8-month-old, Shetland pony, equine (Equus caballus).The lethargic pony was referred to the equine veterinarian in a poor body condition, showing moderate
pallor of mucous membranes, severe dyspnea and diarrhea. Thoracic radiography revealed diffuse clouding shadows
of lung parenchyma with distinct vascular pattern. The pony died two days after admittance to the clinic.
Main findings during necropsy included severe pulmonary edema with multifocal hemorrhage
and multifocal thrombosis, moderate to severe disseminated granulomatous pneumonia in all lobes as well as
granulomatous lymphadenitis of tracheobronchial lymph nodes.Â Moreover, moderate chronic verminous
endoarteritis of the cranial mesenteric artery with intravascular presence of numerous nematodes consistent with
Strongylus vulgarism was present.Â The ileum and large intestine contained numerous nematodes consistent with
Strongylidae spp.Â and multifocally revealed moderate chronic ulcerative ileitis and typhlocolitis with multiple firm
transmural ulcers of 2-3 cm in diameter, partly constricting the lumen, with hemorrhagic margins as well as
multifocal subserosal hemorrhagic plaques (consistent with hemomelasma ilei).Â Most mesenteric lymph nodes were
moderately enlarged and firm with an irregular surface (granulomatous lymphadenitis).Â Liver and kidneys were
diffusely moderately swollen, pale and soft with sporadic small firm white granulomatous foci.
Lung: Expanding and replacing approximately 45 % of the normal lung architecture are multiple discrete up to 6 mm in diameter foci with marked central karyorrhectic debris (necrosis), which are sometimes accompanied by intense eosinophilic material (Splendore Hoeppli phenomenon), and surrounded by numerous degenerate neutrophils, macrophages and fewer lymphocytes and plasma cells.Â Often within necrotic centers few to large numbers of fungal hyphae of 3-6 Î¼m width are present and are characterized by regular septation, thin, parallel walls and dichotomous, progressive acute angle branching.
Multifocal to coalescing alveolar lumina are expanded by homogeneous eosinophilic material (edema), fibrillar eosinophilic material (fibrin) or abundant extravasated erythrocytes (hemorrhage) in varying composition, discontinued by multifocal areas of alveolar distention and ruptured alveolar septae (emphysema).Â Overall, alveolar septae are slightly thickened due to multifocal to coalescing moderate engorgement of erythrocytes (hyperemia) and/ or mild mixed inflammatory cell infiltrates with dominance of neutrophils accompanied by multifocal hyperplasia of type II pneumocytes and a moderate increase of alveolar macrophages (alveolar histiocytosis).
Several small, medium-sized or large arterial and venous blood vessels contain thrombi of different size and extension composed of homogeneous (serous) to fibrillar (fibrinous) eosinophilic material, erythrocytes, necrotic debris, degenerate neutrophils, occasionally admixed with the fungal hyphae described above, accompanied by moderate to marked infiltration of vessel walls by fibrinous to necrotic debris, degenerate neutrophils and sporadically by fungal hyphae.Â Multifocally, but not present in all slides, there is segmental to circumferential necrosis of bronchial/bronchiolar walls with sloughed epithelial cells, mixed inflammatory cells, erythrocytes, fibrin and necrotic debris within the lumina in varying composition and sometimes admixed with the fungal hyphae described above.Â Subpleural, interlobar, perivascular, as well as, peribronchial/peribronchiolar interstitial connective tissue is moderately separated by clear space or homogeneous eosinophilic material (edema) with few mixed inflammatory cell infiltrates.
1.Â Lung: pneumonia, severe, multifocally extensive, acute to subacute, necrosuppurative to pyogranulomatous with intralesional hyphae consistent with Aspergillus sp.Â and bronchitis/ bronchiolitis (not present in all slides), moderate to severe, multifocal, acute, necrosuppurative.
2.Â Lung: vasculitis, severe, multifocal, acute, fibrino-necrotizing with intramural hyphae consistent with Aspergillus sp., thrombosis, alveolar and interstitial edema as well as pulmonary hemorrhage.
