Case Report Limbal Conjunctival Plaques and Idiopathic Eosinophilic Conjunctivitis J

EQUINE VETERINARY EDUCATION / AE / AUGUST 2010 375

Case Report Limbal conjunctival plaques and idiopathic eosinophilic conjunctivitis J. E. Wolfe*, M. E. Utter, A. G. Boyle and P. L. Habecker Department of Clinical Studies New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, USA.

Keywords: horse; equine; eosinophilic; conjunctivitis; plaques; keratoconjunctivitis

Summary et al. 2007; Maggs 2008). Eosinophils are not normally present in conjunctival scrapings and the presence of A 4-year-old Welsh pony crossbred gelding was examined even a few is considered significant (Giuliano and Moore for acute onset of blepharospasm, epiphora and corneal 2002). Eosinophilic infiltration of the conjunctiva and oedema. Ophthalmic examination identified 2 conjunctival surrounding tissues is found in cases of eosinophilic keratitis plaques located near the superior limbus of the left eye. The (EK), eosinophilic keratoconjunctivitis (EKC), eosinophilic plaques did not resolve following treatment with topical conjunctivitis (EC), ocular onchocerciasis and cutaneous triple antibiotic ointment, topical atropine ointment and oral habronemiasis (Fadok and Mullowney 1983; Yamagata flunixin meglumine and therefore a conjunctival biopsy was et al. 1996; Pusterla et al. 2003, Brooks 2005). Eosinophils are performed. Histology of the biopsy resulted in a diagnosis of inflammatory cells associated primarily with allergic and eosinophilic conjunctivitis, which was treated with a topical parasitic infections. Most of these diseases are diagnosed corticosteroid ointment. Plaques resolved after 73 days of through careful examination, clinical signs, cytology and therapy but lesions did not improve with concurrent histopathology of lesions and response to treatment. treatment with anthelmintics early in the course of therapy. Equine EK and EKC are diagnosed based on clinical Failure to identify an aetiological agent led to a diagnosis of signs and cytological findings of eosinophils, a few mast idiopathic eosinophilic conjunctivitis. The conjunctival cells, lymphocytes and plasma cells (Ramsey et al. 1994; plaques have not recurred in the 10 months following Yamagata et al. 1996). Cases of EK and EKC are discontinuation of therapy. characterised by white to pink raised plaques involving the cornea alone or the cornea and conjunctiva (Ramsey et al. Introduction 1994; Yamagata et al. 1996; Brooks 2005). The pathogenesis of EK and EKC is currently unknown but it is thought that eosinophilic infiltration may be associated with an immune, Conjunctivitis in the horse is a manifestation of ocular allergic or parasitic cause. Long-term corticosteroid disease (Brooks and Matthews 2007). It can be either therapy is required to obtain resolution of lesions and primary, or secondary to infectious or noninfectious recurrence is common (Yamagata et al. 1996, Brooks 2004). diseases of the lids, cornea, sclera, anterior uvea, orbit and Eosinophilic ocular diseases are well documented in the cat nasolacrimal system (Brooks and Matthews 2007). Causes and may offer insight into the disease process in the horse. In of conjunctivitis include allergic and immune reactions, cats, EC, EK and EKC have been documented. These 3 conjunctival foreign bodies, parasitism, neoplasia and feline diseases are differentiated by the location of lesions viruses (Brooks and Matthews 2007). Diagnosis is made and clinical signs. through a combination of clinical signs, culture, cytology Parasitic diseases are suspected causes of conjunctivitis and histopathology. in the horse. Two major parasitic diseases that can involve On cytology preparation, a normal equine conjunctiva the conjunctiva include cutaneous habronemiasis and scraping yields sheets of desquamated epithelial cells and ocular onchocerciasis (Fadok and Mullowney 1983; goblet cells accompanied by lesser numbers of Pusterla et al. 2003; Brooks and Matthews 2007). When erythrocytes, lymphocytes, monocytes, plasma cells and ocular tissues are involved in both of these diseases, neutrophils (Giuliano and Moore 2002; Bourges-Abella eosinophilic infiltration of ocular tissues is characteristic. This case report describes the clinical and *Corresponding author email: [email protected] histopathological findings and treatment of a young

gelding with idiopathic EC. A definitive aetiology for the conjunctival plaques and eosinophilic infiltration present in this case was not identified. Through histopathology and response to various systemic and ocular treatments, known causes of conjunctival eosinophilic infiltration were ruled out. To the authors’ knowledge no previous reports of EC have been documented in the horse. Case details

