii var. luriei,
and Sephadex fractions of whole protoplasm from mycelial and yeastlike
schenckii were injected in 0 .1-ml volumes intradermally into rabbits
homologously and heterologously infected. Cell walls of mycelial form
S. schenckii were injected as a 1:5 (v/v) suspension. A 1:100 (v/v)
suspension of the same merthiolate-killed whole mycelial cells and of
whole yeastlike cells was found optimum for skin testing. Protein and
carbohydrate determinations were made on soluble antigens used for skin
testing. Skin reactions were observed at 2, 6, 12, 18 , 2h, U8 , 72, and
96 h. A positive reaction was one that was obviously greater than that
which occurred in an uninfected rabbit. Reactions were read as simply
positive or negative.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER III
RESULTS
Purity of Cultures
Mycelial and yeastlike forms of schenckii and schenckii
var. luriei when streaked on brain heart infusion agar plates were
found to have no contaminating fungi or bacteria.
Protein and Carbohydrate Content of Whole Protoplasms
Whole protoplasms used in skin testing are shown in Tables 9
and 10. Antigens used for skin testing contained from 23 to 6l pg
of protein and from 9 to hè> p.g of carbohydrate per injection.
Sephadex G-200 Chromât, -graphy of l#ole Protoplasms
Absorbance at 280 nm of Sephadex G-200 fractionation of yeast
like and mycelial whole protoplasms is shown in Figure 1. In both fractio]
tions a high peak is observed at the void volume and elution patterns
are similar until the last fractions eluted, where there is another
high peak in the fractionation of yeastlike protoplasm, but not in that
of mycelial protoplasm.
Subsequent chemical assays of Sephadex G-200 fractions of
yeastlike whole protoplasm (Figures 2 and 3) reveal that the first ma
jor peak of absorbance at 280 nm is due to a high concentration of pro
tein, but that it also contains a substantial amount of carbohydrate.
There are secondary peaks of protein concentration at tubes 57-70,
13
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CD ■ D O Q. C g Q. Figure 1 ■D CD Comparison of Absorbance at 280 nm of Protoplasms of schenckii
C/) Fractionated by Sephadex G-200 Chromatography 3o' O
8
(O' ïeastlike Cell Protoplasm
Mycelial Cell Protoplasm
3. 3 " CD ■DCD O CO CQ. CM a Eh 1.0 o 3 ■D O
CD Q.
■D CD
C/) C/) « ****** 4*. •
* * —* •••• a # a f ^
60 70 80 90 100 110 120 ELUTION TUBE NUMBER CD ■ D O Q. C g Q. Figure 2 ■D CD Protein Content of Sephadex Fractions of Whole
C/) Protoplasm of leastlike S. schenckii C/)
1.0
0.9
0.8 -I
0.7
"OCD I 0-6 -I O Q. g C a | o . 5 3O "O O m o.k
CD Q. 0.3 J
■D 0.2 CD
C/) * â • • C/) 0.1 1
0 — ------1------— --- r - 10 So 60 70 80 90 100 110 ELUTION TUBE NUMBER va CD ■ D O Q. C g Q. Figure 3 ■D CD Carbohydrate Content of Sephadex Fractions of Whole Protoplasm of ïeastlike S. schenckii WC/) 3o" O
8
(O'
0.6 3. 3 " CD ■DCD O Q. C a o 3 ■D O
CD Q.
■D CD
C/) C/) 50 60 70 80 90
ELUTION TUBE NUMBER 17
IOU-108, and 112-113. Of those fractions tested for carbohydrate con
centration, tubes 39-L7 contained the largest share. The carbohydrate
content of the last fractions could not be determined because that is
where the sucrose used to suspend the protoplasm was eluted (Figure 3).
The peak with high absorbance at 280 nm seen in the last fractions
eluted does not contain enough protein to explain such absorbance, so
compounds other than proteins are responsible for most of this absor
bance. A greater concentration of nucleic acids relative to other pro
toplasmic constituents would probably be found in yeastlike or conidial
cells than in mycelial cells, which may explain why the peak was not ob
served in the fractionation of mycelial whole protoplasm. It is also
possible that fractionation of mycelial protoplasm was not carried out
long enough to observe the second. It is noteworthy that the concentra
tion of mycelial whole protoplasm fractionated was not as great as that
of yeastlike cells.
Artificial Infection ol’ labbits
Rabbits infected with either S_. schenckii or schenckii var.
luriei developed very prominent intramuscular nodular lesions character
istic of subcutaneous infections at the site of intramuscular injection
of live rQTcelial cells. When injections were discontinued for a month
or more, the lesions became less palpable, but when injections were
again resumed for two weeks or more the nodules became more apparent
when observed grossly and when palpated.
Histology of Sporotrichic Lesions
Both varieties of & schenckii produced granulomatous lesions
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 8
with a central zone of necrotic tissue surrounded by fibroblasts and an
infiltrate of monocytes and neutrophils. Fungal cells were not found
in sections observed from a rabbit infected with schenckii, but were
observed in profusion in tissues of a rabbit infected with the variety
luriei which had died of an apparently unrelated intestinal disorder.