The genus Aspergillus encompasses more than 200 species of which only approximately
19 cause disease in humans.(1) Aspergilli reproduce asexually by forming conidia-bearing multicellular structures
(-ï¿½-ï¿½conidiophores) which release millions of uninucleate cells called conidia into the air that are easily dispersed by
the wind and have a diameter small enough (2.5 to 3.5 Î¼m) to reach peripheral airways.(1,25) Aspergillus conidia
occur in soil, air, water and greatest numbers are found in hay and straw enriched with leaf and grass compost.Â They
are considered to be the main vehicle for infective transmission, and when they get the chance to germinate inside
the body producing branched septate hyphae that invade tissues, different forms of aspergillosis can develop.Â (1,25)
Aspergillosis refers to a variety of diseases caused by several species of Aspergillus, which are relatively uncommon in humans and mammals but represent a major cause of mortality in birds.Â The diseases vary in severity and clinical cause, depending on the species and organs affected as well as the immunocompetence of the host.Â Members of the A.Â fumigatus group cause most cases of aspergillosis.(9,23) However, several other Aspergillus species, particularly A. flavus, A.Â terreus, A.Â nidulans and A.Â niger have also been described as causative agents.(7,9,17,26)
In horses, equine guttural pouch mycosis is the predominant form of aspergillosis and is considered a rare, lifethreatening opportunistic infection, with A.Â fumigatus being most frequently isolated.Â There is no breed or gender predisposition and the pathogenesis remains nearly unknown.Â However, predisposing factors might be soft tissue trauma and environmental conditions such as poor ventilation, high humidity and warm temperatures that encourage conidial germination.Â In the majority of cases, the dorsomedial aspect of the guttural pouch is affected, showing necro-hemorrhagic to fibrinous inflammation accompanied by angioinvasion, erosion of cranial nerves and tissue necrosis as common sequelae.(17,23)
Pulmonary or invasive aspergillosis is very uncommon in horses and usually comprises hematogenous spread of fungal hyphae.Â Typical lesions are multifocal embolic pneumonia, often centered on pulmonary vessels, and include neutrophilic and fibrin exudate in alveoli, hemorrhage, necrosis and leukocytoclastic vasculitis with resultant thrombosis and infarction,(8) all of which is also seen in this case.Â More chronic lesions can appear as classic granulomas with central necrotic cores.Â Although hyphae are often present within the lesions and may be readily seen with hematoxylin and eosin stains, special stains, such as Periodic Acid-Schiff (PAS) reaction or Gomori- Methenamine Silver (GMS) stain are helpful to visualize their characteristic morphology.Â In this case, we used PAS reaction to show the characteristic frequent dichotomous branching and hyphal morphology, which is described in table 1.
Important predisposing factors for the development of invasive aspergillosis in horses seem to be prolonged antibiotic, glucocorticoid or NSAID administration,(13,18,22) immunosuppression associated with leukemia,(5) neutropenia ,(4) pituitary adenoma,(6) heavy exposure to conidia from mouldy environmental material(7,13) or prolonged and intense periods of stress.(14,24) In addition, a compromised intestinal mucosa serving as the site of entry, which has been also found in this case in the form of an ulcerative enterocolitis, is considered to be an important predisposing condition for hematogeneous spread of fungi resulting in invasive, systemic aspergillosis, often with predominant pulmonary manifestation.(4,8,13,18,22,24,26) Also mixed invasive fungal infections, such as concomitant aspergillosis and mucormycosis, have been reported in horses(7,24) and calves.(10)
Besides horses, dogs, cows and dolphins also are particularly susceptible to certain forms of aspergillosis.Â In dogs, canine sinonasal aspergillosis predominantly affects mesocephalic or dolichocephalic breeds, where A.Â fumigates is most commonly isolated, but A.Â terries predominates in cases involving German shepherd dogs.Â Predisposing factors are unknown, and pathology is concentrated on the nasal cavity and paranasal sinuses.Â Sometimes the cribriform plate is invaded and CNS infection may be established.(23) Bovine mycotic abortion due to Aspergillus infection occurs worldwide and sporadically.Â Typically second or third trimester abortion is observed, with highest incidence during winter when gravid cows are indoors and fed with heavily contaminated hay or silage.Â Aspergillus fumigatus accounts for the majority of cases and suggested routes of infection are ingestion or inhalation.Â The pathological hallmark is placentitis with leathery appearance of the placenta (almost pathognomonic), intercotyledonary thickening and hypertrophy of cotyledons.Â (23) Cetacean mycotic pneumonia is the predominant form of aspergillosis in free ranging dolphins and is considered to be a regional disease.Â Predisposing factors are suspected to be a combination of viral and environmental factors.Â (23)