History

A 4-year-old chestnut Welsh Pony Cross gelding was initially examined in the month of December, with an acute history of corneal oedema, blepharospasm and epiphora of the left eye (oculus sinister [OS]). Fig 1: Initial presentation of ocular plaques, corneal oedema, aqueous flare and superficial temporal neovascularisation of the Clinical findings left eye (the eye is not stained).

On examination, the pony had mild blepharospasm and epiphora OS. A neurological exam revealed a positive menace, palpebral, and dazzle reflex OS. Direct pupillary light reflex (PLR) and a consensual PLR from left to right were present. Due to the pony’s temperament, the remaining examination was conducted under standing sedation using detomidine hydrochloride (0.01 mg/kg bwt) and butorphanol tartrate (0.01 mg/kg bwt) i.v. Examination with a slit lamp biomicroscope and tonometer was not available in the field. An auriculopalpebral nerve block was performed using 3 ml (60 mg) of 2% mepivacaine hydrochloride. A direct ophthalmoscope (Welch Allyn 3.5V Standard Ophthalmoscope)1 was used for examination. On examination, 2 small white plaques, 3 mm in size were seen at the dorsal limbus extending onto the bulbar conjunctiva. Corneal oedema was present in the superior portion of the cornea with mild neovascularisation dorsally (Fig 1). Initial evidence of keratitis without plaque Fig 2: Left eye ocular plaques and uptake of Rose Bengal stain 3 days after treatment with topical BNP ointment and autologous involvement of the cornea was evident. On palpation, the serum. Superior corneal oedema is visible. plaques were firm and well-adhered to the underlying conjunctiva. No abnormalities were noted on fundic examination. Mild aqueous flare was observed in the OS) until the pupil was dilated, autologous serum (0.2 ml anterior chamber (Fig 2). Rose Bengal and fluorescein t.i.d. OS), and bacitracin-neomycin and polymyxin (BNP) stains were applied and there was local uptake of both ointment (t.i.d. OS) until further examination. Due to stains over the plaques. The plaques did not appear to suspected bacterial involvement, autologous serum was extend onto the cornea and involved primarily the bulbar used initially for its antiprotease properties. The horse conjunctiva. The right eye (oculus dexter [OD]) was received flunixin meglumine (1.1 mg/kg bwt i.v.) once examined and no abnormalities were noted. Additional following ophthalmic examination, and then was placed relevant history included inadequate deworming one on flunixin meglumine (0.5 mg/kg bwt per os s.i.d.) for month prior to presentation: the gelding received a dose 4–5 days. of ivermectin dewormer below the recommended dose Aerobic culture yielded a coagulase-negative (100 mg/kg bwt). On subsequent examination, 3 days after Staphlococcus group. Staphlococcus species are presentation, a new 5 mm nodule had developed at the commonly cultured from the equine cornea (Brooks 2004; dorsolateral bulbar conjunctiva. Maggs 2008). A scraping of one of the plaques was performed under standing sedation with xylazine Treatment and clinical management (0.45 mg/kg bwt) and topical proparacaine ophthalmic solution 0.5% (15 mg) OS. A cotton swab was used to Culture and sensitivity was obtained of the 2 small plaques. scour the lesion and the larger plaque was easily peeled Initial therapy consisted of atropine solution 1% (0.1 ml s.i.d. off. The swab was then rolled onto a glass slide. Slides

were then stained with Diff-Quik stain. On microscopic paraffin-embedded (FFPE) tissue. The plaque of examination using 100¥ magnification under oil conjunctiva was covered by an intact stratified squamous immersion, sheets of desquamated epithelial cells with an epithelium that was oedematous and infiltrated by abundant number of widely scattered uniformly-sized migrating eosinophils (Fig 3). Eosinophils and neutrophils eosinophilic granules devoid of cellularity were observed. were scattered throughout the stroma. Deeper stroma Ophthalmic treatments were continued until referral after revealed clusters of eosinophils and dilated lymphatics culture and cytology results. The gelding was referred to (Fig 4). No aetiological agents were visible. The the George D. Widener Hospital for Large Animals at the lymphoplasmacytic component of the superficial University of Pennsylvania’s New Bolton Center for stroma was consistent with normal conjunctiva. A further evaluation. histopathological diagnosis of eosinophilic conjunctivitis was made.