Serology
Tube agglutination. Merthiolate-killed whole yeastlike cells
gave tube agglutination end points that were easier to read than those
obtained when mycelial cells or mycelial cell walls were used. Titers
were quite similar with all three antigens (Table 1) and ranged from
160 to 320 . Antigens from schenckii worked equally well with anti
sera from rabbits infected with both varieties of S^. schenckii.
Slide latex agglutination. Mycelial whole protoplasm of S^.
schenckii gave titers of from h to I6, the highest titer occurring in a
heterologously infected rabbit and the lowest titer occurring in a ho
mologously infected one. Slightly higher titers, from 16 to 6J4, were
obtained with the mycelial Sephadex fraction with maximum absorbance at
280 nm (Figure 1, tubes 37-^1). Again, the highest titer occurred in
the same heterologously infected rabbit and the lowest titer in the same
homologously infected one. Undiluted sera from three of four uninfected
rabbits tested gave positive reactions to this fraction. One was posi
tive up to a titer of 2 (Table 2).
Indirect hemagglutination with untanned sheep erythrocytes. Ti
ters ranging from $12 to 16,38U were obtained using mycelial whole
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CD ■ D O Q. C g Q. Table 1 ■D CD Tube Agglutination Titers with Mycelial and leastlike Whole Cells and Mycelial Cell Walls of S. schenckii C/) 3o' O 5 CD 8 Titers To;
(O' 3 " Source of Antisera Yeastlike Mycelial Mycelial i Whole Cells Whole Cells Cell Walls 3 CD
c"n 3- Rabbits infected with CD S. schenckii; 1 320 160 320
CD "O O 2 320 320 320 CQ. a o 3 Rabbits infected with ■D 3 S. schenckii var. luriei: 3 320 320 320 3 " O' 1—H CD I 320 160 160 $ 1—H o c Uninfected Rabbit; 22 0 0 0 "O CD
C/) C/)
\ 0 CD ■ D O Q. C g Q. Table 2 ■D CD Slide Latex Agglutination Test with Mycelial Whole Protoplasm 3 C/) and a Sephadex Fraction of Mycelial Protoplasm of S. schenckii C/) 3o' O 3 8 Titers To:
c q ' 3 " Source of Antisera Mycelial Whole Combined Sephadex Protoplasm i Tubes 37-Ul 3 CD
c"n 3. Rabbits infected with 3 " CD S. schenckii: 1 8 32 ■DCD O 2 u 16 C Q. o' Rabbits infected with "Og S. schenckii var. luriei : 3 8 16 3 " CT 1—H U 16 6U Q. $ 1—H o Uninfected rabbits : FU 0 0 "O CD 3 5 0 0 C/) C/) o' 3 18 0 0
Br 2
1 ND=Not Done ro o 2 1
protoplasm from schenckii adsorbed to formalinized but untanned sheep
erythrocytes. The lowest titer was from a homologously infected rabbit
and the highest titer came from a heterologously infected one. Uninfect
ed rabbits produced titers up to 128 (Table 3).
Indirect hemagglutination with tanned sheep erythrocytes. Whole
mycelial protoplasm of S_. schenckii and S. schenckii var. luriei demon
strated titers of 1021; and 20U8, the highest dilutions tested (Tables h
and 5). On the other hand, sera from uninfected rabbits gave no or very
low titers. One had a titer of 8, two gave titers of U, and iL pro
duced titers of less than U.
Whole protoplasm of yeastlike cells of schenckii produced
results very similar to those of the homologous mycelial protoplasm.
As seen in Table 6, homologously infected rabbits gave titers of Si 2 and
102U. Rabbits infected heterologously with S^. schenckii var. luriei
had titers of at least 20^8, the highest dilution tested. Although the
titers changed slightly for a given I'abbit, the overall results obtained
from uninfected rabbits were similar to those obtained with mycelial
protoplasm, that is, one with a titer of 8, one with a titer of U, and
l5 with titers of less than i;.
Sepnadex G-200 fractions of yeastlike whole protoplasm are de
scribed as antigens in Table 6. In general, those fractions containing
the maximum protein and carbohydrate eluted at the void volume and those
with more than about 0.1 mg protein/ml eluted later (Figures 2 and 3)
provided veiy high titers with antisera from homologously and heterolo
gously infected rabbits. The fraction in tube 108 was not reactive.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ■o o Q. C g Q.
