JPC SYSTEMIC PATHOLOGY

RESPIRATORY SYSTEM

September 2023

P-F06

 

SLIDE A: Signalment (JPC #1945809): Adult male Magellanic penguin

 

HISTORY: This penguin was found dead in its pen.

 

HISTOPATHOLOGIC DESCRIPTION: Lung: Affecting over 50% of the lung, there are multiple granulomas up to 1 mm in diameter predominantly centered on parabronchi and secondary bronchi. Granulomas are characterized by a central core of lytic necrosis admixed with viable and degenerate heterophils and bounded by lymphocytes, epithelioid macrophages, rare multi-nucleated macrophages (foreign body type), fibroblasts, sparse collagen, and sparse fungal hyphae. Hyphae are 5-8 µm in diameter, have thin, parallel walls, display acute angle, dichotomous branching, and have distinct infrequent septations. Adjacent to the granulomas, air capillary septa are lost and replaced by eosinophilic cellular debris (septal necrosis), fibrin, heterophils, lymphocytes, and histiocytes. In less affected regions, blood capillaries are congested, and there are increased macrophages in the atria, and there is abundant eosinophilic homogenous fluid and increased macrophages and few heterophils in parabronchi. The tunica media and tunica intima of blood vessels are infiltrated by the aforementioned inflammatory cells, necrotic debris (vasculitis), hemorrhage, fibrin, and edema, which also expand the pleura up to 100 µm (pleuritis).

 

MORPHOLOGIC DIAGNOSIS: Lung: Pneumonia, granulomatous, heterophilic, and necrotizing, multifocal to coalescing, severe, with vasculitis and fungal hyphae, Magellanic penguin, avian.

 

SLIDE B: Signalment (JPC #1785683): Pregnant 7-year-old Morgan horse

 

HISTORY: This horse was euthanized due to extreme dyspnea.

 

HISTOPATHOLOGIC DESCRIPTION: Lung: Affecting 70% of the lung are multifocal to coalescing, randomly distributed, infarcts up to 5 mm in diameter characterized by loss of differential staining and retention of cellular architecture (coagulative necrosis) and rimmed by radiating fungal hyphae. Fungal hyphae are 5-8 µm in diameter, have thin, parallel walls, display acute angle, dichotomous branching, and have distinct infrequent septations. Infarctions are centered on blood vessels whose walls are effaced by abundant fungal hyphae, degenerate neutrophils, fewer macrophages, lymphocytes, and plasma cells, fibrin, necrotic cellular debris, hemorrhage, and edema (vasculitis). Occasionally, vessel lumina are partially obstructed by fibrin thrombi. There is loss of adjacent alveolar architecture with replacement by eosinophilic cellular debris, previously described inflammatory cells, and fungal hyphae. Less affected alveoli contain abundant eosinophilic homogenous material (edema fluid) and few alveolar macrophages, and septa are expanded by congestion and previously described inflammatory cells. The lumens of adjacent bronchi and bronchioles contain fewer aforementioned inflammatory cells. 

 

MORPHOLOGIC DIAGNOSIS: Lung: Pneumonia, embolic, necrotizing, severe, with infarction, vasculitis, and fungal hyphae, Morgan, equine.

 

ETIOLOGIC DIAGNOSIS: Pulmonary aspergillosis

 

CAUSE: Aspergillus sp.

 

SYNONYMS: Brooder pneumonia (in poultry); mycotic pneumonia; pneumomycosis

 

GENERAL DISCUSSION:  

• Aspergillus spp: saprophytic molds common in air, soil, and animal feed; mucosal commensals that can cause serious infections in debilitated, neutropenic animals and those on prolonged antibiotic therapy
• A. fumigatus, A. flavus, A. niger, A. nidulans and A. terreus most commonly infect mammals and birds
• Mammals: Pneumonia (+/- systemic dissemination), superficial infections of the skin and cornea occur in most mammalian species (typically sporadic)
• Birds, especially those from colder climates, are particularly susceptible to invasive aspergillosis

 

PATHOGENESIS:  

• Transmission: Inhalation of 2 um conidia or entry through the gastrointestinal tract
• Healthy animal: Phagocytosis of conidia leads to rapid destruction
• If phagocyte function is defective à conidia germinate into hyphae à colonization of respiratory epithelium à produce several virulence factors which impair mucociliary apparatus and damage mucosal epithelium à basement membrane exposure à exposure of hyphal binding sites (fibrinogen, fibronectin, C3 fragments, laminin, collagen) à hyphal invasion
• Subsequent hematogenous spread is common: Aspergillus sp. invades blood vessels and forms mycelial emboli; also may spread via leukocyte trafficking of conidia
• Toxins and virulence factors: 
  • Gliotoxin, fumagillin, and helvolic acid: Impair cilia, damage epithelial cells 
  • Oxalic acid (especially in A. nidulans) reacts with blood and tissue calcium to precipitate calcium oxalate 
  • Secrete heparin-like factors, proteases, elastase, endotoxins, and mycotoxins (aflatoxin, which is a hepatotoxin, teratogen, and carcinogen) 
  • A. fumigatus produces molecules that block the killing mechanisms in neutrophils and macrophages including β-glucan, melanin, and others that block reactive oxygen species and phagolysomal acidification 