Referral and biopsy Progression and outcome

On referral to the George D. Widener Large Animal Five days after referral, the recommended dose of Hospital, corneal oedema and mild blepharospasm OS ivermectin (200 mg/kg bwt) was given and topical BNP noted previously had persisted, but there was no epiphora noted. The gelding was sedated with detomidine (0.03 mg/kg bwt i.v.) and butorphanol (0.01 mg/kg bwt i.v.) for ophthalmic examination. No abnormalities were noted OD. The left eye appeared visual and comfortable. Menace response, dazzle reflex, and palpebral reflexes were positive OS. Direct2 and indirect ophthalmoscopy using a Finoff transilluminator2 and 15D Volk indirect lens3 and slit lamp biomicroscopy using a Kowa SL-154 were performed on both eyes (OU). Intraocular pressures OU were measured using a Tono-Pen XL applanation tonometer5. Intraocular pressures were within normal limits at 18–20 mmHg OD and 23 mmHg OS. No significant abnormalities were found OD. PLRs OS were negative, with a fixed and dilated pupil. This was attributed to a previous atropine administration. A yellow raised lesion 2–3 mm in size was located subconjunctivally at the superior limbus, with mild diffuse superior corneal oedema, and faint Fig 3: Histopathology of conjunctival plaque showing lymphocytes superficial neovascularisation associated with the lesion. (closed black arrow), plasma cells (open arrow) and eosinophils Corneal oedema and evidence of keratitis were attributed migrating through the epithelium (arrowhead). Gaps between to sympathetic inflammation secondary to conjunctivitis. epithelial cells are indicative of intercellular oedema. H&E stained, ¥400. No other anterior chamber, lens or fundic abnormalities were noted. Topical proparacaine 0.5% was applied OS prior to sampling of the lesion. Biopsy of the yellow limbal lesion was performed using Westcott tenotomy scissors. The biopsy specimen was submitted for histopathological analysis and cytology. No culture was performed due to previous culture 3 days prior to referral. Diagnosis was a subconjunctival mass with secondary conjunctivitis and keratitis OS. The gelding was discharged post biopsy on topical BNP ointment t.i.d., atropine sulphate 1% ophthalmic solution (0.1 ml s.i.d. OS) as needed, and flunixin meglumine (0.5 mg/kg bwt per os s.i.d.) until recheck in 4–5 days. Topical autologous serum was discontinued due to lack of corneal ulceration and topical antibiotic therapy was used for antibacterial coverage.

Histopathology

Fig 4: Histopathology of conjunctival plaque showing stratified Haematoxylin and eosin (H&E) stained, 5 mm thick squamous epithelium, dilated lymphatics (asterisks) and clusters of histology sections were created from formalin-fixed, eosinophils in the deep stroma (arrows). H&E stained. ¥100.

b) Fig 5: Left eye conjunctival plaque appearance 5 days post-biopsy. Re-emergence of the plaque is evident.