■O CD Table 3 (AC/) O 3 Indirect Hémagglutination Titers with Mycelial Whole Protoplasm of S. schenckii Using Untanned Sheep Erythrocytes CD 8 c5' Source of Antisera Titers to Whole Protoplasm
Rabbits infected with 3 3 " 1 512 CD S. schenckii;
CD ■O 2 O 8,19k Q. C a O Rabbits infected with 3 ■O S, schenckii var. luriei; 3 16,38ii O U 8,192 CD Q. Uninfected rabbits ; Fh 128
"3 5 16 CD
C/) 18 C/î 6k
ro ro CD "D O Q. C 8 Q. Table L
"O Indirect Hémagglutination Titers with Mycelial Whole Protoplasm from £. schenckii, CD and a Sephadex Fraction, Using Tanned Sheep Erythrocytes
C/) 3o ’ O Titers To; 8 Source of Antisera Mycelial Whole Combined Sephadex ci' Protoplasm Tubes 37-^1
Rabbits infected with 3. S. schenckii: 'i 20L8 3 " CD 2 2018^ 20^8
CD Rabbits infected with "D O S. schenckii var. luriei; 3 20U8 ND^ Q. C “ - " ■■ h 20L8 2018 a O 3 ■D Uninfected rabbits; Fit h 0 O S h h 18 0 0 CD Q. 35 8 0 36 I®, 0 13 others <1.'* HD ■D CD 1 C/) C/) Highest dilution tested. 2 Reaction was weak at this dilution.
KD=Not done.
Due to a shortage of sera, several were diluted 1 :J|, therefore 1:14 is the lowest dilution tested. nj Ui 7) TDCD O Q. C g Q. Table S
CD Indirect Hemagglutination Titers with Mycelial Whole Protoplasm from S. schenckii vsr. luriei Using Tanned Sheep Erythrocytes C/) C/) o 5 CD 8 Source of Antisera Titers to Whole Protoplasm
CQ o Rabbits infected with 3 S. schenckii: 1 102k^ CD "n c 2 102U 3"3. CD Rabbit infected with "OCD o S. schenckii var. luriei: k 1021 Q. 1
3 "O Uninfected rabbits : 21 0 O 1—HCT 23 0 CD Q. 1—H$ 3ii 8 o ■O 35 8 CD 3 (/) 50 2 o' 3 1 Highest dilution tested.
ro r î: jle 6
Indirect Hemagglutination Titers with W.iule protoplasm and Sepnadex Fractions of leastlike Cell Protoplasm of S. schenckii Using Tanned Sheep Erythrocytes
Titers To: (/>(/) Source of Antisera Whole Sephadex Fractions in Tubes : CD ProtC'p' asm Q. i 't 56 63 80 83 99 108 113
"O CD Rabbits infected with S. schenckii: 1 ^02h 102ii 20L81 20L8 ND^ 0 0 0 . 0 2 0 0 0 0 Q. 2 3'2 20^8 20^8 20L8 0 C Rabbits infected with g "G3 S. schenckii var. luriei: 3 20li8 20ii8 20ii8 2048 ND 0 NDNDND "O 2 il 2018 20ii8 20ii8 2048 ND 0 0 0 0 Q. 2 Uninfected rabbits ; 5 il 0 0 ND ND ND ND ND ND ■c 22 ND 0 0 ND 0 ND ND ND ND 35 3 32 -==4 ÎJD '=:4 •^4 ^ 4 ■^4 Fil HU ND 0 ND NDND ND ND ND 15 others <"3 ND m ND ND NDND ND ND
1 Highest dilution tested.
^MD=Not done.
3 CO Where ch is indicated, it means that +he serum was diluted 1:ii initially because of a shortage CO
of serum. CD Q. ro "O s3 ~o 2 Q. Q1CD 26
however. Rabbits not infected possessed no or very low titers. Frac
tions Ul, $6, and 63 (Figure 2) produced titers of 102U and 20U8.
Later eluting fractions did not react with antisera from infected rab
bits.
Immunodiffussion. Whole mycelial protoplasm of schenckii re
acted with pooled antisera from two homologously infected rabbits to
form three lines of precipitate (Table 7). An ammonium sulfate precipi
tated antigen from pooled Sephadex tubes 37 to i+1 of this fractionated
protoplasm gave at least two lines of precipitate. With a heterologous
antiserum, five lines of precipitate were obtained with mycelial whole
protoplasm. Pooled Sephadex tubes 37 to Ll from the fractionated my
celial protoplasm produced at least three lines of precipitate to this
heterologous antiserum.
Sephadex fractions of yeastlike whole protoplasm also elicited
precipitin reactions. Using a microtechnique, a line of precipitate
was observed with contents of each of tubes 1^1, 1|.2, and combined tubes
6l to 6S (Table 8).
Leukocyte Adherance Inhibition Test
The procedure of Walter e^ al. (1|3) was not reproducible by
this researcher using schenckii whole cell and protoplasmic antigens.
It was not possible to get the same percent of leukocyte adherance to
a hemacytometer from test to test using leukocytes incubated without
antigen to say nothing of those incubated with antigen.
Skin Testing
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CD ■ D O Q. C g Q.
■D CD
C/) Table 7 C/) Immunodiffusion with Mycelial Whole Protoplasm and Sephadex Fractionation Tubes 37 to U1 of Whole Protoplasm of S. schenckii CD 8
Reaction to;
Source of Antisera Whole Sephadex Ammonium Sulfate 3. Protoplasm Tubes 37-l|1 Precipitate of 3 " CD Sephadex ■DCD Tubes 37-l|1 O Q. C a 3O Pool from two homologously "O infected rabbits *4- + O Rabbit infected with CD Q. S. schenckii var. luriei + ■ f +
■D CD
C/) C/)
ro "OCD O Q. C g Q.
