      
   

Ml

WWWWW

§§ WWIIWHHIIHH‘HIH

.THS

I007

LIBRARY
Michigan State
Universrty

 

 

——i

This is to certify that the
thesis entitled

MORPHOLOGICAL AND ANTIGENIC STUDIES OF
PYTHIUM INSIDIOSUM HYPHAE USING TRANSMISSION
ELECTRON MICROSCOPY

presented by

RICHARD BENJAMIN GARCIA

has been accepted towards fulﬁllment
of the requirements for the

MASTER OF degree in BIO-MEDICAL LABORATORY

SCIENCE DIAGNOSTICS

 

 

I
/

“Major Professor‘s-Signature
9/ 20/ 0 a

Date

MSU is an Afﬁrmative Action/Equal Opportunity Institution

--------------0-.-n-c-.-o—u-o--u-D-o-o---o-o-o-o-I-0---o-a‘0-o-n-o-o---0-.-o-.---r-o-a-n-u---'-o--n--c-I.

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2/05 p:/ClRC/DateDue.indd-p.1

MORPHOLOGICAL AND ANTIGENIC STUDIES OF PYTHIUM INSIDIOSUM
HYPHAE BY TRANSMISSION ELECTRON MICROSCOPY

By

Richard Benjamin Garcia

A THESIS
Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of
MASTER OF SCIENCE
Bio-Medical Laboratory Diagnostics

2006

ABSTRACT

Morphological and Antigenic Studies of Pythium insidiosum Hyphae by
Transmission Electron Microscopy

BY

Richard Benjamin Garcia

For the past century Pythium insidiosum, the etiologic agent of pythiosis.
has been reported causing disease in both humans and animals. While many
diagnostic tests and immunotherapeutic treatments have been developed to
diagnose this parafungal pathogen, the location and characterization of the
antigens expressed during infection have yet to be described. We have mapped
the antigens present in the hyphae of P. insidiosum using an immuno-electron
microscopic approach. During this study new morphological features of the
hyphal structures of P. insidiosum were also described using transmission
electron microscopy. The immuno-electron microscopy data using sera from
infected hosts (bovine, canine, equine, feline, and human) and Protein A colloidal
gold (PACG), shows the speciﬁc binding of protein A colloidal gold particles to
antigens within P. insidiosum’s hyphae only with sera from cows, horse, and
humans with pythiosis. Sera from infected dogs and cats did not show speciﬁc
binding of the gold particles to P. insidiosum’s hyphal structures. The ﬁnding of
novel antigenic components in the cell wall and the cytosol of P. insidiosum’s
hyphae is of paramount importance for the future characterization of these
proteisns to understand the immunopathology of the disease, and for the use of

better immunogens for the immunotherapy of pythiosis.

Dedication

I would like to dedicate this thesis to the faculty of the Medical Technology
Department, my family and friends who supported me through my education at
Michigan State University, and especially Dr. Mendoza for his enthusiasm and
dedication in the parafungal sciences.

ACKNOWLEDGMENTS

I would like to express gratitude to my major advisor, Dr. Leonel Mendoza,
for his guidance, encouragement, and support in my degree program. I would
also like to express gratitude to Dr. Alicia Pastor and Dr. Thomas Schmidt for
serving on my guidance committee and all the helpful meetings and suggestions
for my research. Finally, I would like to thank the Medical Technology Program

faculty and staff for their assistance.

iv

TABLE OF CONTENTS

LIST OF FIGURES ................................................................................. vi

INTRODUCTION .................................................................................... 1

CHAPTER 1

LITERATURE REVIEW ........................................................................... 3
History ........................................................................................ 3
Life Cycle .................................................................................... 4
Taxonomy and Other Characteristics of Pythium insidiosum... ......7
Epidemiology and Distribution ................................................................ 8
Clinical Disease ............................................................................. 9
Human ....................................................................................... 9
Animal” .. .. 10
Diagnosis and SerologIcal Testing" ................................................... 12
Host-Parasite Relationship ............................................................ 14
Treatment .............................................................................................. 16
Introduction to Transmission Electron Microscopy .................................. 17

CHAPTER 2

MATERIALS AND METHODS ................................................................ 19
Strain, Culture, and Sample Strategies ............................................ 19
Transmission Electron Microscopy (T EM) ......................................... 20
Sample Preparation... 20
Sample Preparation for MorphologIcal AnalySIs ....................................... 20
Sample Preparation for Immuno labeling Analysis ................................. 21
Sectioning Procedures .............................................................................. 22
Thick Sectioning ........................................................................................ 23
Thin Sectioning ......................................................................................... 24
Immuno-labeling .......................................................................... 24
Transmission Electron Microscopy Evaluation ......................................... 26

CHAPTER 3

RESULTS ........................................................................................... 27
Electron Microscopic Features of Pythium insidiosum Hyphae .............. 27
Immuno — labeling of Pythium insidiosum’s Hyphal Antigens ................. 37

CHAPTER 4

DISCUSSION ...................................................................................... 68

BIBLIOGRAPHY .................................................................................. 74

LIST OF FIGURES

Figure 1. Life Cycle of Pythium insidiosum .................................................. 6
Figure 2. Distribution of Pythium lnsidiosum ................................................ 9
Figure 3. Orbital Pythiosis ...................................................................... 11
Figure 4. Cutaneous Lesions .................................................................. 11
Figures 5-12. Electron Micrograph of Stained Pythium insidiosum Hyphae ....... 29
Figures 13-15. Negative Control .............................................................. 40
Figure 16. Speciﬁc Binding of Protien A Colloidal Gold Particles ..................... 43
Figures 17. Immuno Feline and Canine ...................................................... 44
Figures 28-26. Immuno Labeling Bovine and Dead Hyphae ......................... 51
Figures 27-30. Immuno Labeling Human ............................................................ 53
Figures 31-33. Immuno Labeling Equine ................................................... 58
Figures 34-41. Immuno Labeling Equine with Long Hyphae ........................... 60

vi

INTRODUCTION

De Haan and Hoogkamer ﬁrst isolated Pythium insidiosum, the etiological
agent of the para-fungal disease pythiosis, in 1901. For decades the treatment
of pythiosis has been limited because of the obscure nature of this pathogen.
Until recently, surgical removal of the infected tissues was the traditional method
of treatment. However, the development of alternative ways for the management
of this infection has been proposed including immunotherapy (Miller 1981). This
novel approach used exo- and endo proteins antigens of Pythium insidiosum
injected in the infected hosts. While this immunotherapeutic approach proved to
be effective and was able to cure chronic and acute cases of the disease,
immunotherapy showed differences in cure rate between host species. Further
more, little is known about the nature of the antigens used for immunotherapy
(Mendoza and Newton 2005).

Different antigens and serological assays have been used and developed
to diagnose and differentiate pythiosis from similar clinical entities (Miller and
Campbell 1982; Hutchens and Mendoza 2002). It is known that an exoantigen is
released from the hyphae of P. insidiosum during infection (Mendoza et al.
1987). However, this antigen has never been characterized. The exoantigens
released during infection seem to trigger a T helper 2 (Th2) response in the
infected hosts, with a coordinated cascade of inﬂammatory cells and cell
mediator proteins, mostly eosinophils, mast cells and lNFv, IL5, and IL4. The

display of these inﬂammatory components, including eosinophil and mass cell

degranulation, are extremely destructive to the host tissue (Miller 1981; Mendoza
et al. 1992b). Since nothing is known about the nature of the antigens expressed
during infection and their possible location in the hyphal compartments, thus this
study has the purpose of mapping those antigens in the hyphae of P. insidiosum
using transmission electron microscopy, immunogold reagent, and sera from
patients with proven pythiosis. The location of the in vivo antigens expressed by
Pythium insidiosum’s hyphae, could answer questions about the immunogens
expressed during infection, which in turn could help in the characterization of

these important immuno—dominant proteins.

CHAPTER ONE

Literature Review

History

During the 1800's unusual cases of horses with cutaneous lesions were
documented from several tropical and subtropical areas of the world (Australia,
Florida, India, and Indonesia). In 1884, Smith ﬁrst published a well-documented
report on pythiosis. Shortly after Fish (1895-96), and Drouin (1896) also
documented new cases of cutaneous granulomas in equines. The etiologic
agent of pythiosis was isolated for the ﬁrst time by Haan and Hoogkamer in 1901
from several horses with the disease, but it was not named. In 1925, J. Witkamp
introduced several serological methods to diagnose the disease. This initial
detailed report also included information on the treatment and the progression of
the disease, later termed pythiosis. Because the report was published in the
Dutch language, this information went unnoticed (Mendoza 2005).

