VDE_296.

fm Page 187 Friday, July 26, 2002 4:09 PM

Veterinary Dermatology 2002, 13, 187 194

PCR-based detection of Pythium and Lagenidium DNA in frozen

and ethanol-fixed animal tissues

ackwell Science, Ltd

NADINE R. ZNAJDA*, AMY M. GROOTERS and ROSANNA MARSELLA*

*Department of Small Animal Clinical Sciences, PO Box 100126, College of Veterinary Medicine, University
of Florida, Gainesville, FL 326100126, USA
Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Louisiana State University, Baton
Rouge, LA 70803, USA
(Received 10 December 2001; accepted 15 January 2002)

Abstract The purpose of this study was to evaluate the application of previously described Pythium insidiosumand Lagenidium-specific nested PCR assays to the detection of oomycete DNA in animal tissues. DNA was
extracted from 15 frozen and 10 ethanol-fixed tissues obtained from six animals with pythiosis, five animals with
lagenidiosis, one animal with nonoomycotic skin disease and two animals without skin disease. First-round PCR,
which utilized universal fungal primers ITS1 and ITS2P, amplified a single product of the expected size for each
of the P. insidiosum- and Lagenidium-infected tissues, but not for tissues obtained from animals without fungal
disease. Second-round PCR using the P. insidiosum-specific primers PI1 and PI2 produced a single 105-bp product
for the P. insidiosum-infected tissues, but not for any of the other tissues. Second-round PCR using the Lagenidiumspecific primers LAG1 and LAG2 produced a single 76-bp product for the Lagenidium-infected tissues, but not
for any of the other tissues.
Keywords: canine, equine, feline, lagenidiosis, PCR, pythiosis.

INTRODUCTION
Pythium insidiosum and Lagenidium sp. are aquatic
oomycetes that cause devastating and often fatal dermatological disease in dogs living in the south-eastern
United States. Although P. insidiosum is well recognized
as a cause of cutaneous and/or subcutaneous infections
in horses, cats and cattle as well as dogs,14 Lagenidium
sp. has only recently been identified as a canine pathogen.5
A presumptive diagnosis of pythiosis is often made on the
basis of clinical presentation in conjunction with supportive histological findings (pyogranulomatous inflammation
associated with broad, irregularly branching, infrequently septate hyphae with nonparallel walls).1,6
However, these clinical and histological characteristics
are also typical of lesions caused by lagenidiosis and
zygomycosis;5,79 in fact, as a result of these similarities,
many pathologists continue to group oomycotic and
zygomycotic infections under the convenient (although
taxonomically obsolete) term phycomycosis.
Because the histological characteristics of pythiosis and
lagenidiosis are not unique, isolation and identification

Correspondence: Dr Amy Grooters, Veterinary Clinical Sciences,

Louisiana State University, Baton Rouge, LA 70803, USA. Fax: +1
(225) 578 9559; E-mail: agrooters@vetmed.lsu.edu
Dr Znajdas current address is Florida Veterinary Specialists, 3000
Busch Lake Blvd, Tampa, FL 33614, USA. Fax: +1 (813) 936 9595;
E-mail: Znajdan@mail.vetmed.ufl.edu. This project was conducted
while Dr Znajda was a visiting scientist in the Department of Veterinary
Clinical Sciences, Louisiana State University.
2002 Blackwell Science Ltd

of the pathogen has traditionally been necessary for a

definitive diagnosis. Unfortunately, this task is difficult.
Because the lesions associated with oomycosis are often
confused with other more common dermatopathies
(such as deep pyoderma or cutaneous neoplasia), veterinarians may fail to submit samples for fungal culture when tissue biopsies are obtained. In addition,
culture may be unsuccessful because many diagnostic
laboratories are unfamiliar with isolation techniques
for oomycetes. Even if the pathogen is successfully
recovered, its morphological identification is challenging. Few medical or veterinary microbiologists are
familiar enough with the morphological characteristics
of these organisms to differentiate either P. insidiosum
or the canine pathogenic Lagenidium species from
other oomycetes. In addition, the sexual reproductive
structures that are commonly used for species identification of oomycetes10 are difficult to produce,6,11 and
the characteristics of asexual reproduction (production
of zoospores by progressive cleavage within a vesicle
that develops at the end of a zoosporangium) are not
specific for either P. insidiosum or the canine pathogenic
Lagenidium species.1214
Molecular techniques have been utilized with
increasing frequency in recent years for the diagnosis
of cutaneous infections caused by organisms that are
difficult or time-consuming to culture, or for which
morphological identification of cultured isolates is
challenging.15,16 More specifically, these techniques
have been successfully applied to the identification of
fungi in skin and nail samples obtained from human
187