■D CD (/) C/)
CD 8 Table 8
Immunodiffusion with Sephadex Fractions of Whole Protoplasm CD of Yeastlike S. schenckii and a Homologous Antiserum
3. 3 " CD "OCD O Fractionation Tube Numbers ; Q. C a 3O U2 56-60 61-65 66-70 71-75 76-80 82-87 98-99 IOii-108’ 112-113 "O O
+ 4- - + — — "" — ™ — — CD Q.
■D CD
C/) C/)
ro oo 29
s. schenckii whole mycelial cells and mycelial cell walls did
not elicit immediate-type reactions, but did elicit delayed skin reac
tions at 2h hours (Table 9)» .Mycelial whole protoplasm from both S.
schenckii and S_. schenckii var. luriei evoked immediate-type reactions
noticeable at two hours as well as an indurated delayed skin reaction
seen at 2h hours (Table 9). Combined Sephadex fractions in tubes 37 to
as well as an ammonium sulfate precipitated antigen from these frac
tions evoked reactions similar to those of mycelial whole protoplasm
(Table 9).
Results of skin testing Sephadex fractions of yeastlike whole
protoplasm of schenckii are illustrated in Table 10. The fraction
in tube 1|1 and most of the fractions found in the second protein pla
teau of the fractionation curve (Figure 2) elicited immediate-type re
actions as well as delayed skin reactions. Pooled fractions lOU to
108 and 112 to 113 evoked delayed skin reactions in the homologously
infected rabbits, but not in the heterologously infected rabbit.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ■oCD O cQ. 3 Q.
■o CD Table S' C/) o' Z5 Skin Testing Rabbits with Various Components 2, of Mycelial Form S. schenckii CD 8 cB' z Skin Reactions2 at: i =3 Antigens Used pg Protein”' Hours CD Injected u 6 18 U8 72 96
CD ■o Whole merthiolate-killed cells : ND^ ------U ND ND 4-4*4‘ 4- 4- 4- 4- 4- ND ND cI a Cell walls; ND - ND ND + 4-4-4- + + + + ND ND 3o ■o o Whole protoplasm: 72 4* + + + ND ND + + + + + + + 4* ND ND
& Combined Sephadex tubes 37-Ul from whole protoplasm: 38 4- + 4- 4- 4" 4- 4* + ND + + + + + + + + ± + ± 1 — — co Ammoninm sulfate precipitate of Sephadex tubes 37-Ul: 33 4* 4- 4* 4- 4- 4- + + ND + + + + + + + + + + (gC/) o'3 Ammonium sulfate precipitate of Sephadex tubes 37-Ul; 10 ND + + + + + + + + + — ND
2Protein content was determined by spectrophotometric absorbance at 260 and 280 nm (UU). ■^Reactions were considered positive when edema and induration obviously exceeded that in an uninfected control rabbit. ' ND=Wot done. ^The first two reactions represent rabbits infected with S. schenckii while the second two reactions represent rabbits infected with S. schenckii var. luriei, CD ■ D O Q. C g Q. Table 10 ■D CD Skin Tests of Rabbits with wliole Protoplasm and Sephadex
C/) Fractions of Protoplas-^ cf Yeastlike S. schenckii 3o" O Antigens Used M Protein pg Carbohy- Skin Reactions at : 8 Injected drate Hours Injected (O' 2 6 12 18 2h h8 72 96
Whole protoplasm: 27 U6 + + + + + + + 4 4 4 4 4 4 4 4 4 4 ■f ^ + 3 Sephadex fractionation 3 " CD tubes : 111 61 23 ’T + 4- 4" 4 4 4 4 ■f* 4 4 4 4 4 4 — + — —
CD ii2 Ui 1 ■h — — ■D O Pooled Sephadex Q. C fractionation tubes: 56- 60 37 20 + + + + + + 4 4 4 4 44 4 44 4 4 4 4 4 + p- — a o 6l - 65 38 20 + + + + + + 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4- — — — 3 ■D 66- 70 32 13 + + + + + + 4 4 4 4 4 4 4 4 4 4 4 4 4 4 + — — — O 71- 75 25 1Q + + + + + + 4 4 4 4 4 4 4 4 4 4 4 4 4 4 + — — 82- 87 32 + + + + + + 4 4 4 4 4 4 4 4 4 4 4 4 4 4 + w — — CD Q. 98- 99 25 +NI^+ +ND + +ND 4 +m 4 + w 4 4ND 4 -ND — -ND - 1 Oil-108 53 + +. + + + + 4 4 4 4 4 - 4 4 - 4 4 - — — - ” — — + 112-113 23 m y +ND^+ +ND +ND — +ND — +ND - -ND - -ND - —ND - ■D PBS Control: CD
C/) C/) Reactions were considered positive when edema and induration obviously exceeded that from similar injec- g tions into an uninfected control rabbit. The first two reactions represent rabbits I'.' cted with S_. schenckii while the tPdrd reaction represents a rabbit infected with S. schenckii var. Inriei. ^kD^kot done. Sucrose used to suspend who]. : ..toplasm for fractionation was eluted in these last frac tions and prevented carbohydrate determination. There was enough of the pool to do three ra-sits. Lu CHAPTER IV
DISCUSSION
Artificial Infection of Rabbits
Rabbits infected artificially by Intramuscular injection of live
mycelial cells of S^. schenckii and S^. schenckii var. luriei possessed
granulomatous lesions very similar to those described for primary cu
taneous disease humans. In human cutaneous infection there is a combin
ation of granulomatous and pyogenic reactions. The basic lesion of the
granulomatous reaction consists of masses of epithelioid histiocytes
which tend to form concentric zones. The central area typically consists
of necrotic material surrounded by an infiltrate of neutrophils and some
pJasi’ia cells and lymphocytes. Demonstration of fungus in tissue sections
is ry ! tficult (3?). In tissue sections from rabbits artificially in-
r^ ci.ed w - !;i the respective variety, cells of schenckii could not be
found, while cells of S. schenckii var. luriei were profuse. It should
be noted, however, that the rabbit from which the Lurie variety was ob
served had died of an apparently unrelated intestinal disorder, and it
is possible that its immunity was compromised at the time of its death
resulting in a decreased ability to phagocytize the fungus. The pre
sence of strong cellular reactions indicates that an infection resem
bling a natural one was achieved.