In the early 20th century, Habronema, a pathogenic nematode with clinical
features in common with pythiosis, was incriminated as the etiologic agent of the
cutaneous granulomas in horses. The clinical similarities between
habronemiasis and pythiosis led to some confusion on the real etiology of
pythiosis. Thus the terms used to describe pythiosis were also indiscriminately
used for equine cutaneous habronemiasis. In 1961 Bridges and Emmons
isolated a ﬁlamentous organism from horses in Texas that they believed to be a

zygomycete. They named the organism Hyphomyces destruens. Austwick and

Copland changed this name into the genus Pythium in 1974, after reporting the
ability of the organism to produce biﬂagellate zoospores when transferred to
water media. However, no scientiﬁc name was proposed. In 1987, de Cook et
al., introduced the specie P. insidiosum, and indicated that many of the proposed
epithets in the past belong to the same species. Recently, Schurko et al. (2003),
using molecular tools conﬁrmed that P. insidiosum is the only etiologic agent in
the genus causing pythiosis in mammals.

Numerous new cases of pythiosis have been reported in humans, horses,
cats, cattle, and dogs from around the world. For instance, in 2005 the ﬁrst
human case of pythiosis was reported in Brazil and the ﬁrst case from Africa in a

dog with pythiosis (de Moraes et al. 2005; Rivierre et al. 2005).

Life Cycle

P. insidiosum inhabits aquatic environments and may require symbiotic
relationship with plants to complete its life cycle in nature (Mendoza et al.1992b;
Shipton et al. 1982). P. insidiosum seems to infect plants inhabiting aquatic
environments. The plant tissue serves as a reservoir for the development of
sporangia and zoospores, which are then released into the surrounding
environment. After the release of zoospores, they encyst developing germ tubes
that reinitiate the life cycle by infecting another plant, or animal tissue (Figure 1).
P. insidiosum has the ability to develop resistant spores that could play a major
role on its capacity to survive in harsh environments. The motile zoospores are

believed to be attracted by injured tissue in susceptible plants or animals. When

chemo-tactically stimulated by a suitable substrate, the zoospores secrete an
amorphous sticky material, which covers their surface, which is used to attach it
to the plant or animal's surface. This substance, which is believed to be a
glycoprotein, has been implicated as a potential virulence factor in establishing
infection (Mendoza et al. 1993). Once attached to the tissue, the zoospores
encyst and developed a germ tube (hypha) that mechanically penetrates

traumatic lesions causing pythiosis in the infected host (Mendoza 2005).

Nature

 

Figure 1: The life cycle of Pythium insidiosum in nature ,(a-e, k, in upper panel). Plant
tissue is ﬁrst colonized by (a) hyphae of P. insidiosum, and then (b-d) the differentiation
of sporangium into mature stages leads to (e) zoospore release. (f) The zoospores swim
to locate another plant or may be attracted by injured animal tissue. The encysted
zoospores (g) were attached to tissue by a sticky substance (circle of dashes). The
zoospores then germinate (h), invade the host (i) and cause pythiosis, (j) histopathology
showing hyphae.

Taxonomy and Other Characteristics of Pythium insldlosum

P. insidiosum has been placed under the new kingdom Straminipila, class
Oomycetes, order Pythiales . The straminipilans, previously classiﬁed in the
Kingdom Fungi, are now considered to be advanced protistal microbes
phylogenetically similar to plants and algae (Dick 2001). The morphological and
genetic features of P. insidiosum are consistent with these ﬁndings.

BELLE P. insidiosum in culture and in the infected tissues develops
sparsely septate hyphae similar to the members of the fungi. This is why many
frequently confuse P. insidiosum with the pathogenic fungi, especially with the
organisms from the order Zygomycetes. This parafungal organism produces
sparsely septated hyphae, which range in diameter between 4 and 10qm with
many tangent branches stretching off the main hyphae. There are usually
enlarged regions of hyphae growth observed (12-28qm in diameter) in culture,
termed apresorium (de Cock et al., 1987).

Zoospores. Cultures incubated in water with some ions, including Ca++ at
28—37°C are able to develop the production of zoosporangia containing
zoospores. The hyphae produce sporangia that, under some cultural conditions,
could form a vesicle (sporangium) where cytoplasm ﬂows from the hypha. At
maturity, the zoospores mechanically break the vesicle’s wall and upon
emergence swim for about 20 minutes and then encyst. The zoospores are
biﬂagellate. The ﬂagella arise from the same point in the zoospore and are

unequal in length. One of the ﬂagella has hair like structures (tinsel type) the

other is of ‘whiplash' type without the hair-like structures. Following encystment
the ﬂagella are detached and the zoospore becomes spherical (cyst), producing
a sticky material covering the entire surface of the cyst. P. insidiosum uses this
strategy for the attachment of the cyst to the host surface (plant or animal tissue).
This phenomenon has also been well documented in plants (Mendoza et al.
1993).

Oogonium. In contrast to other species of the genus, only a few strains of
P. insidiosum have been reported to produce oogonia. Thus, the mechanism
implicated in the process of oogonial formation in P. insidiosum remains obscure
(Mendoza 2005). It is believe that the oogonium plays the role of the resistant
stage in the members of the genus Pythium. Thus, this could be an important

feature for survival during extremely dry conditions in the endemic areas.

Epidemiology and Distribution

While many Pythium sp. are primarily plant pathogens found in soils and
stagnant waters, only P. insidiosum has been reported infecting animals. P.
insidiosum ﬂourishes in the summer months after large amounts of rainfall.
Horses that have contracted pythiosis were usually in contact for long periods of
time in stagnant water. However, some cases in humans, dogs, and horses with
pythiosis never exposed to stagnant water, have suggested that the infection
may be contracted from soil or grass containing the etiologic agent as well.

Rainfall increases stagnant water accumulation, which increases the

number of cases of pythiosis, this fact has been used to name the disease as:

burusattee (meaning rain in India), swamp cancer, leeches, and summer sores.
Pythiosis has been usually reported in most tropical and subtropical areas of the
world (Figure 2). Although unusual, cases in temperate climates, such as Japan

and the United states, have been also reported (Mendoza 2005).

 

 

 

 

 

Figure 2: Distribution of Pythium insidiosum reported throughout the world. So far,
a single case of pythiosis in Africa (Mali) has been reported.

Clinical Disease: Humans

Pythiosis of humans in Australia, Brazil, Haiti, New Zealand, Thailand,
and the United States were all associated with hosts visiting aquatic
environments. Open wounds, thalassemia, and occupational involvement in wet
environments, have been suggested as predisposing factors of pythiosis. So far
the majority of cases of human pythiosis have occurred in Thailand, with more
than 90 new cases reported since the ﬁrst report in 1987 (Mendoza 2005). There
seems to be a correlation between the clinical forms of human pythiosis

observed and the parts of the world where the disease was contracted. For

instance, in Thailand most infections are initiated as dry gangrenous lesions with
pain of the infected subcutaneous tissues, especially on the extremities, with
subsequent dissemination to the nearby arteries. In the United States, orbital
pythiosis in young boys have been recorded (Figure 3) (Shenep et al. 1998).
Similar observations were reported in Australia (T riscott et al., 1993). Other
clinical forms of the disease include, periorbital swellings developing into orbital
tumor, subcutaneous pythiosis, and keratitis (Triscott et al., 1993; Virgile et al.,
1993). Mendoza and Newton (2005) described three main forms of pythiosis: (1)
a superﬁcial pythiosis (keratitis), (2) a granulomatous and ulcerative lesions
involving the skin and subcutaneous tissues of the limbs and face, including the
periorbital area, and (3) a systemic form with vascular involvement leading

vasculitis, thrombosis, aneurysms, and death.

Clinical Disease: Animals

Pythiosis has been reported in different animals from the endemic areas of
the world with new cases reported every year. Horses, dogs, cats, cattle, sheep,
a camel, and even a jaguar have all been reported with active pythiosis. Horses
and dogs are the most frequently affected animals. Horses usually develop
cutaneous and subcutaneous skin lesions in the extremities. Dogs develop a
gastrointestinal form of the disease as well as skin lesions (Figure 4). In addition,
cases involving lungs, bones, intestines, and the nasal cavity have been reported

in different animals (Mendoza and Newton 2005).

10

 

 

Figure 4: Cutaneous lesions in dogs with pythiosis.

Diagnosis and Serological Testing

Early in the 20th century several immuno-assays were developed to
diagnose pythiosis in horses. This initial study demonstrated that there were
speciﬁc antibodies produced against P. insidiosum hyphae. The serological
tests, immunodiffusion (ID), compliment ﬁxation (CF), and skin test, designed to
diagnose pythiosis went largely unknown because the ﬁndings were published in
Dutch, and confusion with reactions involving similar diseases.

