Feline intralenticular Encephalitozoon cuniculi: three cases from California

Case series summary Three domestic shorthair cats from California presented to veterinary ophthalmologists with immature cataracts. Other presenting clinical signs included corneal edema, anisocoria, anterior uveitis, elevated intraocular pressure, blepharospasm and/or lethargy. All patients were immunocompromised due to concurrent diseases and/or immunomodulatory drugs. Diagnostics included serial comprehensive ophthalmic examinations with tonometry, ocular ultrasound, electroretinogram and testing for other causes of feline uveitis. Testing for Encephalitozoon cuniculi included serology, histopathology and/or PCR of aqueous humor, lens material or paraffin-embedded whole eye. Treatments included antiparasitic medication, anti-inflammatory medication and supportive care in all three cases. Surgical treatment included enucleation (one case), bilateral phacoemulsification and unilateral intraocular lens placement (one case) and bilateral phacoemulsification with bilateral endolaser ciliary body ablation and bilateral intraocular lens implantation (one case). Both cats for which serologic testing for E cuniculi was performed were positive (1:64–1:4096). In all cats, diagnosis of intraocular E cuniculi was based on at least one of the following: lens histopathology or PCR of aqueous humor, lens material or paraffin-embedded ocular tissue. The clinical visual outcome was best in the patient undergoing phacoemulsification at the earliest stage of the cataract. Relevance and novel information Encephalitozoon cuniculi should be considered as a differential cause of cataracts and uveitis in cats in California, the rest of the USA and likely worldwide.


Introduction
Encephalitozoon cuniculi is a worldwide microsporidian. 1,2 Spores can be transmitted via respiratory, oral, 3 conjunctival, 4 intranasal, intraovarial or transplacental routes. 5 In veterinary ophthalmology, E cuniculi is predominantly known as a cause of cataracts and phacoclastic uveitis in rabbits; 6,7 after transplacental transmission, spores are speculated to enter the lens via the lenticular blood supply while it is still present. 8 However, a recent study with immunohistochemical evidence of intralenticular E cuniculi after oral infection in 4-month-old, immunocompetent, specific pathogen-free rabbits 9 suggested that alternative mechanisms for lens infections are possible. Reports of ocular involvement in other species are limited and include cataract and uveitis in a snow leopard in France, 10 cataract, uveitis and chorioretinal lesions in dogs in Europe, 11 keratitis and uveitis in an American cat, 12 polyarteritis nodosa and cataract in a blue fox, 13 cataract and neurologic lesions in mink in Norway 14 and keratoconjunctivitis in an American cockatoo. 15 In addition, E cuniculi has been thoroughly investigated as a cause of feline cataract in Austria. 16 This report includes the clinical data from three feline cases of intralenticular E cuniculi in California, USA; one case is discussed at length (case 1), while the other two cases (cases 2 and 3) are summarized in Table 1.