When weekly intramuscular injections of live fungus were dis
continued temporarily, subcutaneous nodules that had been palpable
32
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 33
became less palpable as the host's resistance apparently mitigated the
infection. Repeated injections, however, simulated a pronounced, chron
ic, subcutaneous infection. In humans, most cutaneous lesions become
chronic if not treated (32).
Host Resistance to Infection
The fact that a rabbit or a human produces an intense tissue re
sponse, both locally and throughout the adjacent lymphatics, of suffici
ent intensity to prevent the spread of the infection beyond the limb of
injection demonstrates a strong host resistance to schenckii, al
though not strong enough to effect a complete spontaneous cure. Organ
isms are present and multiplying rapidly just after innoculation, but
begin to decrease in number coincident with the inflammatory response
of the host. There seems to be an immunological resistance elicited by
the organism (U8), but current serological methods using existing anti
gens are not efficient at diagnosing cutaneous forms of the infection
and the only skin test antigens used so far have not been able to dis
tinguish between present and past infection.
Skin Testing
Skin testing has limited value as a diagnostic tool because it
fails to distinguish between past or present infection, but it is valu
able as an epidemiological tool in defining endemic areas of disease. A
positive skin test is of diagnostic value only when a negative skin test
has been recorded prior to the onset of clinical symptoms. A negative
reaction is of greater significance because it shows definite absence of
disease. Negative reactions are also observed in very early stages of
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3U
infection, terminal stages, or where there is a defective immune system
(23).
Immediate-type and delayed skin hypersitivities. Immediate-
type skin hypersensitivities are mediated by circulating antibodies.
Arthus reactions, for example, will not occur unless precipitating an
tibodies are present along with complement (9,10). Since these reac
tions are related to antibody production, they are related to serolog
ical techniques and do not offer any diagnostic information that can
not be obtained from sera. Delayed hypersensitivity reactions, on the
other hand, do not depend on circulating antibodies, but are mediated
by activated or sensitized lymphocytes. In immediate-type reactions
there is an accumulation of neutrophils in response to the fixed pre
cipitate and complement, but in delayed-type reactions lymphocytes
and monocytes accumulate (lU). Cell mediated immunity is a different
immune system and forms in response to different, kinds of ant i ijen'^.^.
usually larger molecules than those involved in immediate-type reac
tions and humoral immunity. Antigens effective in cell mediated im
munity often occur on cell surfaces and are often proteins (1U).
Prior research in skin testing for sporotrichosis. One of the
most frequently cited works concerning skin testing for sporotrichosis
is that of Nielsen (29). He fractionated yeastlike cells into 10 cell
wall and protoplasmic fractions which he then tested for their anti
genic potential. He found that guinea pigs sensitized with merthiolate-
killed yeastlike cells or cell walls were not sensitive to any anti
gens he used. Those animals innoculated with viable yeastlike cells
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3 5 were hypersensitive to both whole cells and cell walls, but not reac
tive to protoplasmic components. It should be noted that he heated
the cells at 90° C to inactivate any autolytic enzymes. Naturally
this treatment could also denature other protein molecules that might
be antigenic. At the time of Nielsen's work the predominant thought
was that polysaccharides were Lhe important antigenic component of
2" schenckii, so he was not particularly concerned with preserving
proteins.
If the major antigenic component of schenckii is a polysac
charide -peptide it is possible that the peptide portion could be re
covered along with polysaccharide by extraction procedures aimed at
isolating polysaccharides. This is perhaps the reason for the popular
thought that polysaccharide is the important antigenic component.