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188

N. R. Znajda et al.

and veterinary patients with cutaneous mycoses.17,18

We hypothesized that similar techniques could be used
for the detection of P. insidiosum and Lagenidium
DNA in tissue biopsies from veterinary patients.
Although PCR-based assays have previously been
developed for the species-specific detection of
P. insidiosum19 and the genus-specific detection of
Lagenidium sp.,20 these assays have only been evaluated
using genomic DNA extracted directly from cultured
isolates, and have not yet been applied to clinical specimens. The purpose of this study was to evaluate the
use of these previously described nested PCR assays
for the detection of P. insidiosum and Lagenidium
DNA in tissue samples obtained from veterinary
patients with suspected oomycosis.

MATERIALS AND METHODS

Tissues
Tissue samples (Table 1) were obtained from patients
that were undergoing biopsy as part of a diagnostic
evaluation for suspected oomycosis or zygomycosis at
the teaching hospital of the University of Florida
College of Veterinary Medicine or the Louisiana State
University School of Veterinary Medicine. After
obtaining client consent, one or two biopsies were
procured in addition to those submitted for routine histological and microbiological evaluation. Samples from
cutaneous lesions were either 6-mm punch biopsies or
similarly sized wedge biopsies, based on the preference
of the clinician managing each case. In addition, one

lung and two gastrointestinal samples were obtained

from animals with suspected oomycosis that were
undergoing necropsy. A total of 16 tissue samples were
collected from three dogs with pythiosis, two horses
with pythiosis, one cat with pythiosis, five dogs with
lagenidiosis and one dog with nonoomycotic dermatological disease. The diagnosis of pythiosis or lagenidiosis in each case was based on isolation of the pathogen
from infected tissues, as well as histopathological findings consistent with oomycosis. A diagnosis of cicatricial alopecia (which was suspected to be secondary to
thermal injury) was made on the basis of histopathology in the dog with nonoomycotic skin disease. In
addition to the samples from clinical patients, nine
skin samples (both punch and wedge biopsies) were
collected postmortem from two dogs without evidence
of dermatopathy that were undergoing necropsy after
euthanasia for noninfectious disease. After collection,
tissues were frozen at 20 C or 80 C, or stored in
95% ethanol at room temperature.16,21
DNA extraction
Stored tissue samples were processed in groups of
four or five (Table 1) within 12 months of their collection. Each group included at least one normal skin
sample, which served as a negative control. For DNA
extraction, a piece of tissue approximately 5 mm3 was
minced with a disposable scalpel and placed in an
Eppendorf tube containing 1000 L digestion buffer
(50 m Tris-HCl, pH 7.2, 50 m EDTA, 1% SDS, 1%
2-mercaptoethanol) and 300 g of freshly thawed proteinase K (Amresco; Solon, OH, USA). After 2 h of

Number*

Diagnosis

Tissue

Species

Storage

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25

Lagenidiosis
Lagenidiosis
Lagenidiosis
Normal
Pythiosis
Cicatricial alopecia
Pythiosis
Normal
Normal
Lagenidiosis
Lagenidiosis
Lagenidiosis
Normal
Normal
Lagenidiosis
Lagenidiosis
Normal
Pythiosis
Pythiosis
Pythiosis
Normal
Normal
Pythiosis
Pythiosis
Normal

Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Skin
Lung
Skin
Stomach
Colon
Subcutaneous granuloma
Skin
Skin
Subcutaneous granuloma
Subcutaneous granuloma
Skin

Canine
Canine
Canine
Canine
Canine
Canine
Feline
Canine
Canine
Canine
Canine
Canine
Canine
Canine
Canine
Canine
Canine
Canine
Canine
Equine
Canine
Canine
Equine
Equine
Canine

95% Ethanol
95% Ethanol
95% Ethanol
95% Ethanol
80 C
80 C
20 C
20 C
80 C
80 C
80 C
80 C
20 C
95% Ethanol
95% Ethanol
95% Ethanol
95% Ethanol
80 C
80 C
80 C
20 C
20 C
80 C
95% Ethanol
95% Ethanol