Case series description
Case 1 A 3-year-old male castrated domestic shorthair cat presented to an emergency service for blepharospasm, anisocoria and lethargy. The patient had a history of chronic upper respiratory infections and diarrhea. It had been rescued from a southern Californian shelter and lived indoors in San Francisco.
One month after initial presentation, the patient developed a large superficial corneal ulcer OS, suspected to be related to herpes exacerbated by topical steroids. Prednisolone acetate was discontinued, and bacitracin neomycin gentamicin ophthalmic ointment (AC Pharmaceuticals, Arroyo Grande CA) was added OU q8h.
Approximately 2 months after the initial examination, the corneal ulcer OS persisted. Aqueous flare was trace OD and 1/4+ OS, with an intraocular pressure (IOP) of 19 mmHg OD and 44 mmHg OS. Aqueocentesis was performed OS, and aqueous humor was submitted for PCR to determine if C neoformans, Bartonella species or feline coronavirus (FCoV) were the cause of uveitis. Because the patient's cataracts appeared to be similar to those described in a previous report, 16 a special request was made to IDEXX to add an E cuniculi PCR test. A contact lens (PureVision BC 8.6; Bausch and Lomb) was placed, and a partial lateral temporary tarsorrhaphy was performed for 2 weeks. Medication administered immediately after the procedures included bacitracin neomycin gentamicin ophthalmic ointment (OU q8h) and dorzolamide HCl/timolol maleate (OS q8h; Bausch and Lomb). Oral medications included ronidazole (for diarrhea), buprenorphine (Hikma 0.5 mg/ml), robenacoxib (6 mg PO q24h for 3 days [Onsior; Elanco]) and famciclovir (250 mg PO q12h; Neogen). Aqueous humor cytology (Veterinary Diagnostics) showed increased cellularity, with 68% mixed (mostly mature) lymphocytes, 15% quiescent to vacuolated macrophages and 17% nondegenerate to slightly poorly preserved neutrophils (see Tables 1 and 2). Given the positive aqueous humor PCR result for E cuniculi, treatment with fenbendazole (50 mg/kg PO q24h for 3 weeks) was initiated.
Three months after initial presentation, ophthalmic examination indicated similar signs of uveitis with punctate fluorescein positivity OS only, and IOP was 16 mmHg OD and 8 mmHg OS. Topical 0.1% nepafenac ophthalmic suspension (OU q12h; Nevanac Alcon) was initiated.
Four months after initial presentation, aqueous flare had resolved with normal IOP without any dorzolamide/ timolol in the previous 3 days. Given the anticipated difficulty in controlling uveitis medically and the likelihood that cataracts would progress in the long term, cataract surgery was considered. Owing to the patient's positive FCoV status, thoracic radiographs (unremarkable), abdominal ultrasound (splenomegaly and mild mesenteric lymphadenopathy) and ultrasound-guided aspiration of a mesenteric lymph node (cytologically normal) were performed (see also Table 1).
Postoperatively, topical anti-inflammatories were tapered over several months, and dorzolamide/timolol was eventually discontinued. On ophthalmic recheck 17 months postoperatively, the patient was visual, comfortable, normotensive and PLR positive OU. There was mild anisocoria, dyscoria and mydriasis OD, aphakia OD, pseudophakia OS, no aqueous flare OU, minimal capsular opacity and an unchanged retinal examination with numerous punctate gray lesions in the dorsal retina OU. Medications consisted of 0.1% Nevanac (OU q24h). At home, vision was reportedly very good.