 

TYPICAL CLINICAL FINDINGS:  

• Serosanguineous to mucopurulent nasal discharge
• Lethargy and dyspnea
• CNS infection: neurologic signs
• Guttural pouch mycosis: Epistaxis
• Chickens, horses: Corneal ulceration and opacity (mycotic keratitis)
• Skin lesions

 

TYPICAL GROSS FINDINGS:  

• Lungs, other affected organs: Solitary or disseminated gray to yellow, discrete, irregular nodules often surrounded by a rim of hyperemia and hemorrhage
• Compact mycelial mass (“aspergilloma” or “fungal ball”) may form in respiratory tract cavities
• Caseous to green-black plaques in air sacs, nose, or sinuses
• Cotton-like plaques (bread-mold appearance) produced by conidiophores may be present on air-exposed sites (especially in birds)
• Guttural pouch mycosis: Usually unilateral; initially mucosal surfaces covered by plaque; over time progresses to confluence mass; may result in invasion of vital structures including the internal carotid artery leading to epistaxis or mycotic emboli causing cerebral infarcts; glossopharyngeal nerve leading to dysphagia; cranial cervical ganglion and sympathetic fibers leading to Horner’s syndrome; or laryngeal nerves leading to laryngeal hemiplegia

 

TYPICAL LIGHT MICROSCOPIC FINDINGS:  

• Hyphae are 3-6 µm in width, regularly septate, parallel walled, with dichotomous, acute-angle branching; in chronic lesions, the hyphae may form short globose and distorted hyphae up to 12 µm wide
• Vascular invasion of fungal hyphae results in vasculitis, thrombosis, and infarction
• Multifocal, nodular, pyogranulomatous to granulomatous pneumonia with areas of necrosis and granuloma formation 
• Conidiophores (fruiting bodies): Only on surfaces with high-oxygen tension (lung, sinus cavities); central flask-shaped, hemispherical or globose vesicle with one or two rows of peg-like sterigmata which form unbranched chains of conidia on the distal end
• Local depositions of birefringent calcium oxalate crystals may be present 
• May be surrounded by Splendore-Hoeppli material
• Guttural pouch mycosis: Necrotic inflammation of the mucosa and submucosa with widespread vasculitis and fungal hyphae

 

ADDITIONAL DIAGNOSTIC TESTS:  

• Cytology: 
  1. Rhinitis/sinusitis may be neutrophilic, macrophagic, or mixed in nature; hyphae have globose terminal ends and are often in thick areas of the smear; conidiophores with cap (phiadiles) and bluish green spores (conidia) are uncommon; presence of hyphae does not rule out underlying neoplasia
  2. Branching septate hyphae may be apparent in tracheal washes/bronchoalveolar lavage or FNA of nodules; may contain calcium oxalate crystals
  3. Hyphae may stain intensely or be negative staining
  4. Neutrophilic pleocytosis noted in CSF samples in dogs with Aspergillus meningitis
  5. Can be identified in urine (systemic mycosis with mycotic cystitis), feces
• Culture or PCR is required for species identification
• Special stains: GMS, PAS, or Gridley
• Immunoassays: Immunohistochemistry, ELISA, AGID

 

DIFFERENTIAL DIAGNOSIS:  

Other pathogenic fungal hyphae:

• Zygomycetes (Mucor): Hyphae are broader (up to 15 µm), infrequently septate, non-parallel walls which are often collapsed and twisted, orthogonal right-angle branching, well stained by H&E
• Fusarium sp.: Septate but with characteristic right angle, irregularly branching (+/- acute angle branching) and constrictions at branch points
• Pseudoallescheria boydii (scedosporium): More narrow hyphae, usually branch haphazardly rather than progressively and at less acute angles  
• Mortierella sp.: Most common cause of bovine abortions in New Zealand, may result in fibrinonecrotic pneumonia in cow after abortion due to hematenous spread of fungus
• Beauvaria bassiana in reptiles
• Candida sp. form pseudohyphae and budding yeast cells in addition to hyphae
• Ochroconis gallopavum: Lung and/or brain of chicks and poults; dematiaceous fungus (pigmented)

Other causes of granulomatous lesions:

• Mycoplasma gallisepticum: Avian pulmonary granulomas
• Other bacteria that typically cause granulomatous lesions: Actinomyces sp., Mycobacteria sp., Nocardia sp.