Shimonaka e^ ad. (36), of Japan, obtained a crude polysaccharide com
ponent from the defatted mycelial cells of schenckii which was
8 7 .5^ carbohydrates and 12.5^ peptide. Proteolytic treatment of this
fraction removed the ability to evoke a delayed hypersensitivity re
action. They found that immediate-type and precipitin reactions were
prevented by treating the fraction with periodate to degrade the car
bohydrate moiety. Barker e_t ad. similarly isolated a peptide galacto-
mannan from Trichophyton mentagrophytes which gave immediate and de
layed reactions (2). Shimonaka e^ cited Blank and coworkers (36)
who isolated pure galactoraannans from mentagrophytes which were
active in complement fixation and precipitin tests, but had no ca
pacity to elicit delayed skin reactions. Cano and Taylor (7) had
success in skin testing infected rabbits with partially purified
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 36
protoplasmic proteins of Chrysosporium parvum and C_. parvum var.
crescens. From these reports it appears that destruction of peptides
is a logical reason for loss of ability to evoke delayed skin reactions.
Skin testing results of the present study. The present study
confirm Nielsen's finding that whole yeastlike cells and yeastlike cell
walls are capable of producing delayed skin reactions. They did not
produce immediate-like reactions (Table 9). Whole protoplasm and Sepha
dex G-200 fractions thereof are capable of producing both immediate-
type and delayed-type skin reactions. The Sephadex fractions which
elicited skin reactions were those which contained moderate amounts of
protein (Table 10 and Figure 2). It is particularly interesting that
two Sephadex fractions, tubes 10J4. to 108 and tubes 112 to 113 , which
were protein peaks in the later elution pattern of the elution of yeast
like cell protoplasm of schenckii evoked delayed-type skin reac
tions only in homologously infected rabbits. It may be that these
fractions possess an antigen unique to S^. schenckii and not found in
S. schenckii var. luriei. Since only one rabbit infected with the
Lurie variety was tested, further testing is warranted before conclu
sions can be made.
While skin testing is valuable as an epidemiological tool, it
will not be a valuable diagnostic tool unless past and present infec
tions can be differentiated. Serology is necessarily studied, then, in
an effort to obtain a sensitive diagnostic method.
Serological Testing
Tube agglutination with yeastlike cells. The commonly used
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 37 whole yeastlike cell antigen in tube agglutination tests has been
shown to be specific (5, 21, 32, i;6). But, while it is accurate in
diagnosing disseminated, articular, and pulmonary forms of the disease,
it often fails to detect cutaneous forms. Blumer et (5) reported
that they were able to detect 53 of 55 previously known cases of local
ized cutaneous-subcutaneous sporotrichosis, but they accepted a titer
of 16 as positive. Karlin and Nielsen (21) found four of 1U cutaneous-
ly infected patients with titers of 20 or lower. Roberts and Larsh (3U)
reported eight of 18 cases of cutaneous disease had no titer and two
more had titers of only 20. However, Welsh and Dolan (^6 ) reported
that some patients without sporotrichosis exhibited agglutination titers
titers of 20 and UO. They concluded that only titers of 160 or higher
are diagnostic and that a titer of 80 might be presumptive, but a titer
of UO or less should be considered only supportive evidence of sporo-
trichotic infection. Similarly, Karlin and Nielsen concluded that a
titer of LO or less should be used with caution unless circumstances
exist which might knowingly influence the amount of antibody present.
Thus, although specific, yeastlike whole cell antigen is not suffi
ciently sensitive clinically.
Using the method of Welsh and Dolan, agglutination titers of
160 and 320 were demonstrated in infected rabbits, while no titer was
detected in an uninfected rabbit. As might be expected, cell walls
reacted similarly to whole cells in these agglutination reactions.
Although the titers would be significant in human infections (L6 ),
they nevertheless are not striking.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 38
Soluble antigens used previously in serological testing. In
searching for a serological antigen that is as specific, yet more sen
sitive than whole cells and, therefore, better able to detect cutan
eous forms of sporotrichosis, several investigators have looked to cul
ture filtrate antigens of both yeastlike and mycelial cells of
schenckii (5, 21, 2k) • Neither immunodiffusion, nor complement fixa
tion has proved to be very sensitive using culture filtrate antigens
(5, 21). Using immunodiffusion Blumer et al. (5) have been able to
detect only 23 of 55 cases of cutaneous sporotrichosis and Karlin and
Nielsen reported only ’J0% to 80^ accuracy (21).
Whole protoplasm as antigen. Roberts and Larsh (3U) investi
gated whole protoplasm from yeastlike cells as an antigen in immunodif
fusion and complement fixation tests. Of 18 cases of cutaneous infec
tion, four were reactive in complement fixations and only two in im
munodiffusions. They freed whole protoplasm from yeastlike cells by
submitting a 20^ suspension to four hours of treatment by an ultra
sonic probe. (in the present research an ultrasonic probe was not
found to be an effective way of breaking cells of S. schenckii.) It is
possible that Roberts and Larsh did not have a sufficient antigen since
they did not concentrate the supernatant fluid after the cell frag
ments were removed. Tables 7 and 8 show that whole protoplasm as well
as Sephadex G-200 fractions were precipitated in immunodiffusion assays
of immune rabbit sera. Although not investigated here, it is probable
that more refined fractions of whole protoplasm may be more sensitive
than whole protoplasm, and crude culture filtrates presently used
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 39
clinically in immunodiffusion.