*Tissues are numbered in the order that DNA extraction was performed; groups for DNA
extraction were as follows: group 1 (tissues 1 4), group 2 (tissues 5 8), group 3 (tissues 9 13),
group 4 (tissues 14 17), group 5 (tissues 18 21), group 6 (tissues 22 25).
2002 Blackwell Science Ltd, Veterinary Dermatology, 13, 187 194

Table 1. Tissues evaluated in this study

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PCR detection of oomycete DNA in tissues

incubation at 65 C, an additional 300 g of proteinase
K was added. The sample was vortexed briefly, incubated
at 65 C for an additional 24 h, and centrifuged at
10 000 g for 15 min at room temperature. Protein and
cellular debris was precipitated from the supernatant
with the addition of an equal volume of chloroform:phenol:isoamyl alcohol (25:24:1) followed by
centrifugation. DNA was precipitated from the supernatant with the addition of isopropanol in the presence
of sodium acetate. The pellet was washed twice with
ethanol, dried, resuspended in 100 L sterile ddH2O,
and stored at 20 C. Prior to use as template in PCR,
DNA was diluted at 1:10 in sterile ddH2O.
Oligonucleotide primers
Oligonucleotide primers were synthesized by the
Louisiana State University Gene Probes and Expression Systems Laboratory on an automated synthesizer
(ABI 392 Nucleic Acid Synthesizer, PE Applied
Biosystems; Foster City, CA, USA). Universal fungal
primers ITS1 (5-TCCGTAGGTGAACCTGCGG-3 )
and ITS2P (5-GCAGCGTTCTTCATCGATGT-3 )
are based on conserved areas of the 18-s and 5.8-s regions
of the ribosomal RNA (rRNA) gene, and amplify the
entire internal transcribed spacer 1 (ITS1) region. The
ITS1 primer is identical to that originally described by
White and colleagues,22 whereas the ITS2P primer is a
slight modification of Whites ITS2 primer designed by
one of the authors (AMG) to allow better amplification
of oomycete DNA. Pythium insidiosum-specific primers
PI1 (forward primer, 5-TTCGTCGAAGCGGACTGCT-3) and PI2 (reverse primer, 5-GCCGTACAACCCGAGAGTCATA-3 ),19 and Lagenidium-specific
primers LAG1 (forward primer, 5-CTTGTTTTGTGCGCGAATGC-3 ) and LAG2 (reverse primer,
5-TCAGTCAATGCTAGCTTTCGCC-3 )20 are based
on heterogenous areas of the ITS1 region, and are
internal to the ITS1 and ITS2P primers.
PCR amplification
Previously described nested PCR assays for the amplification of P. insidiosum19 and Lagenidium species20
were applied to DNA extracted from each of the tissue
samples. The P. insidiosum assay had previously been
shown to be species-specific, whereas the Lagenidium
assay had been shown to be only genus-specific, amplifying DNA from the mosquito pathogen L. giganteum
as well as from the canine pathogenic Lagenidium species, but not from Pythium, Conidiobolus, Basidiobolus
or Aspergillus species.
For each assay, first-round PCR amplified the ITS1
region using universal fungal primers ITS1 and ITS2P.
Amplification reactions were performed in 50-L
volumes containing 20 pmol (0.4 ) of each primer,
20 each of dATP, dCTP, dGTP and dTTP, 1.5 m
MgCl2, 50 m KCl, 10 m Tris-HCl (pH 8.3), 5 L of
diluted DNA template and 2.5 units of Taq DNA
polymerase (Promega Corporation; Madison, WI, USA).
Reactions were assembled in a laminar flow hood using
dedicated pipettors and aerosol barrier pipette tips.