Miller and Campbell (1982) developed speciﬁc antigens to diagnose
pythiosis in horses based on Witkamp’s ﬁndings. Miller and Campbell (1982)
also reported that select antigens from P. insidiosum were beneﬁcial for the cure
of some infected horses. Three antigens were used for the ID, CF, and skin test.
A trypsinized antigen for the ID test detected 100% of horses with pythiosis. A
freeze-thawed antigen for CF test detected 82% of infected horses. Finally a
soluble precipitated antigen for skin test detected 62% of horses studied. After
treatment and resolve of the disease, Miller and Campbell (1982) reported that
anti-P. insidiosum immunoglobulins could not be detected by the ID test in
horses. However, CF and the skin test did detect the anti-P insidiosum
immunoglobulins after resolve of infection. Later, evidence of cross-reaction with
the antigens was reported with Basidiobolus haptosporus. Other ID tests using
different antigens were developed over the years, but it soon became evident

that the ID test was not always effective in other animals with pythiosis. A more

12

sensitive assay was needed for the diagnosis of pythiosis. Mendoza el al. 2003,
developed a ﬂuorescent-antibody assay that was speciﬁc for the hyphae of only
P. insidiosum, that did not cross react with any other similar organism. Other
speciﬁc tests like the indirect immuno-peroxidase assay were developed to
speciﬁcally detect P. insidiosum in tissue (Brown et al. 1988). The immunoblot
test used for diagnosis was also used to observe the antigens of P. insidiosum
detected by host antibodies (Mendoza et al. 1992a). The ELISA test was used to
diagnose pythiosis because of the sensitivity and speciﬁcity of the test, which
many of the previous tests lacked. Humoral responses to pythiosis hyphae were
also observed using the ELISA test (Mendoza et al. 1997; Rosa 1993). An onsite
assay, called the latex agglutination test, was recently developed for rapid
diagnosis of pythiosis to assist farmers’ diagnoses of pythiosis (Hutchens and
Mendoza 2002). These immuno-assays have contributed to the proper diagnosis
of pythiosis. However, medical personnel familiar with this pathogen have to be
trained to further improve the diagnosis of this disease. Because the diagnosis
of pythisois is difﬁcult, it is possible that uncommon clinical manifestations, such
as ocular pythiosis, may have been under diagnosed. The disease has high
morbidity, as it is evident by the many amputation or enucleation cases among
the patients at Ramathibodi Hospital with active pythiosis (Krajaejun et al. 2004).
Thus, early detection and an effective treatment (surgery, immunotherapy,

chemotherapy) are key for successful management.

13

Host-Parasite Relationship

The immune response to pythiosis has been studied in a rabbit model
(Miller and Campbell 1983). When rabbits were administered a suspension of
motile zoospores of Pythium sp. isolated from a horse in Queensland by
subcutaneous, intraperitoneal, and intravenous routes they developed clinical
signs of pythiosis. Some of the rabbits were cortisone treated, but they
developed the same infection to that in the control group. It has been suggested
that one of the predisposing factors towards infection of opportunistic fungi is
decrease of the hosts’ immune response (Rippon, 1982). Cortisone treated and
immuno-competent rabbits both were highly susceptible to P. insidiosum from all
routes of inoculation. The histopathology of the skin lesions of the rabbits
administered subcutaneously showed acute necrosis, with eosinopils and fungal
hyphae in abundance, just like the changes observed during natural infection.

P. insidiosum cannot penetrate normal animal skin. However, an open
wound could provide entrance for the development of pythiosis. The entrance of
the pathogen initiates a humoral immune response involving a massive attack of
eosinophils, giant cells, mast cells, macrophages, plasma cells, and
lymphocytes. This inﬂammatory reaction is also similar to that triggered by
different fungal pathogens including Conidiobolus and Basidiobolus (Mendoza
2005). The ‘Kunkers’, a mass of necrotic tissue caused by massive

degranulation of eosinophils over the hyphae of P. insidiosum, differentiate

14

pythiosis from similar fungal infections. It is interesting to note that the hyphal
elements of P. insidiosum are usually found in the center of eosinophilic micro-
abscesses. This feature has been used by some investigators to postulate that a
Th2 subset is probably activated by the immunogens expressed by P. insidiosum
in the infected host (Mendoza & Newton 2005). Eosinophilic degranulation is the
major factor in the pathogenesis of the disease. However, mast cells and other
immunological mediators also contribute to tissue damage. The up-regulation of
the inﬂammatory cells leads to eosinophilic degranulation that is responsible for
tissue damage during pythiosis in animals and humans. Wachiwanawin et al.,
1993 investigated some of the inﬂammatory regulators in a human with arterial
pythiosis. They found that interleukin-4 (IL-4) and lL-5 were elevated, supporting
the hypothesis that the immune system mounts a Th2 response against P.
insidiosum during natural infection. This study suggested the rise of lgE levels,
responsible for eosinophilic response, conﬁrm the postulated Th2 response to
pythiosis infection.

It is believed that P. insidiosum expresses a soluble exoantigen during
infection that might be responsible for the Th2 response. In 1987 Mendoza el al.
conﬁrmed the expression of an antigen released outside the cytoplasmic
compartment of P. insidiosum hyphae. Using ﬂuorescent antibodies, these
investigators found that the ‘kunkers’ present in the tissue of horses with
pythiosis ﬂuoresced, indicating that an antigen, detected by labeled anti-P.
insidiosum immunoglobulin, had been released from the hyphae. Apparently,

some of the antigen is retained in the ‘kunker’, from where it is released to the

15

surrounding tissues and then presented to local dendritic cells (Mendoza et al.
1987)

The pathogen is always presenting this antigen to the host dendritic cells
resulting in high levels of lgE, which in turn triggers tropisms of eosinophils and
mast cells to the site of infection, locking the immune system into a Th2
response. This immune response enables the pathogen to hide inside the
inﬂammatory responses restricting certain antigens from being fully presented to
the host immune system. The degranulation of eosinophils and mast cells are
responsible for the clinical and pathological changes observed during infection.
Based on the destructive effect of the inﬂammatory response triggered by this
pathogen on the infected hosts, and the fact that it is the only mammalian
oomycete pathogen, it could be speculated that P. insidiosum only recently
developed the ability to survive in mammalian tissues, a belief also supported by

current phylogenetic analysis (Mendoza and Newton 2005).

Treatment

Since most of the antifungal drugs (iodide and amphotericin B) are
ineffective against P. insidiosum, and are very toxic, surgical removal of the
infected tissue has been the main treatment for pythiosis (Mendoza 2005). Most
recently a new non-invasive treatment termed immunotherapy has been
introduced (Mendoza et al., 2003).

Surgical removal has been successful in numerous cases of equine

pythiosis for over a century. Lesions and kunkers are removed followed by

16

cauterization (Habbinga 1967; McMullan et al. 1977). These surgical procedures
are extremely invasive and in some cases lead to death (Fisher et al. 1994).
Side effects and disease reoccurrence are commonly associated with surgical
removal of infected tissue. Drastic surgical procedures like amputation of
extremities have been used on humans with pythiosis. This last resort is rarely
successful usually leading to death (Sathapatayavongs et al. 1989;
Wanachiwanawin et al. 1993).

Immunotherapy includes a treatment of products produced by P.
insidiosum. Immunization using these products was reported to have curative
properties in the early eighties (Miller 1981). lmmuotheropy using Miller’s
vaccine was only 53% successful in horses. Using both exo and endoproteins
precipitated from P. insidiosum hyphae Mendoza et al. 2003, developed a new

more effective immunotherapeutic vaccine to treat both humans and animals.

Introduction to Transmission Electron Microscopy (TEM)

The locations of the antigens produced by Pythium insidiosum hyphae
have yet to be mapped. The use of immuno electron microscopic approaches for
identiﬁcation of the antigen positioning of hyphal organisms has been
successfully used in the past by some investigators (Herr et al., 1999; Sandoval
el al., 1996). The identiﬁcation of the antigens within P. insidiosum hyphae is
essential for a better understanding of the immunopathology and for
development of a more speciﬁc immunotherapeutic approach (Mendoza and

Newton 2005). Thus, this study has the following objectives: To evaluate the

17

morphological structures of Pythium insidiosum hyphae cultured at 37°C using
the Transmission Electron Microscope (T EM). To investigate the antigen-
antibody reaction of host sera with sections of Pythium insidiosum hyphae, using

Protein A Colloidal Gold (PACG) particles as a detection system.