Slide latex agglutination test. At present, one of the most
widely respected serological methods for diagnosing sporotrichosis is
the slide latex agglutination using culture filtrate of yeastlike cells
(2U)- A titer of U is considered presumptive evidence for sporotrich
osis. In the present study whole mycelial protoplasm and the Sephadex
fraction, in tubes 37 to Ll, of this protoplasm were tested as anti
gens fo- slide latex agglutinations. The Sephadex fraction gave slight
ly higher titers than whole protoplasm (Table 3), but the maximum titer
obtained was only 6U. Since titers of k to 128 are expected from in
fected humans, the whole protoplasm and the Sephadex fraction are
equivalent to culture filtrates as antigens using this method. Slide
latex agglutinations pose technical problems occasionally, however.
Polystyrene particles used cannot be washed after they are sensitized.
It is therefore necessary to leave the particles in the antigen sus
pension used to sensitize them. If too much antigen is used for sen
sitizing the latex particle 3 there will be too much free antigen to tie
up antibodies and no agglutination of latex particles will occur. Con
sequently one must adjust antigen concentrations carefully (16).
IndlrecL hemagglutination. Unlike polystyrene particles, eryth
rocytes can adsorb antigen and retain it after being washed. It is
therefore possible to add antigen just short of the point where non
specific or spontaneous agglutination occurs. This seems to be an ad
vantage over latex agglutination. No reports were discovered of in
vestigations of indirect hemagglutination as a diagnostic tool for
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UO
sporotrichosis.
Indirect hemagglutination using untanned sheep erythrocytes
and whole protoplasm of mycelial cells yielded very high titers
(Table 3), but some uninfected rabbits also gave titers up to 128.
One infected rabbit produced a titer that was only two 2-fold dilu
tions greater than that obtained from an uninfected rabbit.
Untanned erythrocytes bind antigens of various molecular spe
cies, but generally do not bind protein (6 ). This suggests that anti
gens that attach to them are something other than protein or are
other molecules complexed with protein. If, for instance, a polysac-
charide-peptide were antigenic, the polysaccharide portion of the
molecule could attach to the erythrocyte and leave the peptide por
tion exposed to attach to antibodies. Therefore, the observation that
an antigen can be adsorbed to untanned erythrocytes does not rule out
the possibility that a peptide plays a part in antigenicity.
Indirect hemagglutination with tanned sheep erythrocytes.
Most techniques of tanning erythrocytes are based on the work of
Boyden who pioneered the technique of indirect hemagglutination with
tanned erythrocytes to adsorb protein antigens (6 ). Using tuberculin
PPD (purified protein derivitivej, he compared indirect hemagglutination
using the direct sensitization technique and that of sensitizing tan
ned erythrocytes. Tuberculin PPD contained S% polysaccharide, 1% nu
cleic acids, and 88 ^ protein. In general, Boyden found that sera that
were positive by the direct sensitization technique gave higher titers
when erythrocytes were tanned first. In addition, he found a lack of
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correlation between the two methods ;
The lack of correlation between the agglutination titers obtained by these two methods suggests that antibodies of more than one kind are involved, perhaps directed against different antigens or differing in some of their other properties, although directed against the same antigen.
There appears to be a different antigen-antibody system involved when
using erythrocytes sensitized directly than when erythrocytes are tan
ned prior to sensitization.
In this study, sheep erythrocytes were formalinized to save
time washing and standardizing cells. A constant source of cells re
moves the variable introduced by different lots and dilutions from
test to test. It has been reported that formalinized erythrocytes
can be sensitized and remain stable for at least 60 days (18) and
even for six months (30). Furthermore, formalinized cells are more
resistant to mechanical injury and are resistant to complement and
hypotonic salt solutions. They are also more spherical than non-
formalinized cells and therefore more apt to form a compact button
when agglutinated (1 2 ).
Indirect hemagglutinations with tanned erythrocytes gave high
titers with infected rabbit sera and low titers with sera from unin
fected rabbits when whole protoplasms and Sephadex G-200 fractions
of whole protoplasms were used as antigens (Tables h and 6 ). Titers
could often be read in as few as 1^ hours. As with all serological
methods tested, certain Sephadex G-200 fractions of whole protoplasms
were as effective as whole protoplasm and may prove to be more advan
tageous as serological antigens because they have perhaps lost compo
nents not unique to S^. schenckii which might be cross-reactive with
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antibodies to other fungi.
Using both slide latex agglutination and indirect hemagglutin
ation with untanned sheep erythrocytes there was nonspecific agglutin
ation by uninfected rabbits. This problem was overcome with indirect
hemagglutination using tanned sheep erythrocytes. This is definitely
the most promising of all methods tested in this work.