189

Amplification was carried out in a DNA thermal cycler

(GeneAmp 9700, PE Applied Biosystems, Foster City,
CA, USA) with the following cycling parameters:
initial denaturation at 94 C for 3 min, 30 cycles of denaturation at 94 C for 45 s, annealing at 63 C for 45 s,
and extension at 72 C for 45 s, and final extension at
72 C for 10 min. A negative control (containing all
reaction components except DNA template) and positive controls (genomic DNA extracted from pure cultures of P. insidiosum and Lagenidium sp.) were included.
For second-round PCR, first-round amplification
products (including the positive and negative controls)
were diluted 1:50 in sterile ddH2O, and 5-L aliquots
(for P. insidiosum assay) or 2-L aliquots (for Lagenidium assay) of diluted product were used as template in
second-round PCR. Amplifications for second-round
PCR were performed using the same reaction mixture
as described for first-round PCR, with the following
cycling parameters: initial denaturation at 94 C for
3 min, 15 cycles of denaturation at 94 C for 30 s,
annealing at 65 C for 30 s, and extension at 72 C for
30 s, and final extension at 72 C for 10 min. A 15-L
aliquot of each reaction for both rounds of PCR was
electrophoresed through an agarose gel (3% TBE) that
contained ethidium bromide. Amplification products
were visualized and photographed under UV light, and
their size was determined by comparison to a DNA
standard. To ensure that positive results were not the
result of carry-over contamination, only those DNA
extraction groups in which the normal skin samples
(negative control tissues) produced no amplification
products were included in the analysis.

RESULTS
The results of first- and second-round PCR amplification are shown in Figs 1 and 2, respectively. Firstround amplification using universal fungal primers
ITS1 and ITS2P produced an amplicon of the expected
size (322 bp for P. insidiosum; 266 bp for Lagenidium
sp.) for each of the tissues from animals with pythiosis
or lagenidiosis. No product was visualized for either
the normal skin or nonoomycotic dermatopathy tissue
samples. Second-round PCR using the P. insidiosumspecific primers PI1 and PI2 produced a single amplicon of the expected size (105 bp) for each of the tissues
from animals with pythiosis, but did not produce
amplicons for any of the other tissues. Second-round
PCR using the Lagenidium-specific primers LAG1 and
LAG2 produced a single amplicon of the expected size
(76 bp) for each of the tissues from animals with
lagenidiosis, but did not produce amplicons for any of
the other tissues.

DISCUSSION
In the early 1980s, work by Miller23 and later by Foil2 identified Pythium sp. as a cause of canine phycomycosis
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N. R. Znajda et al.

Figure 1. Results of first-round PCR using universal fungal primers ITS1 and ITS2P to amplify the ITS1 region of the ribosomal RNA gene.
Lane M = 100-base pair (bp) molecular weight marker; lanes 125 correspond to products amplified from DNA template extracted from tissues
125 as listed in Table 1.

Figure 2. Results of second-round PCR using P. insidiosum-specific primers (a) PI1PI2 and (b) LAG1-LAG2. Lane M = 100-base pair (bp)
molecular weight marker; PC = positive control lane; NC = negative control lane; lanes 125 correspond to products amplified from DNA
template extracted from tissues 125 as listed in Table 1. Lanes corresponding to tissues obtained from animals with pythiosis are marked with
a bar over the lane number in Figure 2(a), and those corresponding to tissues obtained from animals with lagenidiosis are marked with a bar in
Figure 2(b).

(a nonspecific clinicopathological syndrome characterized by pyogranulomatous gastrointestinal or

subcutaneous inflammation associated with broad,
irregularly branching, poorly septate hyphae). At that
time, Foil and colleagues proposed that, instead of
phycomycosis, the term pythiosis be used to describe
the disease when culture of the aetiologic agent allowed
Pythium infections to be differentiated from those
caused by zygomycete fungi in the genera Conidiobolus
and Basidiobolus, which produce similar histological
lesions.2 Unfortunately, since that time, difficulties
associated with the isolation and morphological identification of P. insidiosum have often prevented definitive culture-based diagnoses from being made. As a
result, the majority of P. insidiosum-, Lagenidium- and
zygomycete-infected veterinary patients continue to be
labelled with a presumptive diagnosis such as suspected pythiosis, suspected zygomycosis or even
phycomycosis on the basis of histopathologic findings. Furthermore, the term pythiosis is occasionally
applied to these patients with no qualifying terms to
denote the presumptive nature of the diagnosis. In fact,
even in recent literature, some canine patients have
2002 Blackwell Science Ltd, Veterinary Dermatology, 13, 187 194

been classified as having cutaneous pythiosis on the

basis of histological findings alone.24
Although the presumptive diagnosis of pythiosis
is likely to be correct in a majority of cases (simply
because it is more common), the limited specificity of
this clinicopathological designation is troublesome
for several reasons. First, the failure to differentiate
zygomycosis from pythiosis and lagenidiosis may have
implications for treatment and prognosis. Because of
differences in cell membrane composition between
these taxonomically different classes of organisms,
zygomycotic infections should theoretically be more
likely than oomycotic infections to respond to traditional antifungal agents (such as amphotericin B or
itraconazole) that target ergosterol. Secondly, the
rarity of culture-based diagnoses of zygomycosis has
resulted in a paucity of information in the current
veterinary literature regarding the clinicopathological
characteristics, prognosis and epidemiological features
associated with this disease in small animal
patients.7,25,26 Finally, and perhaps most importantly,
the recent identification of canine pathogens in the
genus Lagenidium5 suggests that the spectrum of

