Equine Keratomycosis: Diagnostic Cytology and Therapy

Elizabeth M. Lennington, DVM; Matthew J. Chandler, DVM; and Kenneth S. Latimer, DVM, PhD

Class of 2002 (Lennington), Department of Small Animal Medicine (Chandler) and Department of Pathology (Latimer), College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-7388

Introduction

Keratomycosis is a serious disease in the horse that may be challenging to diagnose and treat successfully. The normal corneal defense mechanisms consist of tear lysozymes, secretory immunoglobulins, leukocytes, and the normal ocular flora. The normal flora usually coexists peacefully with the horse and prevents the establishment of pathogenic organisms.¹ The normal flora of the healthy equine eye varies somewhat by geographical region but may include Gram-positive nonpathogenic bacteria, Gram-negative cocci (Neisseria sp.), Gram-negative bacilli, and various environmental fungi.² Because of the placement and size of the horse’s eye, the cornea may easily be traumatized, allowing normal flora to advance past the epithelial defect into the corneal stroma.³

Keratomycosis is caused by the invasion of commensal ocular fungi or environmental pathogenic fungi into the corneal stroma subsequent to trauma or preexisting bacterial infection. After releasing collagen-digesting proteases, the fungi rapidly invade the stroma.¹

Clinical Signs and Gross Appearance

Signs of keratomycosis include blepharospasm, miosis, epiphora, and photophobia.¹ There is often a history of trauma to the eye, with either a lack of response or worsening of the ulcer after routine antibiotic therapy. Previous administration of topical corticosteroids commonly precedes fungal invasion of the corneal. Therefore, topical treatment with corticosteroids should be avoided when treating corneal ulcers to prevent secondary infections. Keratomycosis also has regional and seasonal predilections, with the majority of cases presenting in humid regions during the late summer and fall.⁴

Keratomycosis has five general clinical appearances (Fig. 1). The most common presentation is an obvious, bacterial-like ulcer with surrounding corneal edema and neovascularization in conjunction with secondary anterior uveitis, flare, and hypopyon. Another presentation is superficial fungal keratitis that appears as a cluster of punctate opacities that are easily debrided from the underlying corneal epithelium. Conversely, some fungal ulcers may be surrounded by a peripheral furrow delineating the infected and noninfected corneal stroma. These ulcers typically cause extreme pain. Another appearance of keratomycosis is an ulcer with a thick, flaky zone of necrotic stromal cells surrounding the ulcer. Finally, corneal abscesses may appear as stromal opacities that are surrounded by corneal edema and neovascularization; the overlying corneal epithelium is intact. The depth of the abscess is variable. Corneal abscesses are thought to result from contaminated, penetrating wounds followed by healing of the overlying epithelium.⁵ Corneal abscesses also may be sequelae to equine recurrent uveitis.⁶

Fig. 1. Horse, keratomycosis. A corneal ulcer is present with a furrowed margin, neovascularization, and edema.

Differential Diagnosis and Scraping Technique

The differential diagnoses for the above clinical presentations include bacterial keratitis, equine recurrent uveitis, indolent ulcer, bacterial stromal abscess, viral keratitis, and corneal dystrophy or degeneration. Diagnosis of keratomycosis is based upon the cytologic evaluation of corneal scrapings.

After applying a topical anesthetic to the cornea, multiple areas from the center and edges of the defect are scraped. The resulting sample should contain numerous corneal epithelial cells. Recovery of fungi often requires a rather deep scraping to adequately sample the corneal stroma. More than one scraping may be needed for diagnosis. When obtaining corneal scrapings, care must be taken to avoid perforating Descemet’s membrane.

Cytologic Features

Romanowsky staining (Wright and Diff-Quik stains) is usually adequate to demonstrate fungal hyphae in cytologic specimens. Upon cytologic examination, masses of intertwining fungal hyphae will often be evident (Fig. 2).⁷ Fungal hyphae, especially of Aspergillus sp., typically have slender parallel walls with septa and branching (Fig. 3).

Fig. 2. Horse, keratomycosis, corneal scraping, Wright-Leishman stain. Masses of intertwining fungal hyphae are present.	Fig. 3. Horse, keratomycosis, corneal scraping, Wright-Leishman stain. Fungal hyphae of Aspergillus sp. have slender, uniform walls with septa and occasional branching.

The hyphae usually appear basophilic if the Romanowsky stain has been adequately bound. Corneal epithelial cells may be observed in most scrapings. These cells are differentiated squamous cells with angular margins. Inflammatory cell infiltrates may be absent, but scattered neutrophils, lymphocytes, plasma cells, and macrophages purulent may be observed occasionally, especially with secondary bacterial infection (Fig. 4). Fibroblasts may occur in foci of neovascularization.⁶ In some cytologic specimens, there is minimal Romanowsky stain uptake by the fungi. They appear pale to ghost-like and are difficult to identify (Fig. 5). In such cases, Gomori methenamine silver (GMS) or periodic acid-Schiff (PAS) staining may readily demonstrate poorly stained or fragmented hyphae (Fig. 6).^4,7

Fig. 4. Horse, keratomycosis, corneal scraping, Wright-Leishman stain. Corneal epithelial cells and a mild infiltrate of neutrophils are present.

Fig. 5. Horse, keratomycosis, corneal scraping, Wright-Leishman stain. Fungal hyphae stain poorly and appear ghost-like in this cytologic preparation.	Fig. 6. Horse, keratomycosis, corneal scraping, GMS stain. Fragment of a fungal hyphae is readily identified as black structure against a fast green-counterstained background.