18

CHAPTER TWO

Materials and Methods

Strain, Culture, and Sample Strategies

P. insidiosum strain H9 was selected based on the principle that it is the
type strain of this species, and also that it is the most immunologically studied
strain (Schurko et al. 2003). In addition, it is the strain used to prepare the
immunotherapeutic antigens for the treatment of this disease (Mendoza and
Newton 2005) (ATCC 58643; CBS 574.85; PIMT 19; originally known as H9
strain). The strain was kept at 30°C in the Medical Technology Program culture
collection and then subcultured onto Sabouraud agar slants (2% agarose, 2%
sucrose, and 1% peptone, in distilled water) just before transferring to broth
cultures. The subculture strain was incubated at 37 °C for 1 to 2 weeks until
white/clear growths were observed. A small section, around 1 cm of growth, was
transferred to separate ﬁaks 500 mL ﬂasks containing 300 mL of Sabouraud
broth (2% sucrose and 1% peptone in distilled water) using a sterile loop. The
ﬂasks were incubated 37°C while agitated using a shaker at 150 rpm for 2 to 4
weeks until the inoculated ﬂasks showed a substantial growth of hyphae. After
incubation, a spherical large mass of hyphal growth was removed from the
Sabouraud broth and placed in a sterile Petri dish. The hyphal mass was then
transferred into a 50 ml glass ﬂask and approximately 10 mL of 2% melted
(~45°C) water agarose was added to each ﬂask containing the hyphal mass.

After the agar solidiﬁed, the samples were placed into two separate sterile 15 mL

19

plastic sample tubes. The samples were ﬁxed by adding approximately 5 ml of
2.5% formaldehyde (Electron Microscopy Sciences, EMS, Fort Washington, PA.)
to each tube. The samples were then stored at 4 °C for 2 to 7 days until they

were prepared for morphology or immunolabeling.

Transmission electron microscopy (TEM)

Thin sections using traditional ﬁxative and stains in TEM were prepared to
evaluate details of the cell wall and intracellular structures of P. insidiosum’s
hyphae. A different approach was also used to evaluate the sample preparation
using immuno reaction of the thin sections containing hyphae with the infected
and non-infected sera from patients with proven pythiosis, and sera from
apparently healthy hosts. The following are the details of the procedures used to

achieve the objectives of this study.

Sample Preparation
Sample preparation for morphological analysis

The samples in solidiﬁed agar were placed in a Petri dish and aseptically
cut into approximately 1 cm cubes using a sterile scalpel. Fixative (2.5%
glutaraldehyde and 2.0% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4)
(EMS, Fort Washington, PA.) was added in vials containing small section
samples to stabilize cellular components, especially proteins, and then stored at
4°C for 5 days. Samples were then rinsed in cacodylate buffer (0.1 M, pH 7.4)

(EMS, Fort Washington, PA.) three times for 15 minutes per rinse. A 2% solution

20

of osmium tetroxide in 0.1M cacodylate buffer (EMS, Fort Washington, PA.) was
added as a postﬁxative agent in order to ﬁx and preserve lipids and maintain
three-dimensional structure prior to dehydration. After postﬁxation the samples
were rinsed with cacodylate buffer for three 15 minute cycles. The samples were
dehydrated in a series of acetone dilutions; 10%, 15%, 30%, 50%, 70%, 100%
for 10 minutes and ﬁnally 100% acetone for 1 hour. After the acetone
dehydration, sample sections were inﬁltrated with a graded series of Poly/Bed
812 and acetone solutions; 3:1, 2:2, 1:3, and pure resin (EMS, Fort Washington,
PA.). To provide support during ultramicrotomy and retain spatial organization
of the specimen section on the TEM grid, samples were placed in a silicone
mold, embedded in Poly/Bed 812 resin, and polymerized in an oven (Blue M

Electric Co., Blue Island, IL.) at 60 °C for 24 hours.

Sample preparation for lmmgno-la_beling analysis

The samples in solidiﬁed agar were placed in a Petri dish and were
aseptically cut into approximately 1 cm cubes using a sterile scalpel. The
samples were then washed three times with TTBS (0.05% Tween 10 in Tris-
buffered-saline pH7.4) using a dropper. Three drops of TTBS were administered
to the surface of the cubes in the Petri dish and then rotated with forceps and
washed again. After the wash, the samples were placed in small vials and, using
a dropper, the samples were dehydrated in a series of ethanol dilutions; 10%,
15%, 30%, 50%, 70%, 100% for 10 minutes and ﬁnally 100% ethanol for 1 hour.

The samples were suspended in the ethanol solutions while slowly rotated using

21

an inclined rotation device. The samples were dehydrated for optimal resin
penetration. The ethanol was removed from the vials before inﬁltration process.
The dehydrated samples were inﬁltrated with LR White resin (EMS, Fort
Washington, PA.) in a stepwise gradient; 25% for 2 hours, 50% for 2 hours, 75%
for 10 hours, and over night in 100%. The samples were inﬁltrated with resin to
maintain the integritylstructuraI-organization and provide support during
sectioning. The samples were embedded and positioned near both ends of the
pill-shaped mold gelatin capsules to facilitate sectioning. The samples were cast

into fresh LR resin and polymerized in the oven at 60°C, for 24 hours.

Sectioning procedures

Before the samples were sectioned, the hard outer shells on the blocks
were removed using a razor blade. Each pill shaped block was secured onto an
MTX ultra-microtome chuck and chuck holder (RMC, Boeckeler Instruments.
Tucson, A2), for trimming. A razor blade was used to trim the sample embedded
in the block. The oculars attached to the MTX ultra-microtome were used to view
the location of the sample in the block during the trimming process. Manipulating
the chuck holder, the top and surrounding area of the block was shaped into a
pyramid-like structure in order to expose the sample. To preserve the hyphal
antigens the samples were not post-ﬁxed with osmium tetroxide during sample
preparation and appeared opaque/white. This made the positioning of the

sample difﬁcult to see, so the block was held under a bright light to determine

22

sample location. Once the block was shaped into a pyramid it was ready for

thick sectioning.

Thick Sectioning

The trimmed exposed sample was placed in the MTX ultra-microtome
geared arch segment mount and adjusted for sectioning. The sample was out
into thick sections (500-1000 nm) using a glass knife, by adjusting the knife
holder and geared arch segment mount. Once the knife and block were aligned,
distilled water was added to the ‘boat’ of the glass knife. The water almost
touched the inside edge of the glass knife. A cutting window and the thickness
were set for the motion of the cutting arm. As the sample was automatically cut,
the newly formed sections would fall and ﬂoat in the water of the ‘boat’. The
samples were retrieved from the glass knife using a long stick with a concave
indentation carved near the end like a skinny spoon. The retrieved sections were
placed on a glass slide in a drop of distilled water and dried on a hot plate for
approximately 30 seconds. The thick sections on the slide were stained with a
drop of 1% toluidine blue and 1% boric acid mixed (1:1) and placed brieﬂy on the
hotplate for 15-30 seconds. The slide was washed with distilled water to remove
the excess stain, and viewed with a microscope (Olympus CX41RF, Optical Co.
Philippines) at 10x, 20x, and 40x to evaluate the presence, absence, and position
of the hyphae located in the resin. Thick sections containing stained hyphae
were also viewed for sample quality. Once positioning and quality of the sample

was acceptable, it was ready for thin sectioning.

23

. __- ,

Thin Sectioning

The blocks were secured to the MTX ultra-microtome chuck and chuck
holder and re-trimmed with a razor using the oculars to eliminate excess resin.
Once all excess resin was removed and only sample of interest remained, the
block was secured to the geared arch segment mount and adjusted. Thin
sections were cut using a diamond knife (Delaware Diamond Knives Inc.,
Delaware). The diamond knife was secured to the knife holder and adjusted.
After the diamond knife and the sample were aligned, the cutting window and
thickness were set. Distilled water was added to the ‘boat’ of the diamond knife
to collect the sample. As the block was cut, the sections ﬂoated into the “boat” of
the knife and viewed through the oculars of the MTX ultra-microtome. The
thicknesses of the sections obtained were 70nm in thickness. During thin
sectioning a plastic platform covered the ‘boat’, knife, and sample to shield the
sectioning from any environmental disruption. Thin sections were transferred to
formvar and carbon coated nickel grids (EMS, Fort Washington, PA.), by
touching the coated side of the grid to the sections in the boat using forceps.
The Ni grids were air-dried section side up on ﬁlter paper for 5 minutes until

ready for immuno-labeling.

