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Keywords:

  • chickens;
  • mycotic keratitis;
  • scedosporium apiospermum

Abstract

  1. Top of page
  2. Abstract
  3. History
  4. Discussion
  5. References

Investigation of ocular disease in a flock of layer pullets identified corneal infection with Scedosporium apiospermum, a fungus of emerging significance in medicine and veterinary medicine.


History

  1. Top of page
  2. Abstract
  3. History
  4. Discussion
  5. References

A small proportion (50–100 of 32 000) of 42-day-old birds in a layer pullet flock was noticed by the farm manager to have ocular abnormalities. The birds had been placed at day old on a deep litter of softwood shavings that was freshly laid for this flock, but at the time of investigation was rather dusty. Mortalities in the shed had not increased, and there was no evidence of a snick (sneeze) or respiratory noise in the shed. Affected birds were not depressed, but moved away with the rest of the flock when stimulated. Only when cornered or ‘blindsided’ against the wall where they easily identified and caught. Clinical examination of affected birds showed unilateral keratoconjunctivitis and apparent exopthalmos (Fig. 1), with head shaking and scratching at the affected eye. Affected eyes of most severely affected birds had a thick, cream apparently fibrinous discharge that could be ‘shelled out’ with a cotton tipped applicator. No other clinical abnormalities were detected.

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Figure 1. Clinical appearance of pullets with corneal clouding, conjunctivitis and apparent protrustion of the eye caused by the abrupt thickening of the cornea.

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Antemortem blood was taken, and serology for Mycoplasma gallisepticum and Mycoplasma synoviae was negative. Chlamydiaceae OMP 1 PCR of cloacal and choanal swabs from all examined birds were also negative. Yolk sac inoculation of 6-day-old fertile eggs with Chlamydiaceae transport media from pooled conjunctival swabs was overgrown with a fungus on day 5, despite the presence of 5 μl/ml of Amphotericin B in the transport media. Fungal samples were forwarded to the Microbiological Diagnostic Unit of the University of Melbourne, and this fungus was identified there as Scedosporium apiospermum.

Autopsy was performed by the field veterinarian (SB), who noted that macroscopic lesions were confined to the eyes. The damaged eyes had opaque, cream-white corneas that bulged abruptly to a height of 1 to 2 millimeters above the limbus. Grossly, the corneas were thickened, and in some birds, the anterior chamber was obliterated by gray-white fleshy tissue. Iris stroma of these birds was discernible only as irregular pigmentation within this tissue, and iridocorneal angles could not be identified macroscopically. Lens changes varied from mild focal anterior opacification to severe extensive opacity with the anterior lens capsule not smooth but irregularly rough. Nasal passages, sinuses, and viscera including respiratory tract were grossly unremarkable.

Histologically, lesions were again confined to the eye, with only one eye from each bird affected. There was complete loss of the epithelium over much of the cornea.(Fig. 2) The stroma was expanded by a severe superficial and midstromal, to full thickness in the central cornea, infiltrate of granulocytes, lymphocytes, plasma cells, and histiocytes, the latter occasionally forming prominent multinucleated giant cells.(Fig. 3) Granulocytes predominated in the superficial layers and plasma cells in the deeper layers. In some birds, the inflammation was full thickness to penetrating across most of the cornea. The stroma itself showed patchy to extensive necrosis, and keratocytes were activated, with enlarged, pale nuclei in moderate cytoplasm, or proliferative. Within the inflammation, particularly near areas of necrosis in the superficial and mid stroma, there were few to moderate numbers of branched, septate fungal hyphae. Staining with both PAS and GMS highlighted the fungi within areas of necrosis and within multinucleated cells (Fig. 4).

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Figure 2. There was extensive loss of corneal epithelium (*), inflammatory cell infiltration in the corneal stroma (C)and adhesion of iris leaflets (I) to the endothelium. H&E L = lens.

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Figure 3. The corneal reaction consisted of marked granulocytic (arrows), lymphoplasmacytic (open arrow heads) and histiocytic (arrow heads) infiltrates, including giant cell (*) formation within which negative hyphal images were sometimes visible, within a vascularised and oedematous stroma. H&E.

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Figure 4. Periodic Acid Schiff stain highlights fungal hyphae (arrow) in areas of necrosis and within multinucleated cells (*).

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Endothelium and Descemet's membrane were missing from the central corneal region at the area of full thickness inflammation. There was a wedge of fibrous tissue bridging the gap. In worst affected birds, Descemet's membrane, which is very thin at this age, could not be detected. Where present, endothelial cell density was normal.

In birds with obliteration of the anterior chamber, there was extensive adhesion of the iris to the posterior cornea, particularly at the areas of full thickness corneal inflammation. In these birds, the iris was only identifiable by the lines of pigment. In other birds, there was moderate to severe infiltration of the iris generally becoming mild to minimal in the ciliary body with inflammatory cells of a similar type to those in the cornea. Granulocytes predominate with small numbers of giant cells.