Similar Antigenicity with Yeastlike and Mycelial Cells
Results from neither serological, nor skin tests revealed any
antigenic differences between yeastlike and mycelial cells of
schenckii. Landay et showed with Coccidioides immitis that diag
nostic antigens may be produced by one morphological phase of a given
species but not by another morphological phase of the same species (25).
That does not appear to be the case with S. schenckii. It can be dem
onstrated that conidia when grown with shaking grow short sympodulae
which then bear secondary sympodulospores (39). It is possible that
"yeastlike" cells are actually conidial cells and that "yeastlike"
cells and conidia differ in size only because of differences in growth
conditions (l+O). If this is true then mycelial cells with a reasonable
amount of conidia should be antigenically similar to "yeastlike" cells
because they are the same.
Similar Antigenicity with d. schenckii and S. schenckii var. 'uriei
With the possible exception of Sephadex G-200 fractions in tubes
101+ to 108 and 112 to 113 of yeastlike cell whole protoplasm of
schenckii when used as skin test antigens, there was no antigenic dif
ference observed between S. schenckii and _S. schenckii var. luriei in
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. U3 either serological or skin tests. This is in general agreement with
Karlin and Nielsen (21), who tested 20 different isolates of
schenckii and found no siginificant differences in antigenicity.
Protein Important for Antigenicity
Sephadex G-200 fractions of whole protoplasm of S_. schenckii
which contained relatively large quantities of protein were found to
react as well as the whole protoplasm from which they were derived in
both serological and delayed-type hypersensitivity skin tests. In
preparing skin test inoculations, protein was the one component that
had to be present in certain quantities to effect a skin reaction.
Protein appears to be an essential component in the antigenicity of
whole protoplasm, but other components, such as polysaccharide, may be
necessary in minimal quantities for reactions to take place.
Directions for the Future
Obviously further work is needed to describe what molecules
are antigenic, but fractionation of whole protoplasm appears to be a
reasonable starting point. Indirect hemagglutination with tanned
erythrocytes has been shown to be the most sensitive tool to use in
measuring the potential of antigens. This points to protein as the
best starting point for further fractionation.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER V
SUMMARY
Iminmioserologic diagnosis of sporotrichosis in humans as well
as in other mammalian species is presently based on recommended proce
dures leading to positive complement fixation, precipitation, agglutin
ation, and/or skin test reactions. Although serological procedures
have been found to be of some value in the laboratory diagnosis of non-
cutaneous forms of sporotrichosis, the more common cutaneous and lymph-
ocutaneous cases are usually serologically negative, or, less often,
react within apparently normal ranges. Among the antigens used for
serological diagnostic purposes have been whole cells and cell extracts
of S^. schenckii. Whole cell protoplasm has shown promise for diagnosing
more severe but less common infections, but has failed to react in tests
for inapparent, atypical, and common cutaneous forms of infection. Skin
testing, because it has not been able to differentiate present infection
from past exposure to schenckii using whole yeastlike cell antigens,
is of more epidemiological value than diagnostic.
The purpose of this study was to prepare and fractionate whole
cell protoplasm from yeastlike and mycelial cells of schenckii by
Sephadex G-200 chromatography and to investigate the diagnostic poten
tials of such antigens with laboratory anin.als realistically infected
with schenckii as experimental models. Two rabbits were infected
intramuscularly with schenckii and two with S. schenckii var. luriei.
UU
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The infections and immune responses of these animals were studied com
paratively. Cell protoplasms or protoplasmic fractions were used as
antigens in various serological procedures including immunodiffusion,
tube agglutination, slide latex agglutination, indirect hemagglutin
ation with formalinized and untanned sheep erythrocytes, and indirect
hemagglutination with formalinized and tanned sheep erythrocytes. A
leukocyte adherence inhibition test and skin testing were employed to
study the potential of these antigens to elicit cell-mediated reactions.
Both whole protoplasms from either mycelial or yeastlike S^.
schenckii cells and certain fractions of protoplasms isolated by
Sephadex G-200 chromatography were found to be equivalent to or some
what more sensitive antigens in detecting sporotrichic infections in
rabbits when recommended sérodiagnostic methods were used. However,
when pr.. toplasm or protoplasmic fractions were used as antigens in
indirect hemagglutination reactions with formalinized and tanned sheep
erythrocytes, titers of at least 20U8 wei-e demonstrated in infected
animals while those in uninfected animals did not exceed 8. The test
appears to be as specific as presently recommended methods, but is far
more sensitive. The most useful protoplasmic fractions were those with
relatively more protein. Two slowly eluting components demonstrated
high varietal specificity in delayed skin tests by eliciting positive
reactions in animals infected with the homologous variety but not in
those infected with the heterologous variety. No antigenic differences
were detected between the mycelial and the yeastlike forms of
schenckii.
Further research should proceed in the direction of fractionation
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o f protoplasmic proteins. Indirect hemagglutination with tanned eryth
rocytes is the most useful tool to employ in studying the potential of
more refined proteins of schenckii. Sephadex fractionation of whole
protoplasm has yielded fractions that certainly warrant testing with
sera from human patients suspected of possessing sporotrichic infection.
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