Fungal Culture and Histopathology

If cytologic examination is equivocal, keratomycosis also may be diagnosed by fungal culture of corneal tissue on blood agar or Sabouraud’s agar and by corneal biopsy. Pathogens isolated from a corneal lesions in horses with keratomycosis often include filamentous, septate fungi such as Aspergillus, Alternaria, Penicillium, Fusarium, or Cladosporium spp. The species distribution may vary somewhat by geographic region, but Aspergillus sp. is isolated most frequently. Histologic examination of keratectomy or enucleation specimens also may provide a definitive diagnosis, but are more expensive and invasive techniques.⁴

Treatment and Prognosis

Early, aggressive therapy is imperative for successful treatment of keratomycosis in horses. Many cases are best managed with surgical intervention early in the course of the disease, particularly if small lesions are present. Surgical techniques commonly used are conjuntival grafting and penetrating corneal grafting.⁸ Medical therapy includes regular and gentle debridement of the affected stroma, allowing medications to reach deeper sites of fungal colonization.⁶ A subpalpebral lavage system is recommended to facilitate treatment and to medicate the lesion reliably. Topical antifungal agents include Miconazole (1% solution or cream), Natamycin (5% suspension), and Itraconazole/DMSO combinations. Each of these drugs differs in penetrability into the cornea and in efficacy against the fungi.⁸ During the first three days of therapy, the antifungal medications should be administered topically every six to eight hours. If given more frequently, these drug concentrations may kill organisms in sufficient numbers to induce an acute, intense iridocyclitis. Starting with the fourth day of therapy, the antifungal drug should be administered every four hours.³

To prevent secondary bacterial infections, a topical antibiotic such as gentamycin with neomycin, polymyxin, and bacitracin should be included in the therapy. To control uveitis, a systemic nonsteroidal anti-inflammatory drug such as flunixin or phenylbutazone should be administered at the normal anti-inflammatory dosage. Flunixin appears to be more efficacious than phenylbutazone in the management of uveitis.⁸ Corticosteroid administration is absolutely contraindicated.

Another important component of therapy is topical atropine (1% solution). Atropine maintains mydriasis to prevent the formation of synechiae and relaxes the ciliary muscles to avoid painful spasms. During atropine therapy, the horse should be observed for systemic effects such as mydriasis of the opposite eye and signs of colic.³ Finally, topical autologous serum or acetylcysteine (10%, diluted with 0.9% sodium chloride) will decrease collagenolysis in the corneal stroma.¹

Proper patient management also is important in treating keratomycosis. Using an eye protector cup/hood, feeding all hay on the ground rather than in a hay rack, and turning the horse out in a supervised pasture environment will aid in protecting the healing eye from further injury and contamination. Removing the horse from stalls and other sites of heavy fungal contamination may help prevent superinfection by environmental fungi. The horse should at least be kept in as clean and dust-free an area as possible.^2,7

Successful therapy for keratomycosis often requires six to eight weeks of intensive and expensive treatment. If the ulcer is very deep, lacks response to medication, or worsens despite therapy, then surgery is indicated. A keratectomy or conjunctival graft, with a resumption of medical therapy, may afford a greater chance of success for the retention of sight in the affected eye.³

The overall success in retention of sight varies with different studies. Commonly, 50% of affected eyes will retain acceptable vision. ³ With proper treatment, superficial keratitis has an excellent prognosis. Keratomycosis with necrotic, flaky, dry material has a good prognosis. Bacterial-like ulcerative lesions have a fair to good prognosis; however, ulcers with a visible surrounding furrow and stromal abscesses have guarded to poor prognosis.⁵ In addition to the risks of loss of sight or loss of the eye, potential complications of keratomycosis include superinfection, corneal scars and pigmentation, corneal rupture, uveal prolapse, uveitis, synechiae formation, cataract formation, endophthalmitis, and phthisis bulbi.⁷

Keratomycosis can be a devastating disease when recognized late or treated inadequately. Cytology is a rapid, inexpensive, and reliable technique to diagnose fungal infection, allowing prompt institution of appropriate therapy. Effective disease diagnosis and aggressive (medical and surgical) therapy provide the best opportunity to combat equine keratomycosis.

References

1. Andrew SE, et al: Equine ulcerative keratomycosis: Visual outcome and ocular survival in 39 cases (1987-1996). Equine Vet J 30: 109-116, 1998.

2. Moore CP, et al: Prevalence of ocular microorganisms in hospitalized and stabled Horses. Am J Vet Res 49: 773-777, 1988.

3. Gelatt KN: Essentials of Veterinary Ophthalmology. Lippincott Williams and Wilkins, Philadelphia, 2000, pp. 353-361.

4. Grahn B, et al: Equine keratomycosis: Clinical and laboratory findings in 23 cases. Prog Vet Comp Ophthal 3: 2-7, 1992.

5. Gaarder JE, et al: Clinical appearances, healing patterns, risk factors, and outcomes of horses with fungal keratitis: 53 cases (1978-1996). J Am Vet Med Assoc 213: 105-112, 1998.

6. Hamilton HL, et al: Histological findings in corneal stromal abscesses of 11 horses: correlation with cultures and cytology. Equine Vet J 26: 448-453, 1994.

7. Barton MH: Equine keratomycosis. Comp Cont Edu 14: 936-944, 1992.

8. Chandler, MJ: Personal communication, 2001.

Acknowledgement

The cave painting of a horse in Lascaux, France, circa 15,000-10,000 BC, is on several sites on the web.

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