Immuno-labeling
The Ni grids containing the sectioned hyphae of P. insidiosum were
placed section side down on a drop (15 pL) of the blocking solution TTBS-BSA

(0.05% TWEEN, TRIS-buffered-saline, 1.0% w/v bovine serum albumin, and

24

0.02% sodium azide pH7.4) and incubated for 15 minutes on a piece of para-ﬁlm.
The grids were dried using the corner pieces of ﬁlter paper by touching the
outside edges of the grid to absorb the buffer. The Ni grids, section side down,
were washed by placing three 15 pL drops of TTBS on para-ﬁlm for 1 minute and
they were then transferred to the next drop using forceps. Host sera from animals
(bovine, feline, canine, equine and human) with pythiosis conﬁrmed by culture,
histopathology, and serological assays such as immuno-diffusion, agglutination,
ELISA, and Western Blot were selected to be used. Sera from each of the
selected species used in this study (apparently healthy animals) were used as
negative controls. Following the wash the Ni grids were transferred to a 15 (IL
drop of each host sera diluted 1:1 with TTBS-BSA facing section side down at
25°C for 1 hour. To prevent the samples from drying out, a Petri dish was placed
over the para-ﬁlm and samples with a small piece of moist paper towel.
Following the binding, the Ni grids were washed with TTBS as mentioned above
and were placed on a 15 pL drop solution of 10% protein A colloidal gold (Ted
Pella, Redding CA) (20 nm in diameter) in TTBS-BSA and incubated at 25 °C for
1 hour. Again, to prevent the samples from drying out, a Petri dish was placed
over the para-ﬁlm and samples with a small piece of moist paper towel. The
sections were washed with TTBS as above. A ﬁnal wash included a series of
three 15 pL drops of water placed on para-ﬁlm, the sections were transferred
between drops after one minute. The samples were dried with the ﬁlter paper as
above, and then stained with one drop of 2% uranyl acetate in 50% ethanol

(EMS, Fort Washingyon, PA.) for 5 minutes. Samples were then washed with

25

distilled water as above. Using Sodium Hydroxide pellets to create an carbon
dioxide free environment, the samples were stained with 15 uL of Reynolds
formulation (EMS, Fort Washingyon, PA.) for 15 minutes. The samples were
washed with distilled water as mentioned above and left to dry on ﬁlter paper for

5 minutes or until ready for Transmission Electron Microscopy.

Transmission Electron Microscopy Evaluation

The stained sections were then evaluated using a JEOL 100-CX
Transmission Electron Microscope (JEOL, Tokyo, Japan) at an accelerating
voltage of 100 W. The TEM was used to locate and evaluate the samples at
different magniﬁcations (5,000X-450,000X). Samples were photographed using a

Mega View 2 digital camera (Soft Imaging Systems, Lakewood, Colorado).

26

CHAPTER THREE

Results

Electron Microscopic Features of Pythium lnsidiosum Hyphae

The presence of intact Pythium insidiosum hyphae and its structural
components in the stained samples were documented on several digital images.
These digital images revealed numerous healthy hyphae with a distinct cell wall
and multiple internal organelles.

The majority of hyphae observed in the sections possessed an intact cell
wall structure followed by a combination of inner layers. The cell wall had a very
smooth proﬁle of the plasmalemma which surrounded the entire hyphae (Figure
5). The inner layers beneath the cell wall interlaced and meshed with the other
layers forming a coarse thick inner layer (Figure 6). Cytoplasmic vesicles
appeared to be transporting proteins to the cytoplasmic membrane, outside the
inner layer, and then integrating them into the thick inner layers. Following
integration, the cytoplasmic bilayer membrane maintained its integrity.

Within the cytoplasmic membrane many internal structures were
observed. Large vacuoles were characterized by the presence of electron dense
bodies (EDBs). The EDBs appeared as large dark black structures within the
large vacuoles (Figure 7). Numerous small vacuoles were also observed
dispersed through out the entire hyphae. These vacuoles ranged in size and

shape, many posses a ﬁnely granular content (Figure 8). Numerous

27

mitochondria with tubular cristae were observed in some of the stained samples
(Figure 9). All observed mitochondria possessed intact tubular cristae. Multiple
nuclei were observed in some hyphae, each surrounded by a bilayered
membrane (Figure 8). The chromatin appeared granular within the membrane.
Several golgi bodies were also observed throughout the hyphae (Figure 10), with
numerous golgi vesicles pinching off from the golgi apparatus (Figure 10 and 11).
Relatively large microtubules were observed dispersed throughout the hyphae
(Figure 6 and 10). Endoplasmic reticula (ER) were sometimes observed near the
mitochondria, multiple ribosomes were also present around the ER (Figure 12).
The mergence of multiple golgi vesicles and the cytoplasmic membrane
near the cell wall were observed (Figure 6). Golgi vesicles were also observed

integrating into the intermediate cell wall layer (Figure 6).

28

 

Figure 5: Electron Micrograph of Stained Pythium insidiosum Hyphae; Displays a
deﬁned cell wall and inner layer structures The components of the hypha are as follows:
Cell Wall (CW), Inner Layer (IL), Mitochondria (M), Large Vacuole (LV), Electron Dense
Body (EDB), Golgi Body (G), Golgi Vesicle(GV).

29

 

Figure 6: Electron Micrograph of Stained Pythium insidiosum Hyphae. Showing the
integration of the golgi vesicles into the cytoplasmic membrane and Inner layers. The
components of the hypha are as follows Cell Wall (CW), Inner Layer (IL), Cytoplasmic
Membrane (CM), Electron Dense Body (EDB), Golgi Vesicle (GV). Microtubule (M).
Panel B is an enlargement of Panel A represented by a box.

30

 

 

Figure 7: Electron Micrograph of Stained Pythium insidiosum Hyphae;

Panel A, shows Cell Wall (CW), Inner Layer (IL), Large Vacuole (LV), Electron Dense
Body (EDB), Golgi Vesicle (GV), Microtubules (MT). Panel B, depicts also several
Mitochondria (M), and Small Vacuoles (SV).

31

 

Figure 8: Electron Micrograph of Stained Pythium insidiosum Hyphae; This ﬁgure depicts
the presence of Small Vacuoles (SV), Nucleus (N), Nuclear Membrane (NM), Cell Wall
(CW), Inner Layer (IL), Cytoplasmic Membrane (CM).

32

 

union"-

Figure 9: Electron Micrograph of Stained Pythium insidiosum Hyphae;

Panel A and B contain Mitochondria (M), Large Vacuole (LV), Electron Dense Body
(EDB), Cell Wall (CW), Inner Layer (IL), Small Vacuole (SV), Panel B is an enlargement
of Panel A represented by a box. Note the presence of mitochondria (M) with tubular
cristae.

33

 

Figure 10: Electron Micrograph of Stained Pythium insidiosum Hyphae; The micrograph
shows the presence of Cell Wall (CW), Inner Layer (IL), Cytoplasmic Membrane (CM),
Microtubule (MT), Large Vacuole (LV), Electron Dense Body (EDB), Golgi Body (G),
Golgi Vesicle(GV).

34

 

Figure 11: Electron Micrograph of Stained Pythium insidiosum Hyphae; This ﬁgure
shows the presence of Golgi Vesicle (GV), Cytoplasmic Membrane (CM), Inner Layer
(IL), Cell Wall (CW). A well developed Inner Layer is clearty observed in this ﬁgure.

35

 

Figure 12: Electron Micrograph of Stained Pythium insidiosum Hyphae; This ﬁgure
depicts Endoplasmic Reticulum (ER) with Ribosomes ®, Nuclear Membrane (NM),
Nucleus (N).

36

Immuno-labeling of Pythium insidiosum’s Hyphal Antigens

Digital images of the immuno-labeled structures in the hyphae of Pythium
insidiosum were photographed for interpretation of the binding immuno-globulins
present in the sera from the above hosts to PACG particles. P. insidiosum
hyphae used for immuno-labeling were not osmium stained to preserve the
antigenic properties of the immuno-reactive hyphal proteins. Thus, many of the
structures observed in the unstained samples for immuno-labeling were not
clearly deﬁned.

A series of negative controls (sera from apparently normal bovine,
canine, equine, feline, and human) were ﬁrst tested to validate the speciﬁcity of
the immuno-labeling. The control samples showed some gold particles in each
of the tested sera randomly bound throughout the examined ﬁelds (Figures 13
and 14). Control samples with only protein A colloidal gold showed few randomly
bound gold particles after rinsed with TTBS buffer (Figure 15).

The antigenic proteins in the cell wall of mature hyphae reacted with the
Bovine, Human, and Equine sera containing anti-P. insidiosum antibodies, as
shown by the binding of protein A colloidial gold to those speciﬁc areas. All of
the examined mature hyphae cell wall structures were found to contain great
quantities of bound PACG particles. In those samples, random PACG binding
were rarely observed (Figure16). In samples reacted with sera from infected
feline and canine resulted in random distribution of the PACG particles with no

speciﬁc binding (Figure 17).