ophthalmic ointment (t.i.d. OS) was continued. Topical atropine sulphate 1% solution was discontinued. Subsequent examinations at 5 days post biopsy/referral (Fig 5), 10 days and 24 days post treatment with ivermectin (Fig 6a,b) revealed continued plaque formation and growth. Resolution of corneal oedema was evident on the day of ivermectin treatment (Day 10 after presentation) and aqueous flare resolved 10 days after treatment with ivermectin (Day 20). A repeat culture was performed and Streptococcus equi ssp. zooepidemicus was isolated. The isolate was susceptible to many antimicrobials c) including Neomycin, and Polymixin B. Streptococcus species are common conjunctival isolates (Maggs 2008). A scraping of one conjunctival plaque was performed. The plaque was easily removed and the scalpel blade sample wiped across a slide, stained with Diff-Quik and then submitted for cytological evaluation. Cytology revealed a moderate number of desquamated epithelial cells, and degenerate neutrophils, monomorphic cocci and several eosinophils were present. No intracellular bacteria were noted. Due to continued clinical signs in the face of antimicrobial and antiparasitic treatment, steroidal therapy was instituted 29 days after biopsy. The gelding was placed on topical dexamethasone, neomycin and polymixin (DexNP) ointment (b.i.d. OS). Re-examination 2 and 5 weeks following initiation of steroid therapy Fig 6: Ocular conjunctival plaque appearance at: a) 10 days post oral ivermectin, b) 24 days post oral ivermectin, c) 49 days on revealed bulbar conjunctival plaques OS, which were topical dexamethasone, small conjunctival plaque still present. resolving in size and number (Fig 6c). No other ocular abnormalities were present. After 10.5 weeks of topical steroid therapy, conjunctival plaques had resolved (Fig 7). Full ophthalmic examination revealed no ocular or Discussion globe abnormalities OS other than mild conjunctival scarring at site of plaque formation. The gelding has Conjunctivitis is a commonly occurring ocular been examined since resolution of conjunctival plaques manifestation of disease with numerous causes. In this at bimonthly intervals and plaques have not recurred in case, EC was diagnosed. Causes of eosinophilic infiltration the last 10 months. of the conjunctiva include EK, EC, EKC, and ocular

Further evidence against parasitic causes for this gelding’s lesions included histopathological differences. Histologically, habronemiasis lesions are characterised by foci of coagulative necrosis surrounded by granulomatous infiltration with eosinophils, with or without nematode larvae in the centre of the foci (Fadok and Mullowney 1983; Scott 1988; Pusterla et al. 2003). In cases of ocular onchocerciasis, fragments or whole microfilaria may be seen as well as cellular infiltration of lymphocytes, neutrophils, plasma cells and large numbers of eosinophils in corneal and conjunctival lesions on cytology and histopathology (Munger 1983; Brooks and Matthews 2007). In the histological sections of this gelding, no microfilariae were seen. We are aware that microfilaria distribute in nests or pockets and can therefore be missed on biopsy (Schmidt et al. 1982; Scott 1988). However, Fig 7: Resolution of lesions after 73 days on dexamethasone. there were no foci of coagulative necrosis located and the eosinophils extended into the deep stroma of the conjunctiva. The cytology samples contained uniformly-sized eosinophilic granules probably from habronemiasis and onchocerciasis. All of these diseases degranulated eosinophils. are diagnosed based on history, clinical signs, histology Treatment of parasitic diseases is similar and and cytology. To the author’s knowledge, no previous response to ivermectin