Lens epithelium was irregularly multilayered and showed fibrous metaplasia centrally and at one pole in more moderately affected birds. The worst affected birds had wrinkles and sometimes fragmented lens capsule outlining a pocket of proliferative epithelial cells. These birds had marked lymphocytic inflammation of the pecten, but the posterior segment in all birds was otherwise unremarkable. There was moderate to severe lymphocytic conjunctivitis in some birds more evident in ventral lids, in the birds that had it, than in the dorsal lids. No inflammation or other abnormalities were evident in the orbital tissue or on any viscera. Nasal passages and sinuses were not examined histologically.

The diagnosis was made of severe chronic mycotic keratitis and severe chronic active iritis or iridocyclitis, with extensive anterior synechia formation and cataract, and pectenitis secondary to the anterior segment inflammation.

Discussion

  1. Top of page
  2. Abstract
  3. History
  4. Discussion
  5. References

Reports of fungal keratitis in poultry are few, and the isolated agent has been Aspergillus fumigatus in all previous cases, confirmed by fungal culture. Itakura et al., (1973) reporting on natural cases, showed that infection was related to the environment of the birds; morbidity coincided with the introduction of chip litter.[1] Reis (1940), (reported in[1, 2]) described lesions involving much of the anterior segment in birds 2–5 weeks of age, which were reproducible by dropping a suspension of Aspergillus fumigatus onto a deeply scarified cornea. Corneal lesions caused by ammonia fumes may permit fungal colonization in chickens.[3]

Mycotic lesions arising in the cornea may extend to involve the anterior segment, but rarely progress into the posterior part of the eye, in contrast to the endophthalmitis associated with respiratory disease, in which retina and vitreous contain the major lesions and cornea is unaffected. Corneal infections are almost always unilateral.[1]

Cytological and histological distinction of Scedosporium apiospermum from Aspergillus spp is difficult. The two genera are closely related and share antigenic epitopes in formalin-fixed tissues.[4] Taxonomy of S. apiospermum. has been confused; recent phylogenetic studies using multilocus genetic investigations have shown that S. apiospermum and Pseudallescheria boydii, once considered to be the teleomorph, or sexual stage, belong to a complex of at least eight different species, including these as two separate species.[5-7] Among clinical isolates, there are now three species of major interest within this group; S. apiospermum, P. boydii, and the newly described Scedosporium aurantiacum.[7] Molecular typing was not available for the isolate from these birds.

In humans, the species in which mycotic keratitis is best described, damage to the corneal surface, including surgery and extended contact lens wear, is a major risk factor.[8] Infection by S. apiospermum complex following penetrating injury and implantation of infectious material is also well described.[9] S. apiospermum has been isolated from sinus infections in psittacine birds,[10] but ocular disease was not evident in these cases. Newton (2012) recently described S. apiospermum keratomycosis in a dog, without known trauma, but apparently associated with prolonged antibiotic therapy.[11] Both S. apiospermum and P. boydii have previously, although rarely, been isolated from canine keratitis,[12, 13] and subconjunctival mycetoma caused by S. apiospermum. is reported in a horse.[14] Zoonotic risk is not reported.

S. apiospermum is a filamentous fungus of widespread distribution, recently described as ‘one of the clinically significant emerging mycoses.’[4] It has, however, a long history, being first recognized in human otitis in the late 19th century and in the mycetoma known as ‘madura foot’ in the early 20th.[15] Its tolerance to cold, low oxygen tension, and high salinity make it a survivor in polluted environments.[15] Cortez et al. (2008) consider recovery of the species from unpolluted environments to be rare;[15] Keltseis et al. (2009) recovered Scedosporium and Pseudallescheria spp. from agricultural soils, but found that the frequency of recovery was higher in urban and industrial environments.[9] An affinity with manure enrichment has been noted.[9] The increasing rate of environmental detection of S. apiospermum suggests that infection may become more common in domestic species and poultry as it has in humans.

Management conditions in the shed in which these affected birds were housed were routine for Australian layer sheds, and there was no evidence at the time of field investigation that there had been increased moisture in the litter or more than usual faecal contamination. It is possible that the rather dusty conditions caused mild irritation, although there was generally little conjunctivitis or other evidence of this, and eyes and adnexa from a clinically normal bird were also histologically unremarkable. The source and pathogenesis of the lesion remain uncertain in these birds.

In summary, we report unilateral keratitis in layer pullets caused by Scedosporium apiospermum, a fungus of emerging medical and veterinary significance.

References

  1. Top of page
  2. Abstract
  3. History
  4. Discussion
  5. References