 

COMPARATIVE PATHOLOGY:  

Aspergillus sp. in other species:

• Chickens, turkeys, game birds: Brooder pneumonia due to A. fumigatus or A. flavus; typically localized to respiratory tract but can disseminate (especially to the brain, posterior chamber of the eye)
  1. Embryonic infection occurs when fungus penetrates egg hell or is injected via needle puncture
  2. Infected eggs break in hatchery à massive contamination à outbreaks in young birds < 3 weeks of age
  3. Adult birds infected via inhalation; disease occurs when host defenses depressed
• Pet/Aviary Birds (i.e. psittacines) and birds of prey: Localized (miliary granulomas in lungs or air sacs; rhinitis) or disseminated (coelomic cavity, CNS, liver, intestines, kidney, pneumonic bone, adrenal glands) infection can occur; predisposing factors include immunosuppression, concurrent infection, poor nutrition, antibiotic therapy, migration
• Penguins: The most important disease in captive penguins; typically A. fumigatus; adults more commonly affected; predisposing factors include stress, concurrent disease, environmental temperature and spore load 
  1. Chronic form: more common; disseminated form; plaques and thickening of air sacs; fungal granulomas in lungs and other organs
  2. Acute form: rapidly progressive; targets the lungs, which are edematous and dark red with multiple small white foci
• Flamingo: recent report of pulmonary artery aneurysm secondary to A. fumigatus (Veiga, JCP 2023)
• Sea ducks, eiders, and trumpeter swans highly susceptible; outbreaks have been reported, likely secondary to massive exposure
• Cattle: 
  1. Mostly affects the forestomaches, especially the omasum, causing discrete ulcers colored by diphtheritic membranes; the angioinvasive fungus then spreads resulting in mycotic placentitis, mastitis, and abortion; dermatitis in aborted calves – see R-F01 for mycotic placentitis
  2. Previously though to be associated hemorrhagic bowel syndrome in dair cattle; recent article was not in support of this association (De Jonge, Vet Pathol 2023)
• Sheep and goats: pneumonia and mastitis; rhinitis in goats; gross and microscopic lesions are very similar to conidiobolomycosis and cryptococcosis (Silva do Caroma, JVDI 2020)
• Horses: 
  1. Aspergillus was isolated in two cases of fungal rhinitis in Florida as a superficial copathogen (More, Vet Pathol 2019)
  2. Pulmonary aspergillosis is due to hematogenous spread of fungal hyphae resulting from colitis (consequence of both neutropenia and disruption of the mucosal barrier); recent article by Hensel et. al described fungal mycosis in 6 horses with Salmonella enteritis; etiologic agents identified via PCR included A. flavus, A. fumigatus, A. fumigatus and Fusamrium oxysporum coinfection, Cladosporium spp, and Curvularia spp. (J Comp Path, 2020)
  3. Can cause fungal keratitis
• Dogs:
  • Nasal sinus infection (more common in young, dolichocephalic dogs)
  • Systemic aspergillosis: A. terreus and A. deflectus are the most commonly reported isolates; typically affects young to middle-aged female German shepherd dogs; Systemic A. terrerus can cause fungal discospondylitis (Craig, JKP 2016)
  • A. fumigatus and A. flavus can cause chronic destructive bronchiolitis in German Shepherd dogs; generally do not spread systemically 
• Porcupine: Recent report of fatal A. flavus rhinitis with marked gaseous gastric distension; porcupines are obligate nasal breathers, rhinitis thought to cause dyspnea and death (Jalenques, JVDI 2019)
• Dolphins: sporadic respiratory infections reported in stranded and captive cetaceans; opportunistic infection of respiratory tract with possibility for systemic dissemination 
• Sea fans (Gorgonia ventalina and G. flabellum): aspergillosis (A. sydowii) is an endemic disease of sea fans (“octocoral” disease) in the Caribbean; appears as circular to annular foci of purple discoloration (galls); soft tissue (coenenchyme) loss; fouling of the axial skeleton by secondary pathogens such as cyanobacteria; purple discoloration due to carotenoid pigment within sclerites (microskeleton) that accumulate in response to insult; however, recent study showed that these signs are not specific for aspergillosis or fungal infection (Becker, Vet Pathol 2023)
• NHPs: Sole outbreak of OWMs coinfected with M. tuberculosis at a zoologic institute 

 

References:

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