differentiates habronemiasis and cases of equine EC without corneal involvement have onchocerciasis from each other and other diseases. been documented in the literature. However, feline EC is Habronemiasis and onchocerciasis are treated through well documented and may offer insight into the equine surgical reduction of lesions, systemic ivermectin disease process documented in this case. Through (200 mg/kg bwt), systemic NSAIDS, and systemic and/or thorough treatment and repeat cytological examinations, topical steroids (Fadok and Mullowney 1983; Munger 1983; established causes of eosinophilic infiltration of the Scott 1988; Pusterla et al. 2003). This treatment has led to conjunctiva were ruled-out. resolution of habronemiasis within 5–18 days (Pusterla et al. Ocular habronemiasis and onchocerciasis are diseases 2003). Other recent literature suggests giving 2 doses of resulting from parasitic infestation of the conjunctiva. They ivermectin or moxidectin 21 days apart, and surgical have a seasonal occurrence observed during vector debridement of large lesions (Paterson 2009). Ivermectin is season, summer and autumn, and not during winter months larvicidal and does not kill adult O. cervicalis and thus (Schmidt et al. 1982; Pusterla et al. 2003). Ocular recurrence of lesions within 2–8 months after treatment is onchocercal microfilariae have been found less frequently common (Foil and Foil 1986). Currently, diagnosis of in younger horses (Schmidt et al. 1982). In contrast, our case onchocerciasis is not common in the USA (Brooks and was a 4-year-old gelding that developed clinical signs Matthews 2007). This gelding had resolution of keratitis during the month of December. Ocular habronemiasis before treatment with ivermectin and the lesions did not involves uni- or bilateral granulomatous lesions on the resolve with a single dose of ivermectin. These findings are conjunctiva and medial canthus of the eye (Foil and Foil not consistent with cases of ocular habronemiasis or most 1986; Pusterla et al. 2003). The lesions appear solitary or cases of onchocerciasis. No recurrence of lesions occurred multiple and may be ulcerated, have exuberant 10 months after discontinuation of corticosteroids which granulation tissue, and often have small yellow granules. would further rule out onchocerciasis. With reasonable Yellow, gritty plaques on the bulbar and palpebral clinical confidence, habronemiasis and onchocerciasis conjunctiva can also be present (Scott 1988). The granules were ruled out as possible causes of the conjunctival are necrotic mineralised tissue surrounding the nematode plaques in this gelding. larvae (Pusterla et al. 2003; Brooks and Matthews 2007). The The other major causes of conjunctival eosinophilic lesions seen in this gelding did involve the conjunctiva, but infiltration are EK, EC and EKC. These diseases are were caseous and not granular. Ocular onchocerciasis is differentiated based on location of lesions. Clinical characterised by unilateral or bilateral uveitis, conjunctivitis, signs include: blepharospasm, epiphora, conjunctival keratitis, as well as depigmentation and vitiligo of the lateral hyperaemia and chemosis, copious yellow to mucoid limbus and limbal nodules (Schmidt et al. 1982; Fadok and ocular discharge, and corneal Ϯ conjunctival plaques. The Mullowney 1983; Munger 1983; Brooks and Matthews 2007). limbus is often involved and the disease can be unilateral The plaques in this case were on the bulbar conjunctiva at or bilateral. Corneal ulcerations are present with EKC the limbus and no vitiligo of the temporal conjunctiva (Brooks 2004, 2005). No corneal ulcerations were present in developed. this case. Cytological samples of lesions in both EK and EKC