37

Samples reacted with sera from an infected bovine showed speciﬁc
binding on the cell wall and in the cytosol (Figures 18-24). Some other
indistinguishable internal structures in the cytosol did not display speciﬁc binding
(Figures 18-24). Within the hyphae a substantial number of PACG particles were
speciﬁcally bound to the antigens of the inner layers beneath the cell wall
(Figures 18-24). In addition, gold particles were heavily bound to speciﬁc areas
in the cytosol (Figures 19 and 20). One section of this sample showed a group of
dead hyphae without cytoplasm, but with intact cell wall structures (Figure 25).
The skinny cell wall structure of these dead hyphae showed speciﬁc binding of
gold particles (Figure 26).

The serum sample from an infected human reacted in the same fashion to
that of bovine. The cell wall and the inner layers beneath the cell wall were
heavily bound with gold particles around the entire hyphae (Figures 27-30).
There was a signiﬁcant binding within the cytosol to the hyphal antigens.
However, it was considerably less compared the binding observed in bovine
samples (Figures, 27-30). Most structures within the cytosol were almost free of
gold particles in most samples reacted with the human serum (Figures 27-30).

The binding of gold particles in samples reacted with the horse sera were
signiﬁcant, especially in the cell wall regions of the hyphae (Figures 17, and 31-
41). On the outside of the cell wall surface gold particles were speciﬁcally bound
to the antigens, sometimes forming chains (Figure 32). Other sections of the cell
wall surface showed speciﬁc binding in clusters as well (Figure 33). Cytoplasmic

structures and sections of the cytosol where the structures were embedded were

38

rarely observed bound to gold particles (Figures 17, and 31-41). A long single
hypha approximately 100 um in length was studied in detail (Figures 34). This
long hypha was intact, and possessed a very thick inner layer structure beneath
the cell wall on only one side of the hypha (Figures 34-41). This section of the
hyphae was heavily bound to the gold particles along the entire 100 um length of
the hypha (Figures 34-41). The thick-sided inner layer structures possessed two
layers located immediately beneath the cell wall (Figures 34-41). Both layers
were speciﬁcally bound by the gold particles (Figures 39-41). The opposite side
of the hypha did not possess inner layers and appeared dark. However, it
displayed the binding of PACG particles along its entire surface of the cell wall
(Figures 39-41). The cytosol of the long hypha showed some sections with few

gold particles binding to it (Figures 34-41).

39

 

Figure 13: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from
healthy equine; Protein A colloidal gold particles randomly bound to sections of the ﬁeld.
Panel B is an enlargement of Panel A represented by a box. Sera from all healthy hosts
(bovine, canine, equine, feline, human) displayed the same random binding of gold
particles (not shown).

40

 

Figure 14: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from
healthy equine; Protein A colloidal gold particles are shown randomly bound to some
sections of this ﬁeld. Sera from all healthy hosts (bovine, canine, feline, human)
displayed the same random binding of gold particles (Not shown).

41

 

Figure 15: Electron Micrograph of Pythium insidiosum Hyphae reacted with only colloidal
gold particles. Most gold particles were washed off by TTBS buffer.

42

 

Figure 16: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from
equine with pythiosis. Protein A colloidal gold particles Speciﬁcally bound to cell wall and
inner layer structures on both Panel A and 8. Note the lack of binding in the cytosol (See
also Figure 31).

43

 

Figure 17: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from
Feline Panel A and Canine Panel B with pythiosis. Protein A collodial gold particles
displayed random binding.

44

 

Figure 18: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from
bovine with pythiosis. Protein A collodial gold particles speciﬁcally bound to the cell wall,
inner layers, and cytosol of the hyphae. Note no gold binding outside the hyphae.

45

 

Figure 19: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from
bovine with pythiosis. Panel A shows Protein A collodial gold particles Speciﬁcally bound
to cell wall, inner layers, and cytosol on the hyphae, Panel B is an enlargement of Panel
A represented by a box. Note the large congruity of gold particles bound to both the
cytosol and cell wall of the hypha.

46

 

Figure 20: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from
bovine with pythiosis. Panel A depicts Protein A collodial gold particles Speciﬁcally bound
to cell wall, inner layers, and cytosol on the hyphae, Panel B is an enlargement of Panel
A represented by a box. Note the prominent binding of PACG particles in the cytosol and
cell wall.

47

 

Figure 21: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from
bovine with pythiosis. Protein A collodial gold particles speciﬁcally bound to the cell wall,
inner layers, cytosol, and some internal stmctures of the hyphae. Figure 21 is an
enlargement of this ﬁgure represented by the box.

48

 

Figure 22: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from
bovine with pythiosis. Protein A collodial gold particles speciﬁcally bound to the cell wall,
inner layers, cytosol, and some internal structures of the hyphae, This Micrograph is an
enlargement of ﬁgure 21. Figure 23 is an enlargement of this ﬁgure, represented by the
box.

49

 

Figure 23: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from
bovine with pythiosis. Protein A collodial gold particles speciﬁcally bound to cell wall,
inner layers, and cytosol of the hyphae, This Micrograph is an enlargement of ﬁgure 22.

50

 

Figure 24: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from
bovine with pythiosis. Protein A collodial gold particles speciﬁcally bound to internal
structures, cell wall, inner layers, and cytosol of the hypha.

51

 

Figure 25: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from
bovine with pythiosis. Panel A Protein A collodial gold particles speciﬁcally bound to cell
wall structures on the healthy hypha and dead hypha. (Gold particles are difﬁcult to see
because of low magniﬁcation). Note the presence of cytosol in the healthy hypha versus
the dead hyphae. The dead hypha is characterized by the thin cell wall and lack of inner
layer Panel B is an enlargement of Panel A represented by a box.

52

 

Figure 26: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from
bovine with pythiosis. Protein A collodial gold particles speciﬁcally bound to the cell well
structures on the hyphae and dead hyphae, Note the presence of cytosol in the healthy
hypha vs the dead hyphae. This Micrograph is an enlargement of ﬁgure 25 Panel B
represented by a box.

53

 

Figure 27: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from a
human with pythiosis. Protein A collodial gold particles Speciﬁcally bound to cell wall.
inner layers, and cytosol of the hyphae. Note, no gold particles are observed outside of
the hypha.

54

 

Figure 28: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from a
human with pythiosis. Panel A depicts Protein A collodial gold particles speciﬁcally
bound to cell wall, inner layers, and cytosol of the hyphae. Panel B is an enlargement of
Panel A represented by a box.

55

 

Figure 29: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from a
human with pythiosis. Protein A collodial gold particles Speciﬁcally bound mainly to the
cytosol of the hyphae. Note the heavy binding in the cytosol. Few particles are bound to
the cell wall.

56

 

Figure 30: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from a
human with pythiosis. Protein A collodial gold particles Speciﬁcally bound to two different
inner layer structures on the hypha.

57

 

Figure 31: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from a
horse with pythiosis. Protein A collodial gold particles speciﬁcally bound to the cell wall
structures on the hypha. Note the lack of binding in the cytosol and outside the hypha
(Enlargement of Figure 16).

58

 

Figure 32: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from a
horse with pythiosis. Protein A collodial gold particles Speciﬁcally bound forming a chain
on the cell wall of the hypha. Note the lack of binding in the cytosol and outside the
hypha.

59

 

Figure 33: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from a
horse with pythiosis. Panel A shows Protein A collodial gold particles speciﬁcally bound
to the cell wall and inner layer structures on the hypha. Note the lack of binding in the
cytosol, Panel B is an enlargement of Panel A represented by a box.

60

 

Figure 34: Electron Micrograph of long hypha of Pythium insidiosum reacted with sera
from a horse with pythiosis and Protein A collodial gold particles. Due to the low
magniﬁcation of this ﬁgure, the speciﬁc binding of gold particles to the hyphal structures
could not be clearly distinguished. Figures 35-41 are of a higher magniﬁcation and depict
the speciﬁc binding of gold particles to this long hypha.

61

 

mdt'afi‘ﬁl’m

Figure 35: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from a
horse with pythiosis. Protein A collodial gold particles speciﬁcally bound to the cell wall
and at least two inner layer structures on the hypha. Note the lack of binding in the
cytosol. This micrograph is an enlargement of ﬁgure 34, displaying the thick inner layer
structures.

62

 

Figure 36: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from a
horse with pythiosis. Panel A and B depicts Protein A collodial gold particles speciﬁcally
bound to the cell wall and inner layer structures on the hypha. Note the lack of binding in
the cytosol, This micrograph is an enlargement of ﬁgure 34, displaying the thick inner
layer structures.