In contrast to the rather low incidence of aspergillosis in mammals, avian aspergillosis resembles a major cause of morbidity and mortality in birds and affects animals of all ages, whether immunocompetent or immunosuppressed in captive or free-ranging environments; captive penguins seem particularly susceptible.Â (2) In approximately 95% of the cases A.Â fumigatus is isolated, and A.Â flavus occurs second most frequently.Â Inhalation is the route of infection, with initial colonization in the lower respiratory tract.Â Susceptibility may be attributed to differences in innate and acquired immunity compared to mammals, as well as predisposing anatomic characteristics such as lack of an epiglottis and diaphragm (inability to produce strong cough), limited distribution of pseudostratified ciliated respiratory epithelium, lack of surface macrophages, and different heterophilic killing mechanisms (using cationic proteins, hydrolases and lysosymes rather than myeloperoxidases and oxidative mechanisms).Â A unique feature of avian aspergillosis is the presence of reproductive phases in tissue.Â (23)
The most common forms of human aspergillosis are pulmonic and may be divided into (1) non-pathogenic saprophytic colonization, including noninvasive pulmonary aspergillomas/mycetomas or invasion of necrotic tissue; (2) hypersensitivity-induced aspergillosis, including Aspergillus-asthma, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis (or extrinsic allergic alveolitis), bronchocentric granulomatosis and chronic eosinophilic pneumonia; and (3) invasive disease, including pseudomembranous tracheobronchitis, acute bronchopneumonia, angioinvasive aspergillosis, chronic necrotizing aspergillosis and invasive pleural disease.(1,25)
In general, human patients with pre-existing structural lung disease, atopy, occupational exposure or impaired immunity are susceptible.(1) Therefore, saprophytic colonization is increased in patients with advanced stages of chronic obstructive pulmonary disease (COPD), chronic asthma requiring long-term steroid therapy, primary ciliary dyskinesia syndrome and cystic fibrosis.(25) Aspergillus spp.Â also have a significant potential to act as powerful allergens; thus, hypersensitivity-induced Aspergillus-asthma in lower airways is caused by Type I anaphylactic reaction in atopic individuals upon exposure to Aspergillus conidia or hyphae.Â Allergic bronchopulmonary aspergillosis (ABPA) is caused by hypersensitivity to colonised Aspergillus sp., resembling a complication of asthma, and is immunologically characterized by Type I, Type III and Type IV hypersensitivity.Â Hypersensitivity pneumonitis (or extrinsic allergic alveolitis) occurs primarily in non-atopic individuals.Â It is an inflammatory interstitial lung disease possibly resulting from Type III and Type IV hypersensitivity reactions following persistent or intense exposure to Aspergillus conidia with acute lung injury via complement-dependent neutrophils (due to Type III reaction) or chronic stages such as granuloma formation, interstitial lung fibrosis and distal bronchiolitis obliterans (due to Type IV reaction).Â (25)
In human patients with altered local or systemic immune defense mechanisms, most severe and life-threatening invasive disease may develop.Â (1,25) Invasive pulmonary aspergillosis ranks second to candidiasis in causing systemic fungal infections in immunocompromised human patients, and in most cases lung manifestation occurs with common haematogenous spread to other organs, especially the CNS.Â Hence, immunosuppression is considered to be the major condition with prolonged neutropenia as the leading cause.Â (25)
In healthy, immunocompetent individuals, various elements of the pulmonary innate immune system are involved in recognition and elimination of inhaled Aspergillus conidia, thereby preventing colonization of the respiratory system.Â Ciliated and mucus secreting epithelial cells perform effective mucociliary clearance that is important for entrapment and elimination of inhaled conidia.Â Surfactant, mainly produced by Type II pneumocytes and Clara cells, has been implicated in antimicrobial activity, with surfactant protein A and D serving as collectins.Â Alveolar macrophages represent first line phagocytic defence by intracellular killing of swollen spores and prevention from germination.Â Recruited neutrophils play an essential role by extracellular (degranulation) as well as intracellular (phagocytosis) elimination of Aspergilli.Â Dectin-1, expressed by macrophages, neutrophils and dendritic cells, is an important receptor of innate antifungal defence, being essential for spore recognition and phagocytosis as well as production of oxygenated free radicals (fungicidal activity).Â Additionally, certain Toll-like receptors (TLR) have been found to play a predominant role in the recognition of A.Â fumigatus (TLR2: recognition of spores, TLR4: recognition of spores and hyphae).Â (3)