diseases contain numerous eosinophils, few mast cells, In the case reported here, parasitic causes were ruled lymphocytes and plasma cells, and histologically out with histological sampling and failure to respond to coalescing foci of degenerating collagen fibres infiltrated anthelmintic treatment. The final diagnosis of EC was by eosinophils, neutrophils, lymphocytes, and plasma cells, made through exclusion. The pathogenesis of EC is surrounded by eosinophilic granular material (Ramsey unknown. The gelding will be frequently monitored for signs et al. 1994; Brooks 2004, 2005). In this gelding, a diagnosis of of recurrence. If lesions recur, conjunctival biopsy and EC was made based on lack of corneal involvement and cytology are recommended. Continued research into the cytological and histological evidence of eosinophilic causes of EK, EKC and EC in equids is needed. Treatment infiltration of the conjunctiva and plaques. with immunosuppressive drugs has been used in cats; this is Treatment of eosinophilic diseases involves topical an area of expanding interest for management of these corticosteroids despite presence of corneal ulcers, and ocular diseases in horses. supportive topical antibiotics, atropine and systemic NSAIDS (Brooks 2004). Treatment is often several months in Manufacturers’ addresses duration and superficial keratectomies have been performed to speed healing (Yamagata et al. 1996; Brooks 1Welch Allyn, Skaneateles, New York, USA. 2004). Residual corneal scarring is a common sequela 2Heine USA, Dover, New Hampshire, USA. of EK and EKC (Ramsey et al. 1994). Mean duration of 3Volk Optical, Mentor, Ohio, USA. treatment for EKC is 64 days (45–106 days) (Yamagata 4Kowa, Tokyo, Japan. et al. 1996). No signs of corneal scarring occurred in this 5Mentor, Norwell, Massachusetts, USA. gelding and several weeks (73 days) of topical corticosteroid therapy was required for resolution of lesions References without recurrence. Feline EK, EKC, EC are classified as slowly progressive Allgoewer, I., Schaffer, E.H., Stockhaus, C. and Vögtlin, A. (2001) Feline infiltrative diseases that are uni- or bilateral involving the eosinophilic conjunctivitis. Vet. Ophthalmol. 4, 69-74. cornea, conjunctiva or both. Pathogenesis is unknown, Bourges-Abella, N., Raymond-Letron, I., Diquelou, A., Guillot, E., Regnier, although an immune or allergic cause has been A. and Trumel, C. (2007) Comparison of cytologic and histologic evaluations of conjunctiva in the normal equine eye. Vet. hypothesised (Pentlarge 1991). Diagnosis is made on Ophthalmol. 10, 12-18. cytology and histopathology of lesions with the presence of Brooks, D.E. (2004) Inflammatory stromal keratopathies: Medical large numbers of eosinophils with or without mast cells in the management of stromal keratomalacia, stromal abscesses, conjunctiva (Pentlarge 1991; Allgoewer et al. 2001). eosinophilic keratitis, and band keratopathy in the horse. Vet. Clin. equine Pract. 20, 345-360. Histologically, both equine and feline EC contain Brooks, D.E. (2005) Equine stromal and endothelial keratopathies: eosinophils, lymphocytes and plasma cells. EC in cats medical management of stromal abscesses, eosinophilic keratitis, responds to topical steroids and immunosuppressive drugs calcific band keratopathy, striate band opacities, and endothelitis with resolution in 3–6 weeks however, due to recurrence of in the horse. Clin. Tech. equine Pract. 4, 21-28. ocular lesions with cessation of medications, lifelong or Brooks, D.E. and Matthews, A.G. (2007) Equine ophthalmology. In: Veterinary Ophthalmology, Volume II, 4th edn., Ed: K.N. intermittent treatment is required in most feline cases Gelatt, Blackwell Publishing, Ames. pp. 45, 1191-1192, 1200- (Pentlarge 1991; Allgoewer et al. 2001; Spiess et al. 2009). 1201. The lesions in this case did not return after cessation of Fadok, V.A. and Mullowney, P.C. (1983) Dermatological diseases of treatment. horses. Part I. Parasitic dermatoses of the horse. Comp. cont. Educ. Although feline eosinophilic conjunctivitis is vet. Pract. 5, 615-623. well-documented, equine EC has not been documented. Foil, L. and Foil, C. (1986) Parasitic skin diseases. Vet. Clin. N. Am.: Equine Pract. 2, 403-437. Equine keratitis and keratoconjunctivitis are similar to Giuliano, E.A. and Moore, C.P. (2002) Eyes and ocular adnexa. In: feline keratitis and keratoconjunctivitis in clinical Diagnostic Cytology and Hematology, 2nd edn., Eds: R.L. Cowell presentation, diagnosis and treatment. Thus, it is and R.D. Tyler, Mosby, St Louis. pp 43-57. presumed that feline EC would support an occurrence of Maggs, D.J. (2008) Conjunctiva. In: Slatter’s Fundamentals of Veterinary a parallel disease process in horses. In this case, only the Ophthalmology, 4th edn., Eds: D.J. Maggs, P.E. Miller and R. Ofri, Saunders Elsevier, St Louis. pp 135-150. conjunctiva was involved, initial corneal oedema Munger, R.J. (1983) Equine onchocercal keratoconjunctivitis. Equine resolved 10 days after presentation and no signs of vet. J., Suppl. 2, 65-70. corneal ulceration or plaque involvement were observed. Paterson, S. (2009) Cutaneous habronemiasis. Equine vet. Educ. 21, Initially corneal oedema and aqueous flare were present, 9-10. probably in sympathetic response to surrounding Pentlarge, V.W. (1991) Eosinophilic conjunctivitis in five cats. J. Am. conjunctival inflammation; however, corneal cloudiness is Anim. Hosp. Ass. 27, 21-28. reported in cats with EC (Pentlarge 1991). Equine EC may Pusterla, N., Watson, J.L., Wilson, W.D., Affolter, V.K. and Spier, S.J. (2003) have been diagnosed and treated but not reported in Cutaneous and ocular habronemiasis in horses: 63 cases (1988–2002). J. Am. vet. med. Ass. 222, 978-982. publication. Prior cases of eosinophilic conjunctivitis may Ramsey, D.T., Whiteley, H.E., Gerding, P.A. and Valdez, R.A. (1994) have been misdiagnosed as EKC due to limbal location Eosinophilic keratoconjunctivitis in a horse. J. Am. vet. med. Ass. 205, of lesions. 1308-1311. Continued on page 390 © 2010 EVJ Ltd