63

 

Figure 37: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from a
horse with pythiosis. Protein A collodial gold particles speciﬁcally bound to the cell wall
and inner layer structures on the hypha. Note the lack of binding in the cytosol, This
micrograph is an enlargement of ﬁgure 34, displaying the thick inner layer structures.

64

 

 

Figure 38: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from a
horse with pythiosis. Panel A and B show Protein A collodial gold particles speciﬁcally
bound to the cell wall and inner layer structures on the hypha. Note the lack of binding in
the cytosol. Both Panel A and B are enlargements of ﬁgure 34, displaying the thick inner
layer structures.

65

 

Figure 39: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from a
horse with pythiosis. Panel A and B depict Protein A collodial gold particles Speciﬁcally
bound to the cell wall and inner layer structures on the hypha. Note the lack of binding in
the cytosol. Both Panel A and B are enlargements of ﬁgure 34, displaying the thick inner
layer structures. Note the lack of Inner layers on the left side of the hypha.

66

 

Figure 40: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from a
horse with pythiosis. Protein A collodial gold particles speciﬁcally bound to the cell wall
and inner layer structures on the hypha. Note the lack of binding in the cytosol, This
micrograph is an enlargement of ﬁgure 34, displaying the thick inner layer structures.
Note the binding of gold to the cell wall on the left side of the hypha. No inner layers are
observed in this section of the hypha.

67

 

Figure 41: Electron Micrograph of Pythium insidiosum Hyphae reacted with sera from a
horse with pythiosis. Protein A collodial gold particles speciﬁcally bound to the cell wall
and inner layer structures on the hypha. Note the lack of binding in the cytosol, This
micrograph is an enlargement of ﬁgure 34, displaying the thick inner layer structures.
Note the binding of gold to the cell wall on the left side of the hypha. No inner layers are
observed in this section of the hypha (See also Figure 36).

68

CHAPTER FOUR

Discussion

The electron microscopic data on the morphological features of P.
insidiosum hyphae conﬁrmed previous ﬁndings (Miller et al., 1985). However,
the electron micrographs from this study added new information regarding the
internal morphological components of P. insidiosum’s hyphal structures, not
reported in earlier studies. Internal organelles were identiﬁed based on
comparisons with the internal structures of other Pythium spp. or other similar
hyphal organisms (Hoch, 1987). The development of golgi vesicles were
observed pinching off from the golgi bodies, responsible for modiﬁcation of lipids
and packaging of proteins. The golgi vesicles were also observed trafﬁcking
from the cytosol to the cytoplasmic membrane and then into the inner layers
beneath the cell wall. Several structures involved in transportation such as
microtubules, endoplasmic reticulum, and ribosomes were all observed. It is
believed that key antigens of P. insidiosum are produced within the cytosol,
packaged and modiﬁed by the golgi bodies, and then pinched off from the golgi
bodies into golgi vesicles and transported by the microtubules, endoplasmic
reticulum, and ribosomes through the cytoplasmic membrane, into and through
the inner layers, to the cell wall and possibly to external compartments of the

hyphae. These ﬁndings were conﬁrmed by the binding of gold particles to

69

speciﬁc areas in the cell wall and other internal hyphal components during our
immuno electron microscopic study.

Early studies have demonstrated that most immunological methods have
successfully detected the antigens of P. insidiosum, and thus they proved to be
useful for the diagnosis of pythiosis (Mendoza and Newton 2005). Other reports
have documented P. insidiosum’s lifecycle, ecology, distribution, immunological
effects, and the characterization of pythiosis in both humans and animals. The
recent phylogenetic study of P. insidiosum conﬁrmed its placement within the
members of the genus Pythium using molecular techniques. A novel therapeutic
approach to avoid invasive surgical treatment, termed Immunotherapy, has been
extremely effective (Mendoza, 2005). Although all previous studies have been of
paramount importance to understand this disease, the mapping of the antigens
detected during infection has yet to be achieved. Thus, the location of P.
insidosum’s putative antigens within the hyphae, involved in the pathology and
immunotherapy of the disease remain a mystery. The identiﬁcation and
characterization of the antigens developed by P. insidiosum during infection is
the next logical step to further understand the mechanisms of both the
immunotherapy and the immunopathology of pythiosis (Mendoza and Newton
2005). This immuno electron microscopic study indicates that P. insidiosum
expresses antigens in speciﬁc areas of the cytosol, which then migrate to the
cytoplasmic membrane, and to the inner layers beneath the cell wall and then to
the cell wall itself, where some of them are possibly taken outside of the hyphal

compartment. Similar techniques have been used to evaluate the distribution of

70

antigens of some fungal and protistal pathogens. For instance, the antigens of
the fungal pathogen Paracoccidioides brasiliensis were mapped using
immunoﬂuorescence and a protein A colloidal gold immunolabeling technique for
detection of the hyphal antigens. Their immunocytochemical techniques
indicated that some exoantigens were synthesized within the cytoplasm and then
excreted through the cell wall (Sandoval et al., 1996). Herr et al. (1999) used a
similar approach to evaluate the location of the antigenic components of
Rhinosporidium seeben' in infected host tissue (Herr et al., 1999).

The speciﬁc reactions of the protein A colloidal gold particles were
validated using a series of negative controls. Sera obtained from healthy hosts
(bovine, canine, equine, feline, and human) reacted with the P. insidiosum
hyphae and then labeled with gold particles all showed random binding. This
random binding validates the speciﬁcity of the gold particles located only on the
antigens developed by P. insidosum hyphae. Gold particles reacted with P.
insidiosum hyphae were mostly washed away with the buffer and those few gold
particles left behind displayed random binding, indicating that the gold particles
themselves have no afﬁnity to P. insidiosum’s hyphal antigens.

The binding of the protein A colloidal gold particles, as observed by this
immuno electron microscopic study using the sera from multiple hosts and P.
insidiosum hyphae, varied between hosts. The used sera from infected hosts
with pythiosis, displayed speciﬁc binding of gold particles to several areas of the
hyphae. While some of the hosts displayed very similar speciﬁc binding patterns,

most of the hosts were unique with different speciﬁc binding patterns observed.

71

For instance, reacted samples from both bovine and humans displayed large
amounts of speciﬁc binding in the cytosol, the inner layers, and the cell wall.
However, the equine reacted samples displayed strong speciﬁc binding on the
cell wall and inner layer structures, but showed minimal binding on cytosolic
structures. In contrast, the bovine and the human immunoglobulins showed
similar binding on the cell wall, but it was not as signiﬁcant as that on the equine
samples. During this study a long laterally sectioned hypha (~100 pm in
diameter) (Figures 34-41) was study in detail. This long hypha showed large
amounts of PACG binding to unusually thick inner layer structures and to the cell
wall. This long hypha showed three clearly inner layers, a feature did not
encountered in other hyphal sections reacted with the same equine serum or with
the other host’s sera. Oddly, these inner layers were observed only on one side
of the hyphal cell wall. The other side of the cell wall hypha was devoid of inner
layers. The long hyphae appeared healthy and contained healthy cytosol.
However, few PACG particles were found bound to the cytosol. In addition, the
canine and the feline immunoglobulins apparently did not display speciﬁc binding
in any of the hyphal structures. According to the manufacturer, the protein A
colloidal gold particles used by them have never been tested in cats, dogs, or
horses. Thus, it is possible that the protein A region of the PACG particles might
possess low or not afﬁnity at all for the Fc regions of these hosts
immunoglobulins (cats and dogs).

It is also possible that each of the studied hosts might produce different

anti-P. insidosum immunoglobulins against the hyphal antigens. This would

72

 

explain why the reacted sera samples from multiple hosts showed different
unique binding patterns to the hyphal antigens. In our studies the bovine,
equine, and human, showed good binding afﬁnity for the Fc regions of the
immunoglobulins in these species. But, the canine and feline immunoglobulins
did not react. This could explain why dogs and cats immunoglabulins did not
show speciﬁc biding to the PACG particles. Interestingly, cats and dogs respond
poorly to immunotherapy whereas bovines, equines, and humans did respond
well to immunotherapy and also displayed speciﬁc binding to P. insidiosum
hyphae in this study. Because it is not a clear link between unresponsiveness to
immunotherapy and the lack of gold binding to the hyphae of P. insidiosum in the
sera of canine and feline, we strongly believe that the protein A low afﬁnity for the
cats and dogs immunoglobulins could well explain our negative results with the
sera from canines and felines.