On the other hand, several pathogenicity factors were found in different Aspergillus spp.Â to overcome certain host defense mechanisms, such as endotoxins that inhibit epithelial ciliary activity, as well as a variety of proteases (incl. elastase, collagenase and trypsin) that damage epithelial cells and thus impair effective mucociliary clearance.(1,3) Further, A.Â fumigatus produces a phospholipid capable of decreasing the binding of complement factor C3b to its surface, resulting in disturbed complement activation.Â (3) Also other fungal proteins of A.Â fumigatus are probably related to virulence by promoting mycelial growth into lung parenchyma or structural alterations of conidia that are resistant to host defence mechanisms.(1)
Moreover, it is likely that Aspergillus mycotoxins can work as virulence factors due to direct cytotoxic effects.Â In vitro studies revealed that aflatoxin (produced by A.Â fumigatus) suppresses the function of macrophages, and ochratoxin (produced by A.Â ochraceus) is cytotoxic to lymphocytes and suppresses lymphocytic, monocytic and granulocytic activity.Â Other possible immunosuppressive mycotoxins gliotoxin, fumagillin, fumigacin, fumitremorgin A and Asp-hemolysin are discussed, while different mycotoxins together may have synergistic effects.Â However, further in vivo studies are needed for confirmation of direct relation to Aspergillus pathogenesis.(15)
Beyond that, melanin pigment, mannitol, catalases and superoxide dismutases are suggested as antioxidant defenses produced by Aspergillus.(11,27) Although it seems that certain antioxidant molecules produced by A.Â fumigatus do not directly inhibit the oxidizing activity of phagocytes, inhibition of reactive oxygen species production by macrophages (e.g.Â during corticosteroid treatment) abolishes their ability to kill the spores while phagocytosis continues so that conidia can germinate and proliferate intracellularly.Â (3) However, since pulmonary macrophages and neutrophils constitute a crucial part of first line innate host defense, neutropenia and long-term corticosteroid treatment are generally regarded as major risk factors for the pathogenesis of invasive aspergillosis.(1,3,11,19)
Table 1: Morphological characteristics, pathogenicity factors and manifestations of common mycotic diseases caused by mycelial fungi:
|Species||Hyphal morphology||Septae/Branching||Pathogenicity factors/|
|Aspergillis spp (1,3,9)||hyphae width 3-6 Î¼m; thin parallel walls (conidia not seen in tissue)||regularly septate; dichotomous progressive 45Â° branching||adhesins, antioxidants (e.g.Â melanin), various proteases; mycotoxins (e.g.Â gliotoxin, aflatoxin, ochratoxin)||sinonasal, placental, pulmonary, angioinvasion, systemic|
|Fusarium spp.(9)||hyaline hyphae width 3-7 Î¼m (similar to Aspergillus spp.)||septate, frequent characteristic 90Â° branching (sometimes 45Â°)||mainly plant pathogenic; mycotoxins (e.g.Â fumonisin B1, fusaric acid)||rare mycosis; pulmonary, angioinvasion, dermatitis, keratitis|
|Candida spp.(9,21)||round or oval hyaline budding blastospores 3-5 Î¼m; hyaline hyphae and pseudohyphae (excl.Â C.Â glabrata)||septate, irregular branching||adhesins, formation of biofilm (oxylipin farnesol), different aspartate proteinases, phenotypic switching; no toxins described||mucocutaneous (e.g.Â vaginal, oral), cutaneous, GIT, systemic (e.g.Â pulmonary, renal), rarely angioinvasion|
|Zygomycetes (Mucor spp.Â Rhizopus sp.Â Basidiobolus spp.Â Mortierella spp.) (9,16)||broad hyphae (up to 25 Î¼m width and 200 Î¼m length ), non-parallel, thin walls (often folded, collapsed or twisted); slightly visualized by PAS and GMS stain||infrequently septate; non-dichotomous, irregular branching; frequently bizarre forms, focal bulbous dilatations||free iron & acidosis play central role; adhering (subendothelial matrix), possibly secreted toxins or proteases; endo-symbiotic endotoxin-producing bacteria (Genus Burkholderia)||cutaneous, subcutaneous, angioinvasion, systemic (with or without pulmonary focus), rhino cerebral|
|Pseudallescheria boydii(12,20)||narrow hyphae, parallel walls (similar to Aspergillus spp.)||septate, highly branching at less acute angles, intertwined||Î±-Glucan, proteases, metalloproteases, superoxide dismutases (pho sphatases)||cutaneous, subcutaneous, respiratory tract/pulmonary, angioinvasion|
Lung: Pneumonia, necrohemorrhagic, multifocal to coalescing, severe, with necrotizing
vasculitis, intra-alveolar edema and fibrin, and myriad angiocentric fungal hyphae, etiology consistent with
The contributor provides an excellent summary of the comparative pathology and
pathogenesis of aspergillosis.Â Like the contributor, participants noted the close association of fungal hyphae with
blood vessels and the presence of angioinvasion, and thus favored the use of the term angiocentric in the
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