Discussion
This case series demonstrates that intralenticular E cuniculi is a potential cause of cataracts, uveitis and secondary glaucoma in domestic cats in California, USA.
Although E cuniculi is found worldwide, feline ocular encephalitozoonosis has only been reported in Austria, 16 France 10 and the USA (feline cornea). 12 Factors including climate and animal reservoirs may affect E cuniculi's prevalence and risk to cats. Specifically, environmental spore viability varies by temperature. 3 Given that encephalitozoonosis in rodents has been documented worldwide, 5,17-22 rodents likely spread disease, as corroborated by case 1 and several Austrian cases that tested positive for the mouse strain (strain II). 16 Although E cuniculi is an opportunistic pathogen in immunocompromised people, 23 the role of immunosuppression in feline ocular encephalitozoonosis remains unclear. The cases in this study were immunocompromised due to concurrent diseases (see Table 1) and immunomodulatory drugs (prednisolone and chlorambucil in case 2). This aligns with the current understanding that immunosuppression exacerbates rabbit encephalitozoonosis. 4 However, in the 2011 study published by Benz et al, 16 11 systemically healthy European Shorthair cats also developed cataracts and uveitis from E cuniculi, though 4/11 cats had positive titers for Toxo plasma gondii (IgG 1:4000). In the same study, 2/100 ophthalmologically healthy cats had a positive antibody titer for E cuniculi. Research conducted in North America, 24,25 Europe 18,26,27 and Asia 28-30 has found that E cuniculi prevalence range from 0% to 26.8%, with one paper demonstrating a seroprevalence of 6.1% (18/295) 30 in healthy, asymptomatic cats.
The mechanism by which E cuniculi causes uveitis is unknown. E cuniculi antigens may contribute to the inflammatory response; 31 this is supported by Nell et al 11 and cases 1 and 3, which suggest that focal anterior cataracts due to E cuniculi may be more inflammatory relative to focal cataracts due to other etiologies. Alternatively, E cuniculi may replicate and physically disrupt the lens, leading to lens-induced uveitis. 7 In case 1, aqueous humor PCR screening failed to show any evidence of other intraocular infections and supported E cuniculi being the causative agent for uveitis.
Currently, phacoemulsification surgery, antiparasitic medication and symptomatic treatment are employed to treat intralenticular E cuniculi infections. Phacoemulsification treats cataracts, removes microsporidia and minimizes further pathogen replication and intraocular inflammation. Fenbendazole, often prescribed at ranges of 20-50 mg/kg q24h for 3 weeks (extra-label), targets various pathogen stages. 32 Symptomatic treatment often includes oral and ophthalmic anti-inflammatories to address anterior uveitis. Since systemic immunosuppression facilitates E cuniculi, 33 corticosteroids should be employed at anti-inflammatory doses. The literature and this report suggest that surgical management of intraocular E cuniculi via phacoemul sification, especially early phacoemulsification, 34 can successfully maintain vision and comfort, while medical management alone may more commonly lead to blindness, discomfort and enucleation. 16 Various diagnostic testing is available for E cuniculi (see Table 2). Serology is a non-invasive, low-risk screening tool that is expected to be weakly or strongly positive for E cuniculi in cats with intraocular E cuniculi; however, PCR positivity of ocular fluid/tissues provides more definitive evidence of intraocular involvement. PCR detection of E cuniculi varies based on sample location. In Benz et al, aqueous humor from 10/19 affected cats was PCR positive, whereas lens material was PCR positive in one or both eyes in 11/11 of these cats. 16 Histopathology with hematoxylin and eosin stains can help guide the diagnosis of E cuniculi (see Figure 3a-c); however, the preferred histologic stains for E cuniculi spore detection are modified trichrome and Gram stain with light microscopy and calcofluor white stain with ultraviolet light microscopy, 35 though acid fast trichome can be effective (see Figure 3d); 16 in case 3, Luna stain was helpful.
When feline cataracts are identified, possible causes include chronic uveitis (most common), trauma (especially penetrating trauma), E cuniculi, secondary to glaucoma or lens luxation, congenital, possibly hereditary, nutritional or uncommonly metabolic (hypocalcemia, hyperphosphatemia, diabetes). 36 The cause of feline cataracts can be very difficult to determine, particularly since chronic uveitis commonly causes cataracts and vice versa. Cataracts caused by chronic lens-induced uveitis and those caused by E cuniculi can be very similar in appearance and size (ranging anywhere from incipient to mature in this report). However, in the experience of the authors, E cuniculi cataracts typically originate as focal lesions in the anterior cortex and spread from there to the whole lens. As cases are presented at different stages, the appearance of E cuniculi cataracts can differ in size and stage of maturity. We suspect that smaller (incipient) E cuniculi cataracts can cause disproportionately severe and acute uveitis and/or may progress somewhat more quickly relative to incipient cataracts of other etiologies (with the possible exception of penetrating trauma where an obvious corneal lesion would be expected). In two cases in this report (cases 1 and 3), incipient cataracts were associated with 1/4 or 3/4+ aqueous flare, which is unusual in cataracts not associated with traumatic intralenticular bacterial implantation or long-standing uveitis. However, inflammation caused by E cuniculi cataracts can be variable; in case 2, cataracts were immature and uveitis was initially minimal (although this patient was also on oral prednisolone for intestinal lymphoma). Speed of progression of E cuniculi cataracts can also be variable. In case 2, cataracts were reported to be rapidly progressive, whereas in case 3 the cataract progressed from incipient to mature over 6 years. Furthermore, features of chronic uveitis that may have led to these cataracts (such as chronic iris discoloration or large areas of posterior synechiation) were generally lacking in the cases presented here, except for mild rubeosis in case 3 and very focal synechiation in case 1 (see Figures 1 and 2). Ultimately, serology for E cuniculi is recommended as a screening tool in all cases of feline cataracts for which an alternative underlying cause is not apparent. If E cuniculi serology is positive, then referral to an ophthalmologist for additional (PCR) testing of ocular tissues and more intensive medical and surgical treatment should be considered, as this would be expected to improve the clinical outcome.

Conclusions
This study highlights E cuniculi as a cause of feline cataracts in the USA (and likely worldwide). Study limitations include low case numbers and heterogeneous, incomplete patient data with limited follow-up. Although this series provides clinically relevant information, it does not necessarily represent optimal treatment of feline ocular E cuniculi. Because the literature on feline encephalitozoonosis is somewhat lacking, future studies should more thoroughly evaluate systemic involvement, pathophysiology and/or best treatment practices. E cuniculi should be considered in cats presenting with cataracts, especially those with concurrent anterior uveitis. The authors hope that increased awareness and testing will lead to earlier diagnosis of feline intraocular E cuniculi and improved clinical outcomes.
Author note Case 3 of this series was presented at a specialty conference in 2015. 37