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PCR detection of oomycete DNA in tissues

oomycotic mammalian pathogens is not limited to the
species P. insidiosum, and in fact may include a number
of previously unrecognized taxa. Identification of these
pathogens in the future will be dependent on our ability
to make a species-specific diagnosis whenever possible.
Diagnostic tools currently available for pythiosis
and lagenidiosis that may provide a more specific
diagnosis than routine histology include immunohistochemical staining and serological assays. Although
immunohistochemical antibodies for Lagenidium sp.
are not yet available, immunohistochemistry is commonly used as a diagnostic aid for pythiosis. Polyclonal
antibodies raised against antigens of P. insidiosum have
been utilized in immunohistochemical techniques
developed first by Brown27 and later by Newton.28
These assays have the advantage of being applicable to
archival specimens, and both have been used regularly
since their development as confirmatory tests for pythiosis.2931 However, although staining of Conidiobolus
and Basidiobolus hyphae in equine tissues was not
reported with Browns antibody, some cross-reactive
staining was observed when Newtons antibody was
applied to Conidiobolus-infected tissues in a previously
reported case of canine zygomycosis.9 In addition, mild
to moderate staining of Lagenidium hyphae was noted
by one of the authors (AMG) when Newtons antibody
was applied to tissues from six dogs with cultureconfirmed lagenidiosis (A. M. Grooters, unpublished
observations). Therefore, although the two techniques
have not yet been evaluated side-by-side on duplicate
tissue samples, it seems likely that the PCR assays
evaluated here have the potential for greater specificity
for the diagnosis of pythiosis than the currently available immunohistochemical methods.
Serological assays that have been developed for the
diagnosis of oomycosis in veterinary patients include
immunoblot analysis for canine pythiosis and lagenidiosis, and an enzyme-linked immunosorbent assay
(ELISA) for canine pythiosis. Although western
immunoblot detection of anti-P. insidiosum and antiLagenidium antibodies in canine serum has yet to be
fully evaluated, preliminary assessments indicate that
the technique provides excellent specificity for the
diagnosis of pythiosis and lagenidiosis.32 In addition, an
ELISA for the detection of anti-P. insidiosum serum
antibodies has recently been shown to be highly
sensitive and specific, allowing the differentiation of
pythiosis from lagenidiosis and zygomycosis as well
as from other mycotic infections.33 Although these
serological assays have the potential to offer an easy,
noninvasive and inexpensive means for the differentiation of pythiosis and lagenidiosis from zygomycosis,
they are unlikely to provide the level of specificity that
molecular-based assays can provide, a difference that
may be important if strain or species differences are
identified in the future among mammalian pathogens in
the genera Pythium and Lagenidium.
Results of the study described here suggest that PCR
is a useful technique for the detection of P. insidiosum
and Lagenidium sp. DNA in frozen or ethanol-fixed

191

tissue samples, and support its use for the specific

diagnosis of pythiosis and lagenidiosis when culture
of infected tissues is not successful, or when cultured
isolates cannot be identified on the basis of morphological characteristics. The obvious advantage of this
approach is that it bypasses the obstacles associated
with culture and morphological identification of the
pathogen, and provides the veterinarian with an
opportunity to make a definitive diagnosis in a greater
number of dogs with pythiosis or lagenidiosis. It also
has the potential to provide a more rapid diagnosis
than culture and morphological characterization
(which takes a minimum of 3 days because of the need
to induce reproductive structures for identification). In
addition, because oomycotic DNA can be extracted
from tissues stored for several months at room temperature in 95% ethanol, handling and shipping of specimens for DNA extraction is simpler and easier than for
oomycete culture. Despite these advantages, there are
several limitations to the molecular-based diagnosis of
oomycosis that should be noted.
First, it requires a tissue sample. This necessitates
that the veterinarian either have a strong enough clinical
suspicion of oomycosis to obtain additional specimens
for PCR analysis at the time of initial biopsy, or be
willing to re-biopsy the patient after the potential for
oomycosis has been suggested by histological findings.
However, although application of the PCR assays as
performed here requires that a separate tissue biopsy
be obtained, the fact that both assays were originally
designed to produce small amplicons would permit
their application in the future to amplification of DNA
from paraffin-embedded tissues sections, allowing retrospective evaluation of archival specimens. Secondly,
like culture, the success of PCR depends on the presence of hyphae in the tissues evaluated. Although the
nested design of these PCR assays should provide
excellent sensitivity, because the distribution of hyphae
is scattered in some oomycotic lesions8 it is possible
(especially in horses with subcutaneous pythiosis associated with excessive granulation tissue) to biopsy a
region of the lesion that does not contain hyphae, giving
a falsely negative result. Thirdly, the same nested design
that contributes to the assays sensitivity also makes it
highly susceptible to false-positive results arising from
contamination of samples during DNA extraction or
PCR amplification. For this reason, it is critical that
negative tissue samples be processed concurrently with
clinical samples during DNA extractions, and that
negative control tubes be included in all PCR amplifications. In addition, routine precautions for limiting
contamination during DNA extraction and PCR reaction assembly (such as using dedicated pipettors and
aerosol barrier tips) should be stringently followed.
The final limitations of the PCR-based approach
described here involve the specificity of the primer
pairs for their oomycete targets. Because they are
designed specifically to amplify P. insidiosum (for PI1
and PI2) or a limited number of species in the genus
Lagenidium (for LAG1 and LAG2), these assays will
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N. R. Znajda et al.

not allow the future identification and characterization

of previously unrecognized oomycotic pathogens.
Therefore, obtaining a culture diagnosis whenever possible will always be important to our ability to continue
to learn more about the aetiology and epidemiology
of mammalian oomycosis. Finally, it should be noted
that, because of the extremely close phylogenetic
relationship between the canine pathogenic Lagenidium species and the mosquito larval pathogen
L. giganteum, the PCR assay for Lagenidium will also
amplify L. giganteum DNA, and thus is genus- but not
species-specific.20 This raises concern that the presence
of DNA from nonpathogenic Lagenidium species on
the skin of veterinary patients could potentially result
in a false-positive result. Because the majority of
Lagenidium species are obligates parasites of algae,
fungi, rotifers, nematodes, crustaceans, Daphne and
mosquito larvae, this seems unlikely. However, until
the positive predictive value of the LAG1LAG2 PCR
assay for the detection of Lagenidium-infected tissues
can be better estimated by applying it to a much larger
number of clinical specimens, it would seem prudent to
limit its use to tissues obtained from patients in which
clinical and histological findings support a diagnosis of
oomycosis or zygomycosis.
Limitations of this study are primarily related to the
small number of tissue samples tested. Better evaluation of the specificity of these assays could have been
achieved by including a larger number of samples
taken from patients with mycotic dermatopathies
other than pythiosis or lagenidiosis (especially those
with culture-confirmed zygomycosis). Unfortunately,
because this was a prospective study designed to utilize
clinical specimens accumulated over a 1-year period,
sample collection was limited to the cases presented to
the two referral institutions during that time. However,
because both PCR assays have been shown not to
amplify genomic DNA extracted directly from cultured isolates of Basidiobolus and Conidiobolus,20 we
do not believe that this is a major limitation.
Future work with these molecular tools should focus
on the evaluation of a large number of tissue samples
obtained by random sampling of a population of
animals suspected of having pythiosis, lagenidiosis or
zygomycosis, which would allow estimation of the
assays sensitivity, specificity and predictive values.
Additional studies comparing the sensitivity and specificity of the PCR-based assays to culture, serology
and immunohistochemistry will likely be important as
each of these diagnostic modalities is better characterized and more widely employed. The results presented
here suggest that the use of PCR-based assays will significantly enhance our ability to reach a more specific
diagnosis of oomycosis in veterinary patients. Their
combined application with other sequence-based tools
for the identification of zygomycotic pathogens should
assist veterinarians in making a definitive diagnosis in
cases that were previously relegated to the presumptive
or less specific categories of suspected pythiosis or
phycomycosis.
2002 Blackwell Science Ltd, Veterinary Dermatology, 13, 187 194

ACKNOWLEDGEMENTS
This project was funded by grants from the American
College of Veterinary Dermatology and the College of
Veterinary Medicine, University of Florida. AMGs
work is supported by the Morris Animal Foundation
and the American Academy of Veterinary Dermatology.

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Rsum Le but de cette tude est dvaluer lapplication dune sonde PCR, pralablement rapporte comme
spcifique de Pythium insidiosum- et Lagenidium, pour la detection dADN doomyctes dans les tissus animaux.
LADN a t extrait de 15 biopsies congeles et de 10 biopsies fixes dans lthanol (six animaux prsentant une
pythiose, cinq une lagnidiose, un cas de dermatose non oomycotique, et deux animaux sans dermatose). La
premire replication, qui utilisait les primres fongiques universels ITS1 et ITS2P, a amplifi un seul produit dune
taille attendue pour les tissus infects par P. insidiosum- et Lagenidium-, mais pas chez les animaux ne prsentant
pas de dermatomycose. La seconde rplication utilisant les primres spcifiques de P. insidiosum- PI1 et PI2 a
permis de produire un seul produit 105-bp pour les tissus infects par P. insidiosum-, mais pas dans les autres cas.
La seconde replication utilisant les primres spcifiques de Lagenidium LAG1 et LAG2 a produit un seul produit
76-bp dans les tissues infects par Lagenidium, mais pas dans les autres cas.
Resumen Este estudio se propone evaluar la aplicacin de pruebas de PCR especficas para Pythium insidiosumy Lagenidium previamente descritas, a la deteccin de DNA de oomicetos en tejidos animales. Se extrajo DNA
de 15 tejidos congelados y de 10 tejidos fijados en etanol, obtenidos de seis animales con pitiosis, cinco animales con
lagenidiosis, un animal con una dermatosis nonoomictica y dos animales sin enfermedad cutnea. La primera
serie de PCRs, que utilizaban los primers fngicos universales ITS1 y ITS2P, amplificaban un solo producto del
tamao esperado para cada uno de los tejidos infectados con P. insidiosum y Lagenidium, pero no para tejidos
obtenidos de animales sin enfermedad fngica. La segunda serie de PCRs utilizando los primers especficos para
P. insidiosum PI1 y PI2 produjeron un solo producto 105-bp para los tejidos infectados con P. insidiosum, pero
no para ninguno de los otros tejidos. La segunda serie de PCR utilizando los primers especficos de Lagenidium
LAG1 y LAG2 produjeron un solo producto 76-bp para los tejidos infectados con Lagenidium, pero no para
ningn otro tejido.
Zusammenfassung Das Ziel der Studie war die Evaluierung von bereits beschriebenen, fr Phytium insidiosum
and Lagenidium spezifischen Nested-PCR Methoden fr den Nachweis von Oomyceten-DNA in tierischem
Gewebe. Aus 15 gefrierfixierten und 10 ethanolfixierten Gewebsproben von sechs Tieren mit Phytiose, fnf Tieren
mit Lagenidiose, einem Tier mit nicht-oomykotischer Hauterkrankung und zwei Tieren ohne Hauterkrankung
wurde DNA extrahiert. Der erste PCR-Schritt mit ITS1 und ITS2P als universellen Primern fr Pilze
2002 Blackwell Science Ltd, Veterinary Dermatology, 13, 187194

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N. R. Znajda et al.
vervielfltigte ein einziges Produkt der erwarteten Gre aus jeder der mit P. insidiosum und Lagenidium
infizierten Gewebsproben, jedoch nicht aus den Proben von Tieren ohne Pilzerkrankungen. Der zweite
PCR-Schritt mit den P. insidiosum-spezifischen Primern PI1 und PI2 ergab ein einziges Produkt von 105 bp Gre aus
denen mit P. insidiosum infizierten Gewebsproben, jedoch aus keiner der anderen Proben. Der zweite PCR-Schritt
mit den Lagenidium-spezifischen Primern LAG1 und LAG2 ergab ein einziges Produkt von 75 bp Gre aus
denen mit Lagenidium infizierten Gewebsproben, jedoch aus keiner der anderen Proben.

2002 Blackwell Science Ltd, Veterinary Dermatology, 13, 187 194