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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: delane10@illinois.edu

Veterinary Pathology

50(1) 172-176

ª The Author(s) 2012

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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.

174   Veterinary Pathology 50(1)

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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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