A recent unpublished study using western blots has identiﬁed immuno-
dominate proteins using multiple host sera. Perhaps the identiﬁcation of an
immuno-dominant protein derived from P. insidiosum hyphae would encompass
all the hosts for a more effective universal vaccine. There have also been
reports of allergenic reactions in horses. However, P. insidiosum hyphae were
not found in these animals (Robert Glass, Personal communication). Perhaps
the antigens contained in the cell wall of the dead hyphae be still active and
could mount an allergenic reaction in equines without being infected with the

etiologic agent.

73

The results obtained during this immuno-electron microscopic mapping of
P. insidiosum hyphae revealed that there are multiple immunodominant antigens
located within the cytosolic components of the hyphae, as well as on the inner
layers and cell wall. Colloquial reports indicating that some horses with positive
serology, to the antigens of P. insidiosum, but without the typical lesions of
pythiosis, had developed allergic reaction of the upper respiratory system,
suggested that P. insidiosum could act as a potent allergen on this and other
species. Thus, the ﬁnding in this study that the empty cell wall structures, found
on dead hyphae, retained their antigenic/allergenic properties is rewarding. All in
all, this study not only mapped for the ﬁrst time the location of the
immunodominant antigens of P. insidiosum, but also specify what of the antigens

encountered in Western Blot should be ﬁrst characterized using molecular tools

74

Bibliography

Austwick, P. K. C., and J. W. Copland. 1974. Swamp Cancer. Nature, 250:
84.

Bridges, C. H., and C. W. Emmons. 1961. A phycomycosis of horses caused
by Hyphomyces destruens. J American Vet Med Assoc, 38: 579-89.

Brown, C. C. and J. J. McClure. 1988. Use of immunohistochemical methods
for diagnosis of equine pythiosis. American J Vet Research, 11: 1866-1868.

De Cock, W. A. w. and L. Mendoza. 1987. Pythium insidiosum sp. the
etiological agent of pythiosis. J Clinical Microbiology, 25: 344-9.

De Hann, J. and L. Hoogkamer. 1901. Hyphomycosis destruens. Veeartseniij
BI v Ned Indie, 13: 350-74.

De Moraes, S., L. Mendoza, et al. 2005. Human Pythiosis, Brazil. Emerging
Infectious Diseases, 1 1: 715-718.

Dick, M.W. 2001. Straminipilous fungi: systematics of the Peronosporomycetes
including accounts of the marine straminipilous protist, the plasmodiophorids and
similar organisms. London: Kluwer Academic Publishers.

Fischer, J. R. and L. W. Pace. 1994. Gastrointestinal pythiosis in Missouri
dogs: eleven cases. J Vet Diagn Invest, 6: 380-2.

Habbinga, R. 1967. Phycomycosis in an equine. Southwest Vet, 20: 237-8.

Herr, R. A., L. Mendoza, S. N. Arseculeratne, L. Ajello. 1999.
lmmunolocalization of an endogenous antigenic material of Rhinospon'dium
seeberi expressed only during mature sporangial development. Fed of European
Soc Immun and Med Micro, 23: 205-212.

75

Hoch, H. C. 1987. Freeze-Substitution: A technique that yields improved
ultrastructural preservation of fungi, p. 161-166. In M. Fuller, and A. Jaworski
(Eds), Zoosporic Fungi In Teaching and Research.

Hutchens, M., and L. Mendoza. 2002. Development of a simpliﬁed latex
agglutination test for the rapid diagnosis of the infections caused by Pythium
insidiosum. 102'“ General Meeting of the American Society for Microbiology,
Salt Lake City, Utah; 124.

Krajaejun T. 2004. Ocular pythiosis: is it under diagnosed. Am J Ophthalmol,
137(2): 370-2.

McMullan, W. C., J. R. Joyce, D. V. Hanselka, J. M. Heitmann. 1977.
Amphotericin B for the treatment of localized subcutaneous phycomycosis in the
horse. Journal of the American Veterinary Medical Association 170: 1293-1298.

Mlller, R. 1981. Treatment of equine phycomycosis by immunotherapy and
surgery. Aust Vet J, 57: 377-82.

Miller, R. l., and R. S. Campbell. 1982. Immunological studies on equine
phycomycosis. Aust Vet J, 58: 227-31.

Miller, R. l., and R. S. Campbell. 1983. Experimental pythiosis in rabbits.
Sabouraudia, 21 : 331 -341 .

Miller, R. l., B. M. Olcott, M. Archer. 1985. Cutaneous pythiosis in beef calves.
Journal of the American Veterinary Medical Association 186: 984-986.

Mendoza, L. 2005. Pythiosis. p. 412-429. chap. 22. In R.J. Ha ,W.G. Merz,
(Eds), Topley & Wilson’s Microbiology and Microbial Infections, 10 ed. Arnold,
London.

Mendoza, L., F. Hernandezand, L. Ajello. 1993. Life cycle of the human and
animal oomycete pathogen Pythium insidiosum. Journal of Clinical Microbiology
31 :2967-2973.

76

Mendoza, L., L. Kaufman, W. Mandy, R. Glass. 1997. Serodiagnosis of
hyman and animal pythiosis using an enzyme-linked immunosorbent assay.
Clinical Diagnosis Lab Immunology. 4: 71 5-718.

Mendoza, L., L. Kaufman, P. Standard. 1987. Antigenic relationship between
the animal and human pathogen Pythium insidiosum. J Clinical Microbiology, 25:
2159-2162.

Mendoza, L., W. Mandy, R. Glass. 2003. An improved Pythium insidiosum-
vaccine formulation with enhanced immunotherapeutic properties in horses and
dogs with pythiosis. Vaccine, 21: 2797-2804.

Mendoza, L., and C. J. Newton. 2005. Immunology and immunotherapy of the
infections caused by Pythium insidiosum. Medical Mycology J, 00: 1-9.

Mendoza, L., V. Nicholson, J. F. Precott. 1992a. lmmunoblot analysis of the
humoral immune response to Pythium insidiosum in horses with pythiosis. J
Clinical Microbiology. 30: 2980-2983.

Mendoza, L., J. Villalobus, C. E. Calleja. 1992b. Evaluation of two vaccines
for the treatment of pythiosis insidiosi in horses. Mycopathologia, 118: 89-95.

Rivierre, C., C. Laprie, O. Guiard-Marigny, P. Bergeaud, M. BertheIemy, and
J. Guillot. 2005. Pythiosis in Africa. Emerging Infectious Diseases 11: 479-481.

Rippon, J. W. 1982. Medical Mycology 2'“ Edition. P 335-341, W.B. Saunders
Co., Philadelphia.

Rosa, P. S. 1993. Development and evaluation of serological tests to detect
pythiosis in horses. Master of Science thesis, Louisiana State University. 1-120.

Sathapatayavongs, B., P. Ledachaikul. 1989. Human pythiosis associated
with thalassemia hemoglobinopathy syndrome. J Infect Dis, 159: 274-80.

77

Sandoval, M. P., G. M. 8. Del Negro, M. J. S. Mendes-Glannini, T. De Brito.
1996. Distribution of exoantigens and a 43-kDa glycoprotein in the yeast and
mycelial forms of Paracoccidioides Brasiliensis. J Mycol Med, 6: 1-6.

Schurko, A., L. Mendoza, W. A. M. de Cock, G. R. Klassen. 2003. Evidence
for geographic clusters: Molecular genetic differences among strains of Pythium
insidiosum from Asia, Australia and the Americas are explored. Mycologia 95:
200-208.

Shenep, J. L., B. K. English, L. Kaufman, T. A. Pearson, J. W. Thompson, R.
A. Kaufman, G. Frisch, M. G. Rinaldl. 1998. Successful medical therapy for
deeply invasive facial infection due to Pythium insidiosum in a child. Clinical
Infectious Diseases 27:1388-1393.

Shipton, W.A., R. l. Miller, I. R. Lea. 1982. Cell wall, soospore and
morphological characteristics of Australian isolates of a Pythium causing equine
phycomycosis. Trans Br Mycol Soc, 79:15-23.

Triscott, J. A., D. Weedon, E. Cabana. 1993. Human subcutaneous pythiosis
Journal of Cutaneous Pathology 20:267-271.

Virgile, R., H. D. Perry. 1993. Human corneal ulcer caused by Pythium
insidiosum. Cornea, 12: 81-3.

Wanachiwanawin, W., M. Thianprasit, S. Fucharoen, A. Chaiprasert, N.
Sudasna, N. Ayudhya, N. Sirithanaratkul, A. Piankijagum. 1993. Fatal arteritis
due to Pythium insidiosum infection in patients with thalassaemia. Transactions
of the Royal Society of Tropical Medicine and Hygiene 87: 296-298.

78

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII