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http://vet.sagepub.com/content/50/1/172The online version of this article can be found at:
DOI: 10.1177/0300985812446153
2013 50: 172 originally published online 9 May 2012Vet Pathol M. A. Delaney, K. A. Terio, K. M. Colegrove, M. B. Briggs and M. J. Kinsel
)Tursiops truncatus Occlusive Fungal Tracheitis in 4 Captive Bottlenose Dolphins (
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Wildlife, Zoo, and Marine Animals
Occlusive Fungal Tracheitis in 4 CaptiveBottlenose Dolphins (Tursiops truncatus)
M. A. Delaney1
, K. A. Terio1
, K. M. Colegrove1
, M. B. Briggs2
, andM. J. Kinsel1
Abstract
Respiratory disease is common in dolphins, primarily affecting pulmonary parenchyma and sparing large airways. Over a 10-year
period, 4 captive adult bottlenose dolphins succumbed to chronic, progressive respiratory disease with atypical recurrent upperrespiratory signs. All dolphins had severe, segmental to circumferential fibrosing tracheitis that decreased luminal diameter.Histologically, tracheal cartilage, submucosa, and mucosa were distorted and replaced by extensive fibrosis and pyogranuloma-
tous inflammation centered on fungal hyphae. In 3 of 4 cases, hyphae were morphologically compatible with Aspergillus spp andconfirmed by culture in 2 cases. Amplification of fungal DNA from tracheal tissue was successful in one case, and sequences had
approximately 98% homology to Aspergillus fumigatus. The remaining case had fungi compatible with zygomycetes; however,culture and polymerase chain reaction were unsuccessful. Lesions were evaluated immunohistochemically using antibodies
specific to Aspergillus spp. Aspergillus-like hyphae labeled positively, while presumed zygomycetes did not. These cases representa novel manifestation of respiratory mycoses in bottlenose dolphins.
Keywords
Aspergillus, dolphin, fungi, immunohistochemistry, tracheitis, Tursiops truncatus, zygomycete
Mycotic diseases are well documented in captive and free-
ranging bottlenose dolphins (Tursiops truncatus).5–7 Common
sites of infection include the skin and respiratory tract, the latter
typically in the form of pneumonia sparing large airways.7
Pulmonaryaspergillosis is the mostcommonly reportedmyco-
tic infection in cetaceans in general, with many cases having
occurred in stranded bottlenose dolphins secondary to dolphin
morbillivirus infection.5,7 In captive and free-ranging bottlenose
dolphins, documented pulmonary mycoses include infections
by opportunistic fungi ( Aspergillus spp, zygomycetes, Candida
spp) and endemic fungi ( Blastomyces dermatitides, Histoplasma
capsulatum, and Coccidioides immitis).7 Second to aspergillosis,
zygomycetous infections are the most commonly reported and
can be devastating, with rapid dissemination and death.7
Dolphins and other cetaceans with respiratory mycoses may
present with obvious clinical signs of respiratory disease, or clinical signs may be nonspecific. Hematologic and biochem-
ical alterations are similar to other infectious diseases. Clinical
course can range from insidious to fulminating.7 In some cases,
fungal pulmonary diseases are diagnosed only on postmortem
examination. Fungal identification is usually achieved through
correlation of histopathology and culture and, more recently,
molecular analysis. In chronic lesions, fungal hyphae may be
rare or difficult to distinguish morphologically if degenerate,
and in such cases, ancillary diagnostics are paramount for
fungal identification.
This case series describes a novel manifestation of
respiratory fungal disease in captive bottlenose dolphins. Addi-
tionally, molecular and immunohistochemical characterization
of the fungal pathogens was attempted.
Materials and Methods
From 1998 through 2008, 4 adult captive Atlantic bottlenose
dolphins (T. truncatus) with chronic upper respiratory disease
were submitted to the University of Illinois Zoological Pathol-
ogy Program for postmortem examination (Table 1). All dol-
phins had a similar prolonged (months to years) clinical
history, including one or more of the following: episodic
lethargy and inappetence, respiratory ‘‘wheezes,’’ abnormal
‘‘honking’’ behavior, and intermittent leukocytosis.
1 Zoological Pathology Program, College of Veterinary Medicine, University of
Illinois, Maywood, Illinois2 Michael Briggs, DVM, Professional Corporation, Bolingbrook, Illinois
Corresponding Author:
M. A. Delaney, University of Illinois, Zoological Pathology Program, Loyola
University Medical Center, Building 101, Room 0745, 2160 South First Avenue,
Maywood, IL 60153.
Email: [email protected]
Veterinary Pathology
50(1) 172-176
ª The Author(s) 2012
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DOI: 10.1177/0300985812446153
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Gross necropsies were performed, and a complete set of
tissues was fixed in 10% neutral buffered formalin for
histologic evaluation. Tissues were routinely processed, paraf-
fin embedded, sectioned at 5 mm, and stained with hematoxylin
and eosin. Gomori’s methenamine silver and Gram stains were
utilized on selected sections. Fresh tissues from all cases weresubmitted for aerobic bacterial and fungal culture at time of
necropsy.
Immunohistochemistry (IHC) was performed on sections of
trachea (all cases) and skin (Case No. 3) using a modified strep-
tavidin–biotin–peroxidase complex method. The primary anti-
body was mouse monoclonal anti- Aspergillus spp (WF-AF-1:
AbD Sertotec, Oxford, UK) diluted 1:300 in 0.1 M
phosphate-buffered saline solution.2 Positive controls included
sections of lung from a bird and a dolphin with polymerase
chain reaction (PCR) and/or culture-confirmed aspergillosis
( Aspergillus fumigatus).
DNA was extracted from 45-mm scrolls of paraffin-
embedded blocks of trachea (all cases) and skin (Case No. 3)
using a commercially available kit (RecoverAll Total Nucleic
Acid Isolation Kit, Cat No. 1975) and amplified using 28S
large subunit rRNA generic primers and MicroSeq D2 LSU
rDNA primers (Applied Biosystems, Foster City, CA) per the
manufacturer’s directions.8 Both strands of successful ampli-
cons were sequenced using an automated capillary sequencer
(Applied Biosystems 3730XL) at the University of Chicago
Cancer Sequencing Facility and compared for homology in
GenBank genetic sequence database.
Results
Fungal morphologic, culture, molecular, and IHC data are sum-
marized in Table 1. Consistent findings in all cases were mod-
erate to severe, segmental to circumferential tracheal and/or
bronchial stenosis and multifocal to regional mucosal ulcera-
tion with multiple yellow, green and tan, friable, adherent mats
or plaques (Fig. 1). In Case Nos. 3 and 4, the entire trachea was
affected, extending from the larynx to the carina. Tracheal and
bronchial stenosis ranged from approximately 25% to almost
complete occlusion primarily due to mural fibrosis and inflam-
mation (Fig. 2), with lesser luminal accumulation of caseous
material in some cases (Fig. 1). Histologic lesions were similar
in all cases and included a range of changes: mucosal ulcera-
tion, necrosis, suppurative and pyogranulomatous inflamma-
tion, and exuberant immature to mature fibrous tissue with
partial to complete effacement of tracheal cartilages. In most
cases, extensive regions of tracheal mucosa and superficial sub-
mucosa were absent (ulcerated). Ulcerated regions were cov-ered by layers of clumped fibrin with enmeshed karyorrhectic
(necrotic) cellular debris, intact and necrotic neutrophils,
hemorrhage, variable numbers of fungal hyphae, and multiple
aggregates of bacteria (Case Nos. 1 and 4) (Fig. 3). Adjacent
intact mucosal epithelium often had squamous metaplasia
(Case Nos. 1 and 3). The submucosa contained nodular accu-
mulations of intact and necrotic neutrophils and cellular debris
centered on small numbers of variably apparent fungal hyphae
and surrounded by concentric bands of epithelioid macro-
phages and few multinucleated giant cells (pyogranulomas).
Few sections had evidence of cartilage invasion by fungal
hyphae with associated inflammation (Fig. 4). In Case Nos.
1, 2, and 4, hyphae were generally abundant, 6–15 mm in dia-
meter, septate, and parallel walled with acute-angle, dichoto-
mous branching, consistent with Aspergillus spp (Fig. 5). In
Case No. 3, fungal tracheitis was more chronic with extensive
mural fibrosis and markedly decreased (approximately 55%)
luminal diameter (Fig. 2) and a fistulous tract with rare remnant
intralesional fungal hyphae. Hyphae were 4–9 mm in diameter,
nonparallel walled, aseptate with acute- to right-angle branch-
ing, most compatible with a zygomycete. Also in Case No. 3,
acute dissemination was evident with severe cerebral and cuta-
neous necrotizing vasculitis and infarction with large numbers
of similar intralesional, intravascular fungal hyphae. Fungal
hyphae were positively stained with Gomori’s methenaminesilver in all cases (Fig. 5).
Case Nos. 1 and 4 also had pyogranulomatous and necrotiz-
ing bronchopneumonia with intralesional fungal hyphae and
Gram-positive cocci. In Case No. 4, bacteria were confirmed
as Staphylococcus intermedius by culture. Culture of lung
tissue from Case No. 1 yielded only contaminants. Bronchop-
neumonia in Case No. 4 was subacute. Bronchopneumonia in
Case No. 1 was chronic, with fibrosis surrounding regions of
necrosis and inflammation, though fibrous tissue was of lesser
quantity and less mature than in the trachea. Case No. 2 lacked
significant pneumonia but did have multifocal alveolar
Table 1. Signalment and Fungal Characteristics in 4 Captive Bottlenose Dolphins.
MorphologicFungal Culture
ImmunohistochemistryAnti– Aspergillus spp
Polymerase ChainReaction (FFPE)
Case No. Age, y Sex Weight, kg Identification of Fungus Tissue Result Tissue Result Tissue Result
1 8 F 134.5 Aspergillus Lung Aspergillus spp Trachea Positive Trachea Negative
2 Adult F *200 Aspergillus Bronchus No growth Trachea Positive Trachea Negative3 12 M *220 Zygomycete Lung No growth Skin Negative Skin Negative
Trachea Negative Trachea Negative4 6 M 126 Aspergillus Trachea Aspergillus fumigatus Trachea Positive Trachea Positive
FFPE, formalin-fixed, paraffin-embedded.
Delaney et al 173
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Figure 1. Trachea (fresh); dolphin, Case No. 2. Partial occlusion of tracheal lumen by fibrous tissue and inflammation with cartilage destruction.Figure 2. Trachea (formalin fixed); dolphin, Case No. 3. Severe circumferential thickening and obliteration of tracheal wall by fibrous tissue anddecreased luminal diameter. Figure 3. Trachea; dolphin, Case No. 4. Marked expansion of the submucosa by pyogranulomatous inflammationand fibrous tissue with mucosal ulceration and destruction of cartilage. HE. Figure 4. Trachea; dolphin, Case No. 4. Cartilage destruction bypyogranulomatous inflammation with multinucleated giant cells. HE. Figure 5. Trachea; dolphin, Case No. 1. Fungal hyphae are 6–15 mm indiameter, septate, and parallel walled with acute-angle, dichotomous branching, consistent with Aspergillus spp. Gomori’s methenamine silver.
Figure 6. Trachea; dolphin, Case No. 4. Fungal hyphae positively labeled with anti– Aspergillus spp antibodies. Immunohistochemistry, anti– Aspergillus spp (WF-AF-1), hematoxylin counterstain.
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however, antibodies specific to common fungal pathogens are
not universally available at veterinary diagnostic laboratories.
Fungal pneumonia is the most common respiratory mycotic
disease in dolphins; however, this case series demonstrates that
fungal organisms may localize to invade and obliterate normal
components of the upper respiratory tract, leading to airway
obstruction and respiratory compromise. Complete postmor-tem examinations including histologic evaluation and ancillary
diagnostics (culture, PCR, IHC) have and will continue to pro-
vide valuable insight into respiratory mycoses of dolphins and
other cetaceans. From this case series, it is recommended that
fungal tracheitis be included as a differential in captive individ-
uals with recurrent upper respiratory signs. Additionally, deter-
mining further correlations between clinical and postmortem
findings is critical to better understanding pathogenesis and
manifestations of fungal diseases in dolphins.
Acknowledgements
We would like to thank the veterinarians and support staffs at
respective institutions for providing case materials; Dr. Jennifer Land-
olfi and Stacy Schultz for their technical assistance; and the Histology
Lab of the University of Illinois Veterinary Diagnostic Laboratory for
slide preparation.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, author-
ship, and/or publication of this article.
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